article stringlengths 0 826k | abstract stringlengths 174 4.34k |
|---|---|
obesity increases the risk for hypertension and is a major driver of morbidity and mortality due to cardiovascular diseases ( dustan , 1983 ; poirier et al . , 2006 ) .
studies in rodents with diet - induced obesity ( dio ) suggest that increased sympathetic nerve activity ( sna ) is an important mediator of obesity - induced hypertension as and adrenergic receptor antagonists and renal denervation significantly blunt the rise in blood pressure ( bp ) associated with weight gain ( carlyle et al . , 2002 ;
however , the precise molecular and neural mechanisms that link changes in weight with changes in bp have not been fully elucidated .
circulating concentrations of the adipocyte - derived hormone leptin increase in proportion to adipose tissue mass and fall with weight loss ( considine et al .
, we hypothesized that leptin may be involved in coupling changes in body weight ( bw ) to changes in bp .
leptin regulates energy homeostasis by acting on hypothalamic neuronal circuits expressing the signaling isoform of the leptin receptor ( lepr ) to reduce calorie intake and increase energy expenditure ( halaas et al . , 1997 ;
leptin can increase sna , leading to increases in bp and heart rate ( hr ) ( haynes , 2000 ; mark et al . , 1999 ) . in the arcuate nucleus of the hypothalamus ( arh )
, leptin stimulates the expression of pro - opiomelanocortin ( pomc ) and increases the activity of pomc neurons , which release the pomc peptides ( , , and melanocyte - stimulating hormones [ mshs ] ) that act on melanocortin 4-receptor ( mc4r)-expressing neurons in the paraventricular nucleus of the hypothalamus ( pvh ) and other brain regions to increase sna ( cone , 2005 ; cowley et al . , 1999 , 2001 ;
however , pomc neurons become unresponsive to leptin in obesity , and leptin can act independently of mc4r signaling ( enriori et al . , 2011 ; patterson et al .
, 2011 ; scott et al . , 2009 ) . therefore , leptin s effects beyond the melanocortin circuits need to be investigated .
the dorsomedial hypothalamus ( dmh ) is critical for leptin s ability to regulate brown adipose tissue ( bat ) temperature and the cardiovascular system ( enriori et al . , 2011 ;
; rezai - zadeh et al . , 2014 ) . here , we investigated the development of obesity - induced hypertension .
we demonstrate that , in dio mice , increasing levels of leptin directly lead to an increase in hr and bp and that blocking the actions of leptin reverses these effects via neural circuits originating in the dmh .
furthermore , humans with loss - of - function mutations in leptin and its receptor have normal bp despite severe obesity , suggesting that these mechanisms are likely to be preserved in humans .
the temporal association between weight gain , changes in circulating leptin levels , hr , and bp were first examined .
four - week - old c57bl/6j mice on a chow diet were implanted with radiotelemetric bp probes ; baseline measurements were recorded at 6 weeks . as bw increased ( figure 1a ) , plasma leptin levels also progressively increased ( figure 1b ) .
after 4 weeks of hfd , hr became significantly elevated ( figure 1c ) and remained elevated throughout the 20 week recording period compared to chow fed mice .
systolic blood pressure ( sbp ) and diastolic blood pressure ( dbp ) were significantly elevated after 12 weeks of hfd ( figures 1d and 1e ) , subsequent to the rise in plasma leptin concentration . when hfd - fed mice were returned to a chow diet following 20 weeks of hfd feeding , mice lost 3.9 g in the first week , 3.9 g in the second week , 3.4 g in the third week , and 0.4 g in the fourth week ( figure 1a ) .
dbp reduced after 1 week ( figure 1e ) , and sbp ( figure 1d ) and hr ( figure 1c ) reduced after 2 weeks of chow feeding .
these findings suggest that leptin appears to increase before hr and bp increases in dio , effects that are reversed with weight loss .
we examined whether the effects seen in dio mice are dependent upon leptin signaling , using mice lacking leptin ( ob / ob ) or the lepr ( db / db ) .
hfd - fed ob / ob and db / db mice were significantly ( p < 0.001 ) heavier than hfd - fed dio and chow - fed lean , ob / ob , and db / db mice ( figure 2a ) . despite the increased severity of obesity in the ob / ob and db / db mice , only dio mice exhibited elevated hr , sbp , and dbp ( figures
2b2 g ) , demonstrating that increased bw alone is not the cause of the increase in bp in obesity .
there was a greater difference in hr and bp between dio mice to other mice during the dark period compared to the light period ( figures s1a s1c available online ) .
the increase in cardiovascular parameters in dio mice was not due to increased activity ( figures s1d and s1e ) .
strong correlations were found in c57bl/6j mice between plasma leptin concentration and hr ( figure 2h ) , sbp ( figure 2i ) , and dbp ( figure 2j ) .
no correlation was found between plasma insulin concentration and hr , sbp , or dbp ( figures s1g s1i ) . to examine the direct effect of leptin on bp ,
leptin - deficient ob / ob mice were treated for 11 days with either leptin or vehicle .
after 11 days of treatment , the average plasma leptin concentration of ob / ob mice treated with leptin was 27.7 2.9 ng / ml compared to 36.3 4.9 ng / ml in dio mice and 6.6 1.1 ng / ml in lean mice ( figure s1f ) .
exogenous leptin treatment in ob / ob mice progressively decreased bw ( figure s1 m ) and food intake ( fi ) ( figure s1n ) compared to leptin - treated db / db mice and vehicle - treated ob / ob mice . despite this decrease in bw , leptin treatment increased hr ( figures 2k and 2l ) and sbp ( figures 2 m and 2n ) in leptin - treated ob / ob mice compared to leptin - treated db / db mice and vehicle - treated ob / ob mice .
dbp also increased in ob / ob leptin - treated mice , however , not to the same extent as sbp ( figures 2o and 2p ) .
the magnitude of the increase in hr and bp in leptin - treated ob / ob mice decreased with time , coincident with their substantial reduction in bw .
the 24 hr circadian rhythm of hr ( figure s2j ) , sbp ( figure s2k ) , and dbp ( figure s1l ) of leptin - treated ob / ob mice was increased toward the baseline recordings of dio mice .
peripheral administration of a leptin antibody into dio mice for 5 days caused a significant reduction in hr ( figure 3a ) , sbp ( figure 3b ) , and dbp ( figure 3c ) , although no change in bw was observed ( figure 3d ) .
central administration of a leptin receptor antagonist ( lra ) into the lateral ventricle ( lv ) of hypertensive dio mice over a 7 day period significantly reduced the elevated hr ( p < 0.001 ; figure 3e ) . by the seventh day of lra treatment , hr was comparable to the average hr of lean animals ( figures 3f and s2a ) .
systolic bp also progressively decreased when dio mice were treated with the lra , and by day 5 of lra treatment , sbp was significantly ( p 0.05 ) decreased compared to vehicle - treated dio mice ( figure 3 g ) . by the seventh day of lra treatment , sbp was significantly ( p < 0.05 ) lower compared to the sbp at baseline in dio mice and was comparable to the baseline sbp of lean mice ( figures 3h and s2b ) .
dbp also decreased throughout the lra treatment period ( figures 3i , 3j , and s2c ) .
these changes were seen despite the absence of a significant change in fi ( figure 3k ) , indicating that these changes are mediated by blocking the effects of leptin signaling rather than through changes in fi and bw . to determine whether leptin s effects on bp and hr were mediated by neurons in the dmh , the lra was injected directly into the dmh of hypertensive dio mice daily over 7 days .
lra treatment in the dmh decreased hr ( figures 4a , 4b , and s3a ) and sbp ( figures 4c , 4d , and s3b ) by day 7 of treatment .
no significant change in dbp ( figures 4e , 4f , and s3c ) was observed .
furthermore , injection of an aav expressing a short hairpin rna directed against the lepr ( hommel et al . ,
2006 ) in the dmh of dio mice led to a sustained decrease in sbp ( figures 4 g , 4h , and s3d ) after 4 weeks . utilizing mice in which the lepr is flanked by loxp sites and can be deleted by an aav cre , hfd for 20 weeks induced increased sbp at baseline , whereas aav cre administration into the dmh ( hence deleting the lepr in only the dmh regions ) decreased sbp ( figure 4i ) . additionally , we examined the effects of reactivation of the lepr by injection of an aav - expressing cre recombinase into the dmh of normotensive morbidly obese lepr null mice ( expressing a loxp flanked stop codon in front of the lepr , called lepr transcriptional blocker or
both hr ( figure 4j ) and sbp ( figure 4k ) increased rapidly after lepr expression was reactivated in just the dmh of obese lepr - deficient mice . to determine the electrophysiological effect of leptin on dmh neurons
, we recorded the electrical activity of neurons expressing lepr in lepr - cre - yfp mice ( leshan et al . , 2012 ) .
application of 100 nm leptin induced membrane depolarization and/or an increase in spontaneous action potential firing rate in 38.2% of recorded neurons ( figure 5a ) .
leptin induced a mean peak amplitude depolarization of 4.9 1.0 mv from a mean resting potential of 49.9 3.1 mv to a new steady - state membrane potential of 45.0 2.7 mv ( p = 0.0004 ) .
leptin - induced excitation was associated with an increase in firing rate from a mean control basal level of 0.48 0.28 hz to 0.64 0.34 hz in the presence of leptin ( n = 5 ) , effects that were at least in part reversible after washout of leptin ( p = 0.59 ) . in two neurons ,
voltage - current relations , generated in response to a range of depolarizing and hyperpolarizing rectangular - wave current pulses ( 150 to + 100 pa , 1,000 ms duration ) revealed that leptin - induced excitation was principally associated with a trend for a decrease ( p = 0.86 ) in neuronal input resistance from 841 111 m in the absence to 743 138 m in the presence of leptin .
plots of the voltage - current relations revealed extrapolated reversal potentials for leptin - induced excitation around 35 mv ( figure 5b ) . taken together , these data suggest that leptin - induced excitation is mediated via activation of one or more nonselective cation conductances .
in addition to these effects on membrane input conductance , leptin also induced modulation of intrinsic subthreshold active conductance in a subpopulation of dmh neurons . in dmh neurons ,
membrane depolarization from negative holding potentials ( < 65 mv ) or depolarization at the offset of the membrane response to hyperpolarization from potentials close to resting potential ( 45 to 50 mv ) evoked a transient depolarizing potential consistent with activation of a low - threshold t - type calcium conductance . in the presence of leptin ,
this potential was prolonged ( figure 5b ) , the half - time to decay increasing from 132 59 ms in the absence to 179 61 ms in the presence of leptin .
these data are consistent with leptin modulating intrinsic active conductances in a subpopulation of dmh neurons .
in addition to these postsynaptic effects , leptin induced an increase in spontaneous excitatory postsynaptic potentials ( epsps ) in a subpopulation ( n = 2 ) of dmh neurons ( figure s4b ) .
the mean frequency of spontaneous epsps increased from 0.07 0.02 hz to 0.31 0.11 hz in the presence of leptin .
these epsps could be of sufficient magnitude to reach threshold for firing , suggesting that indirect presynaptic effects of leptin on dmh neurons can contribute to leptin - induced increases in neuronal excitability . in vivo ,
the effects of directly altering the neuronal activity of lepr - expressing dmh neurons were assessed using engineered pharmacologically selective chimeric ion channels for activating and silencing neuron activity ( magnus et al . , 2011 ) .
briefly , this technology requires the injection of a virus , which only infects and replicates in a cre - dependent manner , into cre - expressing mice .
the virus drives the cre - dependent expression of a modified ion channel , containing a psam element .
after injection ( intraperitoneal injection ) , the otherwise inert molecule psem binds to an ion channel that contains a psam element , which opens the ion pore and allows ion flux across the cell membrane , which depolarizes or hyperpolarizes virus - infected , cre - expressing neurons . using male 20-week - old chow - fed lean lepr - cre - yfp transgenic mice ( leshan et al . , 2012 ) , we investigated whether activation of dmh lepr - expressing neurons could increase hr and bp .
lean lepr - cre - yfp mice were injected bilaterally with activator virus , an engineered ionotropic serotonin receptor packaged in an aav2 ( psyn::flex - rev - psam y115f:5ht3 hc - ires - gfp ) into the dmh . in lean mice 21 days after virus injection , twice daily intraperitoneal ( i.p . ) administration of the receptor ligand psem ( i.p .
2x daily [ 5 mg / kg ] ) for 3 days significantly increased hr and bp ( p 0.05 ; figures s4d
lepr - cre - yfp mice , an inhibitory virus , an engineered ionotropic glycine receptor packaged into an aav2 ( aav2 : psyn::flex - rev - psam l141f : glyr - ires - gfp ) was administered bilaterally into the dmh .
compared to vehicle treated mice , psem acutely decreased hr ( figures 5c and 5d ) and sbp ( figures 5e and 5f ) , but no significant change in dbp was observed ( figures 5 g and 5h ) .
sub - chronic daily treatment with psem reduced hr ( by 72.6 5.6 bpm ) , sbp ( by 6.1 1.7 mmhg ) and dbp over 3 days ( figures 5i , 5j , and s4c ) , effects which were reversed once psem treatment ceased .
treatment of mice with psem , prior to virus administration had no effect on hr or bp ( figures s4 g and s4h ) .
cumulatively , these findings indicate that leptin signaling is required for the changes in bp seen in dio and that lepr expressing neurons in the dmh are necessary and sufficient to , cause these effects .
these data also support the premise that the hypertension induced by leptin in the dmh is due to leptin - induced depolarization of dmh neurons .
homozygous complete loss - of - function mutations in the gene encoding leptin ( lep ) are associated with undetectable leptin levels and severe early - onset obesity in humans ( montague et al . , 1997 ) .
we measured bp in the fasted rested state in eight leptin - deficient children and 42 equally obese controls of the same age with normal leptin levels for the degree of obesity studied in the same conditions ( table s1 ) .
sbp was significantly lower in leptin - deficient children compared to age- and bmi - matched controls ( figure 6a ; p < 0.05 ) ; there was no difference in dbp .
a statistically significant attenuation of sbp was also seen in severely obese children who were homozygous for complete loss - of - function mutations in the lepr gene ( n = 12 ) compared to 48 age- and bmi - matched controls ( figure 6b ; p <
there were no significant differences in resting hr between the groups ( data not shown ) .
administration of recombinant methionyl human leptin to individuals with congenital leptin deficiency leads to significant weight loss ( farooqi et al . , 1999 ;
ozata et al . , 1999 ) . in a previously reported experimental paradigm ( galgani et al . ,
2010 ) , three leptin - deficient adults were studied before and after treatment with recombinant leptin for 19 weeks , which was sufficient to cause 15.5 0.5 kg weight loss . in parallel , three age- and bmi - matched controls were studied before and after a diet and exercise intervention , which achieved a comparable degree of weight loss ( 14.8 1.76 kg ) .
the three adult leptin - deficient individuals were found to have normal bp despite severe obesity ( figure 6c ) . whereas the map of the obese control group decreased as expected ( figure 6d ) , the bp of the leptin - deficient adults did not change .
similarly , there was no statistically significant change in bp after recombinant leptin administration to leptin - deficient children ( data not shown ) .
these studies in rare individuals completely lacking leptin or functioning lepr support the assertion that leptin is necessary for the increased bp associated with obesity in humans .
cumulatively , these studies demonstrate that leptin signaling is necessary for obesity - induced increased bp .
we have used multiple convergent methods , including making lean mice obese , by adding leptin systemically and by restoring lepr to the dmh of lepr - deficient mice . in all these studies , bp increased .
similarly , we have studied reduced leptin signaling in leptin- and lepr - deficient mice , neutralized circulating leptin with a systemic antibody , infused lepr antagonist icv and intra - dmh ; used shrna to knock down lepr expression ; selectively deleted lepr ; and inhibited lepr - expressing neurons in the dmh of obese hypertensive mice . in all of these experiments , reducing leptin signaling reduced bp , even in the presence of obesity .
clinical studies in severely obese humans with two different forms of defective leptin signaling show that these observations are relevant to human physiology and pathophysiology .
leptin can acutely increase hr , bp , and bat , even in anesthetized animals , presumably through activation of the sns ( enriori et al .
intracerebroventricular ( icv ) leptin increases sna to numerous organs , including the lumbar , kidney , and bat regions ( haynes et al . , 1999 ) .
central antagonism of lepr caused a reduction in bp and hr in dio hypertensive mice .
although early lesion studies confirm the importance of the dmh for the control of energy homeostasis , little is known about the neurochemical phenotype of the neurons present in the dmh ( elmquist et al . , 1999 ;
the dmh appears to have a critical ability to control thermogenesis and is involved in elevated bat - mediated thermogenesis found in obese mice ( enriori et al .
, 2011 ; fan et al . , 2005 ; morrison et al . ,
2008 ) . in the studies conducted with antagonism or knocking down of the lepr in dio mice
these results suggest that the dmh lepr - expressing neurons are not essential for leptin s effects on bw because the mice lost no additional weight when leprs were inactivated .
although lesion studies have previously demonstrated a critical role of the dmh in the control of bw , we have shown that long - term knockdown of the lepr does not significantly affect bw , despite the significant reduction in bp and previously reported increase in bat temperature in obesity ( elmquist et al . , 1999 ; enriori et al . , 2011 ) .
additionally , acute activation or inhibition of lepr - expressing neurons did not change fi or bw .
contrary to our expectations , we did not observe an increase in bp with the administration of leptin to leptin - deficient patients .
first , the magnitude of leptin s effect on bp may be too small to be detected given the sensitivity of the tools for measurement of bp in humans and the small number of individuals studied .
notably , despite weight loss with continued leptin administration , we did not see a significant decrease in bp in these patients as would be expected in more common forms of obesity .
these effects are comparable to the effects of leptin on total energy expenditure ( tee ) , another phenotype mediated by sympathetic tone .
we have previously shown that , in contrast to weight loss in obese controls where tee decreases , tee does not change with leptin administration in leptin deficiency ( galgani et al . , 2010 ) .
whether leptin increases bp ( to a small degree ) or leptin - deficient individuals respond differently to weight loss can not be deduced from our data . however , our results are in line with the suggestion that the effect of leptin administration may be to prevent the reduction in bp expected in congenitally leptin - deficient individuals as they lose weight . in previous trials of leptin in common obesity , no effects on bp were observed ( heymsfield et al . , 1999 ) .
rosenbaum and leibel have shown that controlled 10% weight loss in an experimental setting is sufficient to reduce leptin levels and is associated with decreased sna ( rosenbaum et al . , 2005 ) .
it is possible that leptin could be producing some of its cardiovascular effects by acting peripherally .
lepr are expressed throughout the brain , including the brainstem ; additionally , lepr are expressed on cardiac myocytes , the kidney , and arteries , including the coronary arteries ( knudson et al .
we treated dio mice peripherally with a leptin antibody and then in a separate experiment , dio mice were treated centrally with the lepr antagonist , and in both experiments , similar responses were observed . in mice lacking the lepr , re - expression of the lepr in only the dmh caused a dramatic increase in bp and hr .
the opposite effects occurred when the leptin receptors were deleted from only the dmh region in hypertensive mice .
thus , although lepr expression in peripheral regions may play a role , the results presented here clearly demonstrate a key role for the dmh lepr - expressing neurons in mediating the changes in bp in obesity .
experiments in rodents and humans support a role for melanocortin signaling in the regulation of bp .
central administration of -msh increases bp and hr in wild - type mice , but not in mc4r mice , which maintain a normal bp despite severe obesity ( kuo et al . , 2003
heterozygous loss - of - function mutations in mc4r are associated with a reduced prevalence of hypertension , low sbp , lower urinary noradrenaline excretion , and reduced peripheral nerve sna ( greenfield et al . , 2009 ; sayk et al . ,
2010 ) . moreover , systemic administration of a centrally active melanocortin receptor agonist acutely increased bp in obese volunteers ( greenfield et al . , 2009 ) .
as such , some of leptin s effects on bp may be mediated by the melanocortin system . however , there are some indications that , in obesity , the pomc neurons in the arh become nonresponsive to leptin ( this has been termed leptin resistance ) , and this may limit how much the actions of leptin can be transduced by the pomc neurons in the arh ( enriori et al . , 2007 ; knight et al . , 2010 ;
previous research has implicated the pomc neurons of the arh in the regulation of obesity - induced hypertension via activation of the ikk/nf-b pathway ( purkayastha et al . , 2011 ; zhang et al . ,
how pomc neurons interact with the dmh lepr - expressing neurons has not been addressed in these studies , but the dmh lepr - expressing neurons do send direct efferent projections to arh neurons ( gautron et al .
it is possible that the lepr - expressing neurons in the dmh express mc4r or that the dmh lepr - expressing neurons act through other neurons that express mc4r ( liu et al . , 2003 ) .
the degree to which leptin s effects on bp in obesity are dependent upon intact melanocortin signaling remains to be determined ( do carmo et al . , 2011 ; harlan et al .
dmh lepr - expressing neurons are known to project to numerous brain regions , including the pvh ( elmquist et al . , 1998 ; gautron et al .
dmh neurons also project to other nuclei , including the raphe pallidus nucleus ( rpa ) and rostral ventrolateral medualla ( rvlm ) ( cao and morrison , 2003 ; horiuchi et al .
microinjection of leptin into the dmh of anesthetized rats can induce an acute increase in hr and bp ( marsh et al . , 2003 ) .
the dmh - rpa connection has already been recognized to be important in the regulation of bat thermogenesis in response to leptin ( zhang et al . , 2011 ) .
the rvlm , however , appears to have a greater control over the regulation of bp , compared to regulation of thermogenesis and hr ( horiuchi et al .
the neurochemical phenotype of the lepr - expressing neuron populations in the dmh is still debated ( lee et al . , 2012 ) .
further studies will be necessary to characterize the dmh circuits that contribute to leptin s effects on blood pressure and to characterize the mechanisms by which these neurons modulate sympathetic outputs to the heart and peripheral blood vessels .
although results here strongly demonstrate the role of leptin in contributing to elevated bp in obesity , a number of other hormones that change in response to weight gain could contribute .
as expected , we found that , compared to lean mice , plasma insulin was elevated in all obese models . despite this increase in plasma insulin , it was only dio mice that exhibited significantly elevated bp , and insulin levels were not correlated with hr or bp , suggesting that insulin contributes little to the chronic elevation of bp seen in obesity . also , insulin sensitivity as measured by hyperinsulinemic euglycemic clamps is comparable in human mc4r deficiency versus obese controls despite a lower bp and reduced urinary catecholamine excretion in mc4r - deficient subjects ( greenfield et al . , 2009 ) .
therefore , our data do not support a role for insulin in mediating obesity - induced increases in bp . in conclusion
, these observations suggest that pharmacological approaches based on the modulation of leptin s effects on specific subpopulations of neurons could represent a potentially useful therapeutic strategy for the treatment of obesity - associated hypertension and for the prevention of some obesity - associated cardiovascular disease .
all mice were housed in a controlled environment in which lights were on a 12 hr light/12 hr dark cycle ; temperature and humidity remained constant . in experiments to examine the development of hypertension , 4-week - old male c57bl/6j mice were implanted with radiotelemetry probes ( dsi , usa , model ta11pa - c10 ) .
these mice were allowed 2 weeks recovery postsurgery and , following baseline recordings , mice were spilt onto either chow ( 4.8% of fat , mouse and rat rodent chow diet , specialty feeds , glen forrest , australia ) or hfd ( 43% of fat , sf04 - 001 , specialty feeds , western australia , australia ) for 20 weeks ( 140 days ) in which recordings were taken every 13 to 15 days .
after 20 weeks ( 140 days ) , the hfd fed mice were swapped onto a chow diet ( 4.8% of fat , sf04 - 001 , specialty feeds , western australia , australia ) for 4 weeks . in all other experiments , male c57bl/6j , leptin - deficient ( ob / ob ) , lepr - deficient ( db / db ) , lepr - deficient ( leprtb ( berglund et al . , 2012 ) , lepr flox , and lepr - cre - yfp mice ( leshan et al . , 2012 )
were placed on either a chow diet or hfd diet at 4 weeks of age and continuing for 20 consecutive weeks , after which mice were used for experiments .
specific experimental procedures for radiotelemetry , pharmacological studies , genetic manipulations , and electrophysiological studies are detailed in the supplemental information .
all human studies were conducted according to the principles outlined in the declaration of helsinki and after approval by local ethical committees .
systolic and diastolic bp were measured in the rested , fasted state using wrist bp monitors ( omron healthcare , hamburg , germany ) in leptin - deficient ( n = 8) and leptin - receptor - deficient children ( n = 12 ) ( farooqi et al . , 2002 , 2007 ) .
these control subjects had been tested for mutations in leptin , leptin receptor , and mc4r .
control subjects were age and bmi matched to leptin - deficient and leptin - receptor - deficient subjects ( n = 42 and 48 , respectively ) ( wheeler et al . , 2013 ) .
leptin - deficient adult patients received pretreatment testing at the pennington center , along with weight- and bp - matched control subjects .
the leptin - deficient patients then received a 3 month treatment of recombinant leptin ( leptin [ amgen inc . , thousand oaks ; galgani et al . , 2010 ] ) .
leptin levels were administered a low - calorie diet ( galgani et al . , 2010 ) for 920 weeks to cause the same weight loss and also returned to the pennington center for posttesting .
the study design was approved by the ethics committee of the pennington biomedical research center .
approval for leptin replacement included ucla irb approval ( april 9 , 2001 ) and fda approval ( ind application number 61690 ) .
p values were calculated by either unpaired or paired two - tailed student s t test , one - way anova with bonferroni post hoc test or two - way anova with bonferroni post hoc test .
p < 0.05 , p < 0.01 , and p < 0.001 . extended experimental proceduresradiotelemetryradiotelemetry probes were implanted into the left carotid artery ( ta11pa - c10 data science international ) under isoflurane anesthesia .
all mice received 2 weeks recovery before any baseline recordings were measured.chronic leptin infusionchow fed , ob / ob or db / db mice were implanted with radiotelemetry probes , and baseline recordings were made .
alzet 14 day osmotic minipumps ( model 1002 , cupertino , ca ) to administer either vehicle or leptin ( peprotech , rocky usa ) ( 30 g / per day in 6 l of solution [ 1.25 g/0.25 l per hour ] ) were implanted into a subcutaneous pocket 3hrs prior to the dark period .
pumps had been prepared and incubated at 37c , 8 - 9hrs prior to implantation .
pumps where removed after 11 days ( prior to the onset of the dark period ) and the mice recovered from treatment while radiotelemetry measurements continued to be recorded ( db / db mice were killed at day 11).immunohistochemistry and radioimmunoassayimmunohistochemistry ( ihc ) for pstat3 and radioimmunoassay ( ria ) measurement to determine plasma leptin concentration were performed as previously described ( enriori et al . , 2007).peripheral neutralization of endogenous leptinto neutralize leptin s actions a goat igg against recombinant mouse leptin ( af498 from r and d systems , minneapolis ) was peripherally ( i.p . ) administered into dio mice for 5 consecutive days , following a sham injection period .
injections of 24 g in 90 l of antibody solution or 90 l of igg control ( antibodies australia , victoria , australia ) . the dose of the antibody was determined from previous research ( konstantinides et al . , 2004 ) .
all mice where randomly treated.central blockade of endogenous leptinto reduce the activation of lepr expressing neurons in the brain , a leptin receptor antagonist ( lra ) ( mouse leptin antagonist mutant l39a / d40a / f41a , 0.5 l of 5g/l , protein laboratories , rehovot , israel ) was used as previously reported ( enriori et al . ,
2011 ) . in dio mice that where implanted with radiotelemetry probes , they were additionally equipped with either a lateral ventricle ( lv ) ( plastics one , 2 mm long 22 gauge , implanted 0.5 mm posterior and 1 mm lateral to bregma , 2 mm below the surface of the skull ) or dmh cannula ( plastics one , 4 mm long , 26 gauge , implanted 1.9 mm posterior , 0.4 mm lateral to bregma , 4 mm below the surface of the skull ) as described previously ( enriori et al . , 2011 ) .
following a 7 day recovery period , mice where all injected for 5 days twice daily with sham acsf injection ( 0.5 l ) into cannulas .
mice then received acsf or the lra , injected directly into cannulas , twice daily for 7 days , immediately following the dark period and immediately prior to the dark period . following the 7 day injection period mice where monitored daily for a 7 day recovery period.chronic knockdown of leptin receptors in the dorsomedial hypothalamusdio mice were implanted with radiotelemetry probes and after 14 days of recovery baseline recordings
mice were injected bilaterally into the dmh region ( coordinated 1.8 mm posterior , 0.3/-0.3
mm lateral from bregma and 5.2 mm below the surface of the skull ) 0.5 l of either an aav scrambled shrna or aav leprshrna over a 20 min period each side ( hommel et al . , 2006 ) .
bw and fi were measurements daily and radiotelemetry recordings were measured periodically throughout the treatment period.reactivation and deletion of leptin receptor in the dmhlepr loxtb mice and lepr flox mice ( berglund et al . , 2012 )
after which mice were implanted with radiotelemetry probes . following the surgery ( 2 weeks ) , baseline recordings where measured .
mice where then injected into the dmh ( coordinates 1.8 mm posterior , 0.3/-0.3
mm lateral from bregma and 5.2 mm below the surface of the skull ) with an aav cre virus .
mice where then monitored and cardiovascular parameters measured throughout.neuron activation and silencing with psam / psem systemsto examine the effect of activation or inhibition of dmh lepr expressing neurons , we injected a virus that only infects and replicates in a cre dependent manner , into lepr - cre - yfp mice ( leshan et al . , 2012 ) .
after injection , the otherwise inert molecule psem binds to an ion channel that contains a psam element which causes opening of ion pore in the channel and allows ion flux across the cell membrane , which causes depolarization or hyperpolarization of cre - expressing neurons .
the psam element only binds psem , this system is similar to dreadds , but uses direct ligand gated ion channels rather than gpcr s to drive neuron specific effects ( magnus et al . , 2011 ) .
these mice where placed on either the chow or hfd for 20 weeks then implanted with radiotelemtry probes and 2 weeks later bp radiotelemetry baseline recordings were made . following this
the lepr - cre - yfp dio mice were injected in the dmh ( coordinates 1.8 mm posterior , 0.3/-0.3
mm lateral from bregma and 5.2 mm below the surface of the skull ) with the inhibitor virus , psyn::flex - rev - psam l141f : glyr - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) , lean chow fed lepr - cre - yfp mice were injected in the dmh ( coordinated 1.8 mm posterior , 0.3/-0.3
mm lateral from bregma and 5.2 mm below the surface of the skull ) with the activator virus , psyn::flex - rev - psam y115f:5ht3 hc - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) .
injection of either of these virus s did not change baseline cardiovascular function , until the peripheral injection of the inert psem ligand , that caused opening of ion pores and activation or inhibition of neurons .
after a 21 day recovery period cardiovascular parameters were re - examined and then mice where sham injected followed by acute or 3 consecutive days of treatment with psem ( 5 mg / kg , i.p .
, twice daily ; apex scientific inc . stony brook , ny ) or vehicle , randomized , crossover design ( magnus et al . , 2011).electrophysiologyexperiments were undertaken on lepr - yfp - cre mice ( leshan et al . , 2012 ) following 20 weeks of chow diet feeding , ad lib . on the day of electrophysiological experiment
the brain was rapidly removed , submerged in ice cold artificial cerebrospinal fluid ( acsf ) and coronal slices ( 200 - 250 m thick ) cut using a vibratome ( leica , vt1000s , uk ) .
slices were maintained at room temperature in oxygenated acsf ( gassed with 95% 02 , 5% co2 ) for at least an hour prior to recording .
for recording , slices were continuously perfused at room temperature with acsf of the following composition ( in mm ) : 127.0 nacl , 1.9 kcl,1.2 kh2po4 , 26.0 nahco3 , 2.0 d - glucose , 8.0 d - mannitol , 1.3 mgcl2 , 2.4 cacl2 , equilibrated with 95% o2 , 5% co2 , ph 7.3 7.4 .
lepr - yfp expressing neurons in the dmh were visualized using fluorescence , fitted with appropriate filters and infrared video microscopy with dic optics and recordings obtained from these neurons with an axopatch 700a amplifier .
patch pipettes were pulled using a horizontal puller ( sutter instrument co. , novato , ca , usa ) from thin - walled , filamented borosilicate glass ( gc150tf-10 ; harvard apparatus ltd ) with resistances between 4 and 8 m when filled with intracellular solution . the pipette solution comprised ( mm ) : 140 k - gluconate ,
the ph was adjusted to 7.3 with koh and the osmolarity to around 310 mosm with sucrose .
data were filtered at 2 5 khz , digitized at 2 10 khz ( digidata 1322 , mds analytical technologies ) and stored on pc running pclamp 9 data acquisition software .
analysis of electrophysiological data was carried out using clampfit 9.2 software ( mds analytical technologies ) .
was determined using unpaired , non - parametric student s t test with p < 0.05 considered as significant .
leptin was made up as a concentrated stock solution and diluted to the concentration required ( 100 nm ) in acsf immediately before commencement of electrophysiological recording .
radiotelemetry probes were implanted into the left carotid artery ( ta11pa - c10 data science international ) under isoflurane anesthesia .
chow fed , ob / ob or db / db mice were implanted with radiotelemetry probes , and baseline recordings were made .
alzet 14 day osmotic minipumps ( model 1002 , cupertino , ca ) to administer either vehicle or leptin ( peprotech , rocky usa ) ( 30 g / per day in 6 l of solution [ 1.25 g/0.25 l per hour ] ) were implanted into a subcutaneous pocket 3hrs prior to the dark period .
pumps had been prepared and incubated at 37c , 8 - 9hrs prior to implantation .
pumps where removed after 11 days ( prior to the onset of the dark period ) and the mice recovered from treatment while radiotelemetry measurements continued to be recorded ( db / db mice were killed at day 11 ) .
immunohistochemistry ( ihc ) for pstat3 and radioimmunoassay ( ria ) measurement to determine plasma leptin concentration were performed as previously described ( enriori et al . , 2007 ) .
to neutralize leptin s actions a goat igg against recombinant mouse leptin ( af498 from r and d systems , minneapolis ) was peripherally ( i.p . ) administered into dio mice for 5 consecutive days , following a sham injection period .
injections of 24 g in 90 l of antibody solution or 90 l of igg control ( antibodies australia , victoria , australia ) . the dose of the antibody was determined from previous research ( konstantinides et al . , 2004 ) .
all mice where randomly treated . to reduce the activation of lepr expressing neurons in the brain , a leptin receptor antagonist ( lra ) ( mouse leptin antagonist mutant l39a / d40a / f41a , 0.5 l of 5g/l , protein laboratories , rehovot , israel ) was used as previously reported ( enriori et al . , 2011 ) .
in dio mice that where implanted with radiotelemetry probes , they were additionally equipped with either a lateral ventricle ( lv ) ( plastics one , 2 mm long 22 gauge , implanted 0.5 mm posterior and 1 mm lateral to bregma , 2 mm below the surface of the skull ) or dmh cannula ( plastics one , 4 mm long , 26 gauge , implanted 1.9 mm posterior , 0.4 mm lateral to bregma , 4 mm below the surface of the skull ) as described previously ( enriori et al . , 2011 ) . following a 7 day recovery period , mice where all injected for 5 days twice daily with sham acsf injection ( 0.5 l ) into cannulas .
mice then received acsf or the lra , injected directly into cannulas , twice daily for 7 days , immediately following the dark period and immediately prior to the dark period . following the 7 day injection period mice where monitored daily for a 7 day recovery period .
dio mice were implanted with radiotelemetry probes and after 14 days of recovery baseline recordings were measured . in a stereotaxic apparatus ( stoelting , usa ) mice were injected bilaterally into the dmh region ( coordinated 1.8 mm posterior , 0.3/-0.3
mm lateral from bregma and 5.2 mm below the surface of the skull ) 0.5 l of either an aav scrambled shrna or aav leprshrna over a 20 min period each side ( hommel et al . , 2006 ) .
bw and fi were measurements daily and radiotelemetry recordings were measured periodically throughout the treatment period .
lepr loxtb mice and lepr flox mice ( berglund et al . , 2012 )
after which mice were implanted with radiotelemetry probes . following the surgery ( 2 weeks ) , baseline recordings where measured .
mice where then injected into the dmh ( coordinates 1.8 mm posterior , 0.3/-0.3 mm lateral from bregma and 5.2 mm below the surface of the skull ) with an aav cre virus .
mice where then monitored and cardiovascular parameters measured throughout . to examine the effect of activation or inhibition of dmh lepr expressing neurons
, we injected a virus that only infects and replicates in a cre dependent manner , into lepr - cre - yfp mice ( leshan et al . , 2012 ) .
after injection , the otherwise inert molecule psem binds to an ion channel that contains a psam element which causes opening of ion pore in the channel and allows ion flux across the cell membrane , which causes depolarization or hyperpolarization of cre - expressing neurons .
the psam element only binds psem , this system is similar to dreadds , but uses direct ligand gated ion channels rather than gpcr s to drive neuron specific effects ( magnus et al . , 2011 ) .
these mice where placed on either the chow or hfd for 20 weeks then implanted with radiotelemtry probes and 2 weeks later bp radiotelemetry baseline recordings were made . following this
the lepr - cre - yfp dio mice were injected in the dmh ( coordinates 1.8 mm posterior , 0.3/-0.3
mm lateral from bregma and 5.2 mm below the surface of the skull ) with the inhibitor virus , psyn::flex - rev - psam l141f : glyr - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) , lean chow fed lepr - cre - yfp mice were injected in the dmh ( coordinated 1.8 mm posterior , 0.3/-0.3
mm lateral from bregma and 5.2 mm below the surface of the skull ) with the activator virus , psyn::flex - rev - psam y115f:5ht3 hc - ires - gfp ( aav2 , unc gene therapy center vector core , nc , usa ) .
injection of either of these virus s did not change baseline cardiovascular function , until the peripheral injection of the inert psem ligand , that caused opening of ion pores and activation or inhibition of neurons .
after a 21 day recovery period cardiovascular parameters were re - examined and then mice where sham injected followed by acute or 3 consecutive days of treatment with psem ( 5 mg / kg , i.p . , twice daily ; apex scientific inc .
stony brook , ny ) or vehicle , randomized , crossover design ( magnus et al . , 2011 ) .
experiments were undertaken on lepr - yfp - cre mice ( leshan et al . , 2012 )
following 20 weeks of chow diet feeding , ad lib . on the day of electrophysiological experiment
the brain was rapidly removed , submerged in ice cold artificial cerebrospinal fluid ( acsf ) and coronal slices ( 200 - 250 m thick ) cut using a vibratome ( leica , vt1000s , uk ) .
slices were maintained at room temperature in oxygenated acsf ( gassed with 95% 02 , 5% co2 ) for at least an hour prior to recording .
for recording , slices were continuously perfused at room temperature with acsf of the following composition ( in mm ) : 127.0 nacl , 1.9 kcl,1.2 kh2po4 , 26.0 nahco3 , 2.0 d - glucose , 8.0 d - mannitol , 1.3 mgcl2 , 2.4 cacl2 , equilibrated with 95% o2 , 5% co2 , ph 7.3 7.4 .
lepr - yfp expressing neurons in the dmh were visualized using fluorescence , fitted with appropriate filters and infrared video microscopy with dic optics and recordings obtained from these neurons with an axopatch 700a amplifier .
patch pipettes were pulled using a horizontal puller ( sutter instrument co. , novato , ca , usa ) from thin - walled , filamented borosilicate glass ( gc150tf-10 ; harvard apparatus ltd ) with resistances between 4 and 8 m when filled with intracellular solution . the pipette solution comprised ( mm ) : 140 k - gluconate ,
the ph was adjusted to 7.3 with koh and the osmolarity to around 310 mosm with sucrose .
data were filtered at 2 5 khz , digitized at 2 10 khz ( digidata 1322 , mds analytical technologies ) and stored on pc running pclamp 9 data acquisition software .
analysis of electrophysiological data was carried out using clampfit 9.2 software ( mds analytical technologies ) .
was determined using unpaired , non - parametric student s t test with p < 0.05 considered as significant .
leptin was made up as a concentrated stock solution and diluted to the concentration required ( 100 nm ) in acsf immediately before commencement of electrophysiological recording .
| summaryobesity is associated with increased blood pressure ( bp ) , which in turn increases the risk of cardiovascular diseases .
we found that the increase in leptin levels seen in diet - induced obesity ( dio ) drives an increase in bp in rodents , an effect that was not seen in animals deficient in leptin or leptin receptors ( lepr ) .
furthermore , humans with loss - of - function mutations in leptin and the lepr have low bp despite severe obesity .
leptin s effects on bp are mediated by neuronal circuits in the dorsomedial hypothalamus ( dmh ) , as blocking leptin with a specific antibody , antagonist , or inhibition of the activity of lepr - expressing neurons in the dmh caused a rapid reduction of bp in dio mice , independent of changes in weight .
re - expression of leprs in the dmh of dio lepr - deficient mice caused an increase in bp .
these studies demonstrate that leptin couples changes in weight to changes in bp in mammalian species . |
hydrogen peroxide - inducible clone 5 or hic-5 is a gene we isolated by subtractive hybridization in 1994 as a cdna clone induced by transforming growth factor ( tgf- ) or hydrogen peroxide . at that time
, we studied tgf- signalling and pursued the possibility that reactive oxygen species ( ros ) function was an intracellular tgf- signal . after isolating the gene , we conducted a number of studies of hic-5 at a molecular as well as cellular level .
its amino acid sequence revealed that hic-5 is a homologue of paxillin , which is a multidomain lim ( lin-11 , isl-1 , and mec-3 ) protein that is localized at focal adhesions and was originally identified as a substrate of the v - src oncogene ( figure 1 ) . together with its family members
( leupaxin specifically expressed in lymphocytes , paxb , an orthologue of paxillin in slime mold , and hic-5 ) , paxillin has now been established as a molecular adaptor that transduces signals in response to changes in the adhesion environment of cells .
a famous example of a molecular adaptor is the grb2-sos system that transduces signals from growth factor receptors to ras .
paxillin transduces signals from extracellular matrix receptors , integrins , to intracellular downstream molecules such as map kinase . of these family members , hic-5 is most homologous to paxillin , and thus , analyses of hic-5 have been conducted in reference to and in comparison with paxillin .
for example , the intracellular localization of hic-5 is , like paxillin , mainly confined to so - called focal adhesion sites where cells adhere to the extracellular matrix via integrins . in terms of expression in tissues and cell types , paxillin is relatively ubiquitously expressed , whereas expression of hic-5 is prominent in the smooth muscle layer of tissues such as the large intestine and uterus .
furthermore , expression of hic-5 is relatively high in the lung and spleen . in cell culture systems ,
high expression of hic-5 is detected in mesenchymal cell lines including fibroblastic and osteoblastic cell lines ; however , it is generally low in epithelial cell lines . in a knockout mouse model , hic-5
was suggested to be inessential for the development and maintenance of homeostasis of the animal , and no remarkable functional abnormality was found under standard rearing conditions .
similar to fibronectin , it exhibits abnormal development of extraembryonic tissues and heart and body segmentation , resulting in death at 9.5 foetal days .
the embryonic lethality of the paxillin knockout mouse means that hic-5 can not substitute the functions of paxillin , at least those associated with development .
these results together with the abovementioned differences in expression patterns indicate that it is most likely that paxillin and hic-5 have different functions in mammals .
the genomic structure of hic-5 features a long intron between the n - terminal and c - terminal domains , a sign that hic-5 evolved from the fusion of two different genes .
accordingly , the protein structure can also be broadly divided from the centre into n - terminal and c - terminal regions .
the n - terminal region comprises four domains , the ld domains , which are rich in leu and asp ; ld1 is deleted in one isoform .
the c - terminal region comprises four lim domains having two zinc fingers ( figure 1 ) .
these features are almost identical to those of paxillin , with minor differences in the number of ld domains in the n - terminal region ( five for paxillin and four for hic-5 ) . given that both the ld and lim domains are protein - protein interacting domains , it is naturally assumed that paxillin family members are adaptor molecules that provide multiple proteins with interfaces to facilitate their interaction and cooperation . based on these structural features ,
, in the case of adaptor molecules , identification of their interacting factors is crucial for inferring their functions .
isolation of new interacting factors was also attempted , and we found an array of proteins that interact with each of the ld and lim domains of hic-5 ( figure 1 ) .
consistent with the localization of hic-5 at focal adhesions , most of the factors identified are those contained in the cytoplasmic domain complex of integrins ( discussed below ) and are involved in integrin signal transduction and/or in the control of actin cytoskeleton dynamics .
more specifically , the factors include signalling molecules such as protein tyrosine kinase 2 beta ( pyk2 ) , c - src tyrosine kinase ( csk ) , and focal adhesion kinase ( fak ) [ 7 , 8 ] . using a yeast two - hybrid method with the n - terminal and c - terminal domains of hic-5 as bait , we originally characterized g protein - coupled receptor kinase interacting arf gap1 ( git-1 ) as an ld3 interacting factor and protein tyrosine phosphatase , nonreceptor type 12 ( ptp - pest ) as an lim3 interacting factor .
vinculin and talin are structural proteins involved in the architecture of focal adhesions and the actin cytoskeleton , and these also interact with hic-5 .
most of the factors that bind to hic-5 also interact with paxillin although paxillin additionally binds src and crk ( v - crk sarcoma virus ct10 oncogene homologue ) , which do not bind to hic-5 [ 2 , 8 ] .
this difference is presumably because of the presence of tyrosine residues ( y31 , y118 , y188 , and y190 ) in paxillin , which do not exist in hic-5 although no definitive answer has been obtained yet .
furthermore , it is known that paxillin binds the cytoplasmic domains of 4 , 9 , and 3 integrins , and hic-5 interacts with the domain of 4 .
no particular pattern of interaction has been identified for the ld and lim domains of hic-5 and paxillin and their corresponding interacting factors .
thus , the study as above identified only factors whose localization and functions are related to focal adhesions .
hic-5 has also been found to be localized in actin stress fibers , while paxillin is not . here , hic-5 appeared to interact with cysteine - rich protein ( crp ) 2 , which belongs to the lim - only group of proteins .
this finding prompted us to search for other lim - only proteins that interact with hic-5 , and we identified a particularly interesting new cysteine and histidine - rich protein ( pinch ) that binds hic-5 .
further investigation demonstrated that lim4 in the c - terminal is important for hic-5 interaction with these lim - only proteins .
in other words , the fourth lim domain located at the endmost c - terminal region has a unique property , whereby it interacts with the same lim domain , thus enabling hic-5 to form lim - lim homo- and hetero - oligomers .
consequently , hic-5 exists as homo - oligomers in cells , which appear to be important for hic-5 localization in the nuclear matrix ( unpublished data ) .
hetero - oligomer formation between hic-5 and pinch / crp2 is essential for the two lim - only proteins to localize at particular sites in cells .
it should be noted that lim4 of paxillin can not form oligomers and does not interact with pinch or crp2 .
another molecular difference is that paxillin is highly tyrosine - phosphorylated during adhesion and growth signalling , whereas hic-5 is not generally tyrosine - phosphorylated to a significant level .
this might be explained by the fact that hic-5 has no tyrosine residues per se to be phosphorylated .
thus , unlike paxillin , hic-5 appears to not have a function in adhesion and growth signalling based on tyrosine phosphorylation although in human platelets , hic-5 has been shown to be tyrosine - phosphorylated with aggregation .
studies at a molecular level have characterized paxillin and hic-5 as being tyrosine - phosphorylated and forming lim - lim oligomers , respectively , which possibly underlies the functional difference between them .
paxillin may transduce adhesion / growth signalling , and hic-5 organizes a fail - safe system for the adhesion dependence of cell growth , as discussed below .
adhesion between cells and the extracellular matrix is achieved by integrins , a group of transmembrane proteins that act as receptors for the extracellular matrix .
the resultant adhesion of cells to the extracellular matrix , in turn , promotes aggregation between integrins , resulting in the formation of a protein complex known as a focal adhesion at the site of adhesion .
importantly , a focal adhesion is not just a physical structure but also a molecular apparatus that senses the adhesion status between cells and the extracellular matrix and sends signals to the inside of cells , thereby coordinating their motility , survival , and proliferation in response to adhesion status [ 14 , 15 ] . during signalling by integrins
whose cytoplasmic domain is very short and contains no enzymatic catalytic domain , multiple proteins assemble at the cytoplasmic domain through protein - protein interactions , forming an undercoat structure .
these proteins include several enzymes such as fak and src , through which the signal is emitted to the inside of the cell [ 14 , 15 ] .
hic-5 and paxillin act as adaptor molecules in this integrin undercoat complex and play roles in controlling enzyme activity by mediating the above - mentioned interactions between binding factors , thereby participating in the regulation of integrin signalling .
a detailed study has shown that hic-5 plays a role in negatively regulating fak by competing with paxillin . however , under normal culture conditions , cellular behaviours including growth and motility are hardly affected by hic-5 expression in most cases .
therefore , hic-5 appears to not be required for any particular function at focal adhesions under normal cellular conditions so long as the adhesion status is maintained .
rather , hic-5 may have a critical function in suppressing excess changes in adhesion and the cytoskeleton structure by antagonizing paxillin when the maintenance of healthy adhesion is in jeopardy under stressful conditions affecting adhesion [ 3 , 16 ] .
in sharp contrast , abnormal cell motility and spreading is observed under normal conditions in paxillin cells in which the focal adhesions are disorganized and integrin signalling by fak and the downstream map kinase is disturbed . in parallel with localization at focal adhesions ,
this fact lead us to investigate the possible function of hic-5 in the nucleus , and a series of study demonstrated that hic-5 was capable of regulating expression of the c - fos and p21 genes [ 17 , 18 ] .
further study demonstrated that hic-5 promotes the formation of a transcriptional complex , specifically with the transcriptional coactivator p300 and transcription factors sp1 and smad3 , on dna .
in other words , hic-5 appears to function as an adaptor for transcription - associated factors in the nucleus by acting as a scaffold for transcriptional complex formation , as is the case for integrin signalling at focal adhesions .
a similar function of hic-5 in the nucleus has been suggested by other researchers who recloned hic-5 as an androgen receptor coactivator 55 kda protein ( ara55 ) , in 1999 [ 19 , 20 ] .
it has also been reported that hic-5/ara55 is a coactivator of ppar and others [ 21 , 22 ] .
taken together , these results indicate that hic-5/ara55 may serve as a scaffold for a fairly wide range of transcriptional activities in the nucleus
. in summary , a growing body of evidence suggests that the function of hic-5 in the nucleus is to facilitate transcription complex formation by interactions with several transcription - associated factors .
however , taking into consideration the fact that under normal conditions , hic-5 is mainly localized at focal adhesions and not in the nucleus at a detectable level , we should be cautious about this conclusion .
rather , we postulate that hic-5 has some roles in the nucleus , primarily under conditions where focal adhesions have been disrupted and that localization of hic-5 in the nucleus then becomes discernable .
our initial observations indicated that hic-5 changed its localization to the nucleus following treatment with hydrogen peroxide , suggesting that besides focal adhesions , hic-5 can also localize in the nucleus based on the cellular conditions .
finally , we found that hic-5 shuttles between focal adhesions and the nucleus constantly in normal adhesion cells and that it does so in company with pinch and crp2 , its lim - lim hetero - oligomer partners .
this molecular - level analysis of the shuttling ability of hic-5 identified nes around the ld3 domain .
a nuclear localization signal is carried by four lim domains in the c - terminal region . of note
, there are specific cysteine residues upstream of the nes consensus sequence in hic-5 that enable nes to sense the intracellular redox state . because of this , nes
is interrupted to stop the export of hic-5 from the nucleus under conditions such as hydrogen peroxide treatment , which results in the accumulation of the protein in the nucleus . in short , hic-5 shuttles between focal adhesions and the nucleus in normal situations , but it accumulates and becomes functional in the nucleus when the intracellular redox state becomes more oxidised . of interest , when cells are deprived of adhesion to a substrate , the ros concentration in the cells increases .
these findings indicate that hic-5 is a shuttle protein and an ros effector , just as the name suggests .
in addition , hic-5 is unique in that it uses ros as a switch for changing its localization by sensing ros in nes .
a group of lim proteins including paxillin , zyxin , llp , and a thyroid hormone receptor - binding factor , trip 6 , has also been shown to communicate between focal adhesions and the nucleus although the controlling mechanisms and significance of this shuttling are poorly understood .
next , we explored the significance of the shuttling of hic-5 between the two compartments .
we focused on the adhesion ( anchorage ) dependence of cell proliferation as an example of a phenomenon that requires such a coupling mechanism .
numerous studies have defined the roles of adhesion signals mediated by integrin - extracellular matrix ( ecm ) interaction in cell - cycle progression , basically , integrin - ecm - mediated signaling potentiates and prolongs the growth factor receptor - mediated mitogenic signalling and is required from mid- to late - g1 phase in various events associated with cell - cycle progression , such as upregulation of g1-phase cyclin - dependent kinase ( cdk ) activity , cip / kips downregulation , association of cyclin e with cdk2 , prb phosphorylation , and cyclin a expression . as a result , loss of adhesion generally causes complete g1 phase cell - cycle arrest in nontransformed cells
; moreover , in susceptible cells , it leads to anoikis , a specific type of apoptosis caused by the detachment of a cell from its supportive matrix , which was first described in epithelial and endothelial cells [ 26 , 27 ] .
in contrast , transformed cells usually circumvent the anchorage requirement in cell - cycle progression .
their anchorage - independent survival and growth is well known as a hallmark of cellular transformation and correlates with tumorigenicity in vivo .
mechanistically , the anchorage - independent growth is considered to be based on an abnormal activation of the g1-phase cyclin cdks uncoupled from anchorage . in general
, an oncogenic pathway activates a robust and/or constitutive mitogenic signal , which is presumed to reduce the requirement for integrin - ecm - mediated signalling and its importance as a booster of growth factor receptor - mediated mitogenic signalling in the transformed cells . among the downstream pathways of oncogenic signals , the activation of the phosphatidylinositol 3-kinase / akt pathway is crucial for the induction of anchorage - independent growth and cell survival . among the events required for
the cell - cycle progression , activation of cdk4/6 by cyclin d is one of the most important events , because it promotes the g1/s transition ; that is , it serves as a determinant of cell - cycle progression along with cyclin e / cdk2 activity .
importantly , activation of this complex is sensitive to adhesion status as well as growth stimuli , and it plays a role in coupling adhesion status with cell - cycle progression .
thus , under conditions of inappropriate adhesion , it operates by circumventing progression of the cell cycle . in other words ,
besides acting as an engine of cell - cycle progression , cdk4/6-cyclin d functions as a security system for the anchorage dependence of cell proliferation .
mechanistically , the expression level of cyclin d is regulated not only by growth signals but also by adhesion signals , and an insufficiency of either signal type decreases its expression [ 28 , 29 ] . the level of expression of other cyclins rather automatically cycles up and down with progression of the cell cycle , as their name suggests .
thus , cyclin d is a key molecule in regulation of the cell cycle , and abnormal cyclin d behaviour is directly linked to aberrant proliferation of cells , which is supported by the fact that cyclin d is a proto - oncogene .
accordingly , not only the amount of cyclin d in cells but also its subcellular localization is rigorously regulated by signalling , and cyclin d localizes in the nucleus only during g1/s phase , when nuclear localization is essential for its function ; it is exported from the nucleus during the other phases . when this does not occur , it becomes oncogenic , as has been shown by observations , where the nuclear localization of cyclin d promoted cell proliferation ; its oncogenic potential is closely related to its nuclear localization ability .
regulation of the nuclear localization of cyclin d is so important that we hypothesized that it may also be regulated by adhesion status .
therefore , we examined the relationship between the nuclear localization of cyclin d1 and the adhesion status of cells . in adherent cells at g1 phase , cyclin d1 is localized in the nucleus .
this result supports the above hypothesis that the nuclear localization of cyclin d1 is also regulated by the adhesion status .
this regulation would be of great help in avoiding unacceptable cell growth when cells are detached ; most interestingly , hic-5 is deeply involved in this mechanism .
basically , hic-5 and cyclin d1 translocate between the cytoplasm and nucleus through the same crm 1-dependent nuclear export system , and for this reason , they compete for this export system .
the presence of pinch with hic-5 is important for the competition between hic-5 and cyclin d1 .
a critical step is that hic-5 stops shuttling in cells on loss of adhesion , the consequence of which is the export of cyclin d1 in place of hic-5 outside the nucleus .
the arrest of shuttling in nonadherent cells is caused by an elevated level of ros , which inactivates nes .
to summarize , the biological significance of the shuttling of hic-5 is competitive localization of cyclin d1 in the nucleus in adherent cells with the aid of pinch .
this results in critical acceleration of the export of cyclin d1 to the cytoplasm and in the arrest of cell proliferation under abnormal conditions .
this mechanism , which is regulated by the on / off status of hic-5 shuttling , functions as a fail - safe system for ensuring the anchorage dependence of cell proliferation ( figure 2 ) .
most recently , we found an additional mechanism involving hic-5 for inducing growth arrest in detached cells .
the mechanism targets p21 , a cyclin - dependent kinase inhibitor that is famous as the downstream target of the tumour - suppressor gene p53 .
this gene is well known to be transactivated by p53 in response to a variety of stresses and to stop g1/s progression by inhibition of cyclin d - cdk4/6 or cyclin e - cdk2 .
of note , when cells are deprived of adhesion , p21 is upregulated and plays a critical role in growth arrest .
our recent investigation suggests that hic-5 participates in the transactivation of p21 in response to loss of anchorage . in this case , hic-5 cooperates with crp2 , another lim - only protein shuttling partner , to transactivate the klf4 and runx1 sites upstream of the gene ( unpublished data ) .
in conclusion , hic-5 has emerged as a critically important molecule for regulation of the anchorage dependence of cell proliferation .
more specifically , hic-5 organizes two types of mobile lim - lim oligomer platforms with pinch and crp2 , which collaboratively prevent anchorage - independent cell growth .
for this purpose , hic-5/pinch regulates the nuclear localization of cyclin d and hic-5/crp2 is operational in the transactivation of p21 ( figure 3 ) .
a series of studies by us and others have suggested interesting possibilities for the function of hic-5 at focal adhesions and in the nucleus .
we also have originally addressed its role as a mobile scaffold for the anchorage dependence of cell growth . however , in terms of an active role of hic-5 under normal adhesion conditions , the evidence presented thus far is not fully convincing for either localization .
we speculate that under normal adhesion conditions , hic-5 senses the adhesion status based on the fact that its nes is protected from attack by ros in the adhesion complex and is able to assist hic-5 shuttling .
once the cells lapse into a state of abnormal adhesion , such as detachment from the matrix , the adhesion structure becomes disrupted along with ros production ; under such conditions , nes of hic-5 is unmasked , becomes exposed to ros , and is modified to stop the shuttling .
when shuttling of hic-5 is stopped , cell growth is arrested through the mechanisms involving cyclin d and p21 . in other words
, hic-5 organizes a fail - safe system that ensures the anchorage dependence of cell growth .
the activation mechanism of this system is oxidative modification of nes of hic-5 in response to increased production of ros as well as the collapse of the focal adhesion complex . at this point
, it should be noted that the interaction between fak and hic-5 is also sensitive to ros .
this is because fak and hic-5 interact through ld3 of hic-5 , which contains nes , and this interaction is regulated by the upstream cysteines in the same manner as for nes . therefore , the interaction between fak and hic-5 is also lost under abnormal adhesion conditions , suggesting that the functionality of nes and the complex formation of hic-5 , at least with fak , in the focal adhesion complex are intimately interrelated . in an earlier section , we discussed the role of hic-5 at focal adhesions and on actin stress fibres . considering that most of these results were observed in cells exposed to stresses such as detachment and stretching but not in cells under normal conditions ,
hic-5 functions can , after all , be interpreted as being closely related to the abnormity of the adhesion environment and devoted to suppression of extreme changes in the cytoskeleton as well as to prevention of proliferation under abnormal adhesion conditions .
a group studying glomerulosclerosis has reported that hic-5 is significantly induced and involved in the induction of cell death in a pathological model , where mesangial cells were cultured on collagen that was of a different type to the original extracellular matrix .
this observation may have captured the essence of hic-5 's function of sensing abnormal adhesion status .
the organization of hic-5 as a fail - safe system for ensuring the anchorage dependence of cell growth is expected to be involved in the cancer development process . in this point
, it should be noted that although hic-5 is actively involved in the growth arrest of fibroblasts via induction of p21 on loss of anchorage , it is not involved in the anoikis or cell death of epithelial cells ( unpublished data ) .
this may be concerned with the fact that p21 , which is the target of hic-5 action and functions in growth arrest , was not induced in normal epithelial cells under detached conditions . instead
, epithelial cells undergo anoikis that is governed by a group of factors specialized for cell death execution .
given these findings together with its relatively high expression in mesenchymal cells , hic-5 might be an important molecule in the development of sarcoma .
another interesting possibility is that hic-5 may be involved in the epithelial - mesenchymal transition , as was pointed out by tumbarello and turner .
the ability of hic-5 to act as a steroid hormone receptor coactivator has led to studies of its involvement in prostate cancer and endometriosis .
furthermore , a study of a human breast cancer cell line showed that both paxillin and hic-5 have distinct impacts on cancer cell phenotypes related to metastatic potential . according to this study , in which sirna was used for knockdown of the genes , both paxillin and hic-5 function in promoting metastatic potential although through different mechanisms .
however , in our recent experiment using shrna , which would be more adequate for long - term observation than sirna , we found contradictory results .
however , hic-5 and cellular transformation remains a controversial issue and awaits further investigation . in our earlier study ,
in contrast , forced expression of hic-5 induced a senescence - like phenotype with decreased proliferative potential after repeated passages in some immortalized human fibroblasts .
recently , it was found that the anchorage - independent growth of cancer cells over a long period of time may be negatively regulated by hic-5 ( unpublished data ) .
thus , the interaction of cell adhesion and the mortality / senescence process would be an interesting subject for future study of hic-5 biology in association with cancer cells . | hic-5 is a multidomain lim protein homologous to paxillin that serves as a molecular scaffold at focal adhesions and in the nucleus .
it forms mobile molecular units with lim - only proteins , pinch , and crp2 and translocates in and out of the nucleus via a nuclear export signal ( nes ) . of note , nes of hic-5
is distinctive in its sensitivity to the cellular redox state .
recently , the mobile units of hic-5 have been suggested to be involved in the regulation of the anchorage dependence of cell growth .
on loss of adhesion , an increase in reactive oxygen species in the cells modifies nes and stops shuttling , which leads to cell - cycle control .
more specifically , the system circumvents nuclear localization of cyclin d1 and transactivates p21cip1 in detached cells , thereby avoiding anchorage - independent cell growth .
thus , the hic-5-lim only protein complex has emerged as a fail - safe system for regulating the anchorage dependence of cell growth . |
numerous systems have been proposed to classify thoracolumbar injuries . among the most influential were the ideas proposed by denis , magerl / ao , and the thoracolumbar injury classification system ( tlics).1
2
3 the denis system was valuable in providing a modern concept of spinal stability and integrating the findings of computed tomography images to advance our understanding of injury morphology.1 although the magerl / ao classification provided a comprehensive morphologic description of fracture subtypes , the multitude of injury subgroups and the lack of guidance for treatment decisions limited its clinical application and adoption.3 the tlics standardized a treatment protocol but has been criticized for its one - size - fits - all approach , which critics have accused of promoting the treatment biases of its creators.3
4 furthermore , the tlics relies heavily on the evaluation of the posterolateral ligamentous complex ( plc ) , which is most commonly performed with magnetic resonance imaging , a modality that does not do so with sufficient specificity and is not widely available or used routinely in many parts of the world .
a classification system must provide sufficiently detailed morphologic descriptions to distinguish between clinically distinct injuries and guide treatment decisions ; both of these purposes are essential but neither alone is sufficient .
the universal adoption of a classification for both research and clinical purposes will likely occur only when a system is developed to classify morphology and guide treatment simultaneously .
the recently described aospine thoracolumbar spine injury classification system combines the strengths of the magerl / ao system and the tlics with respect to morphologic injury classification.5 utilizing the same hierarchical approach of the ao classification scheme initially used to classify long bone fractures , this system classifies injury morphology into three main groups : type a compression ( subgroups a0 to a4 ) ; type b
tension band injury ( subgroups b1 to b3 ) ; and type c translation injuries .
this system also includes the patient 's neurologic status ( n0 to n4 and nx , unknown ) and two clinical modifiers .
this system was reliably applied to classify thoracolumbar injuries by a group of 100 surgeons uninitiated to the system from around the world.6 the requisite next step in the development of guidelines is the creation of a scoring system to accompany the classification system .
the scoring system should accurately reflect the relative injury severity with respect to the need for surgical stabilization and eventually will suggest appropriate surgical thresholds . complicating
the standardization of treatment algorithms is the wide variation of preferred treatment methods currently seen among surgeons in different regions of the world .
the variation in the treatment of thoracolumbar injuries is likely multifactorial , reflecting patient and surgeon biases and risk aversion , treatment cost , surgeon access , and tolerance of temporary disability during the postinjury recovery period.6
7
8
9
10
11 overcoming this clinical equipoise to establish uniform treatment algorithms is hindered by the paucity of data that provides indications for surgery and compares outcomes between different surgical and nonsurgical strategies .
an important first step in establishing treatment algorithms is to be able to grade the severity of injury , considering the patient 's injury morphology and neurologic status .
the purpose of this study was to derive a data - driven thoracolumbar aospine injury score ( tl aosis ) to accompany the aospine thoracolumbar spine injury classification .
this process relies on data obtained from a survey of surgeons on their perception of the relative severity of different types of thoracolumbar injury and neurologic deficits in determining the need for surgical stabilization .
the surgical thresholds to accompany this scoring system will be established later and may reflect cultural differences that play an important role in surgical indications .
the aospine thoracolumbar spine injury classification system was developed under the oversight of a group of surgeons with interest in spinal trauma through a consensus - forming process , which has been previously described.5
12 although the classification system included input from surgeons with a relatively diverse approach to the treatment of spinal injuries , the consensus process was completed by a relatively small number of surgeons .
an instrument that will drive clinical decision making must accurately represent the practice patterns from a wider group of well - informed surgeons from around the world .
because of the need for wider input , the spine injury score described in this article was derived from the results of a worldwide survey of 100 surgeons instead of determined by the consensus of a small group .
a survey was sent to 100 aospine members from all regions of the world including representatives in north america , south america , europe , africa , asia pacific , and the middle east who were not part of the initial classification efforts .
each respondent was asked to numerically grade the severity of each variable of the aospine thoracolumbar spine injury classification system including injury morphology , neurologic injury , and patient - specific modifiers .
a grade of 0 was considered not severe at all , and a grade of 100 represented the most severe injury possible .
the complete results of this survey have been previously described.13
analysis of the results of this survey was used to place the classification system components into a relative hierarchy using point values to represent relative injury severity , keeping in mind that a system that uses noninteger point values for various components would be cumbersome and limit practical usage .
furthermore , the m2 modifier ( representing patient - specific variables ) of the classification system can not be easily assigned a point value as it is incompletely defined by nature , describing a comorbidity that affects surgical decision making and may influence a surgeon either toward or away from surgical intervention . following the initial interpretation of the survey results ,
the original classification group provided limited input to ensure that certain aspects of clinical practice would be captured in the scoring system .
all alterations made in this manner to the scoring system from what would be dictated by strict interpretation of the survey results are specifically described below .
of the 100 surgeons around the world who were invited to complete the injury severity survey , complete responses were received and analyzed from 74 surgeons ( table 1 ) .
although more detailed analysis of this data has been previously published , the aospine tlics morphology subgroups demonstrated a stepwise progression in terms of perception of injury severity through the a , b , and c groups ( i.e. , a0 a1 a2 a3 a4 and b1 b2 b3 ) with nearly even spacing between progressive morphologic subgroups ( table 2).13 a4 and b1 injuries scored within 5 points on a 100-point scale of one another , suggesting these injuries are perceived as relatively similar in severity despite the difference in morphology group ( a versus b ) . based on this input , the groups were consecutively given an additional point starting with 1 point assigned to a1 injury morphology .
importantly , no points were awarded to the a0 group . the point scheme for morphology
because the a4 morphology was graded as slightly more severe than the b1 morphology and closer to the b2 morphology than the a3 morphology , a4 was determined to receive 5 points rather than downgrading b1 to receive 4 points .
the survey suggested the perception of an uneven stepwise increase in injury severity moving from n0 to n4 with a large gap separating n2 ( nerve root injury with radicular symptoms ) and n3 ( incomplete spinal cord injury ) . because of this pattern of perceived severity
, each grade of neurologic injury was progressively given an additional point starting with 0 points for n0 because it does not represent an injured state .
the substantial difference in perceived severity between n2 and n3 injuries was recognized by elevating n3 to 4 points , an action endorsed by the classification group to recognize the urgency for surgical stabilization and decompression of patients with incomplete spinal cord injuries . additionally , both the results of the injury severity survey and the members of the classification group felt that without the ability rule out a neurologic injury , a cautious approach must be taken , and so 3 points were awarded to nx .
the point scheme for neurologic status components and the m1 modifier is presented in table 4 .
as described above , the nature of the m2 modifier makes assigning point values impossible , as it is inherently variable and dependent upon the specific details of the individual patient 's comorbidity .
burns over the potential operative site , for example , may argue against surgery , and a fracture in a patient with ankylosing spondylitis is often a strong indication for surgery given the associated instability .
with respect to the m1 modifier ( an indeterminate injury to the plc ) , the severity survey suggested that the respondents viewed ambiguous plc compromise as intermediate between n2 and n3 but closer in severity to n2 injuries , which would indicate that the m1 modifier should receive 2 points . however , both in the classic literature and in a recent study performed by the aospine trauma knowledge forum,14 the interobserver reliability of indentifying an injury to the plc is only slight to fair , and so the decision was made to lessen the points awarded to the m1 modifier .
although there is no doubt about the biomechanical importance of the plc , it was determined that the inability of the surgeons to reliably determine the integrity of the plc limits the clinical importance of the m1 modifier .
the summation of the score awarded to each variable ( i.e. , morphology , neurologic status , and patient - specific modifiers ) represents the total tl aosis for a specific injury .
although the aospine tlics describes a new scheme that was designed to overcome the shortcomings of previously described classification systems , the description of morphologic injury patterns and associated neurologic injury alone is likely to be insufficient to spur widespread adoption , particularly among clinicians . to provide greater benefit for patients and clinicians , a thoracolumbar injury classification system should provide robust guidance for physicians treating patients with these injuries .
past criticism of the scoring system and treatment algorithm associated with tlics was centered on the perception that tlics largely represents the treatment biases of its creators and does not accurately represent treatment algorithms commonly used in many parts of the world.11
15
16 despite this criticism , there is ample precedence for establishing widely used severity scales based on expert opinion and consensus ; the injury severity scale , its predecessor the abbreviated injury scale , and the glasgow coma scale were all developed through an expert consensus process.17
18
19 nevertheless , because of the amount of previous study of thoracolumbar trauma and the divergent attitudes toward management of these patients , we felt it was critical to use a largely data - driven approach with only gentle editing by the smaller group of surgeons to establish a scoring scheme to accompany the aospine tlics . instead of relying on consensus alone ,
the development of the spine injury score was driven by data and created in consideration of the perception of injury severity of a much larger group of surgeons across all regions of the world to reflect the various aspects of cultural diversity that likely play a role in the treatment algorithms . although the results of the severity survey were allowed to dictate the associated tl aosis point value in nearly all cases , the classification group intervened to increase the score assigned to a4 to give it the same point value as b1 injuries based on the almost identical severity scores of these two injury patterns .
rather than decreasing the b1 score , the a4 score was increased to reflect several considerations .
first , the a4 score was closer to the b2 score ( difference < 10 points ) than it was to the a3 score ( difference > 15 points ) .
second , neurologically intact patients with burst fractures are commonly treated with surgical intervention in many parts of the world , suggesting that surgeons feel patients with these injuries have worse outcome with nonoperative treatment .
although this article does not yet provide treatment guidelines , it is logical to promote such an injury to a higher score rather than demote an injury ( b2 ) that is most commonly treated with surgery to a lower score .
it is important to note that despite an increased score for c injuries compared with b3 injuries , both of these injuries are unstable and require surgical stabilization .
reports of nonoperative treatment of either thoracic extension - distraction injuries or translational injuries are rare , and every previous morphologic classification system has recognized the unstable nature of these injuries and the associated indication for surgery barring extenuating circumstances that absolute preclude decompression and stabilization.1
2
3
5
significant debate was had by the classification group when attempting to determine the point value for a patient who is unable to be examined ( nx ) .
it is likely that patients who sustain an injury severe enough to limit the ability of the surgeon to obtain a proper neurologic exam are polytrauma patients , and so even in a neurologically intact patient , immediate stability is particularly beneficial in these patients .
furthermore , the risk of catastrophic neurologic sequelae from the failure to promptly stabilize a patient can not be ignored .
there is very little variation in grading fracture morphology or in perception of injury severity on a geographic basis .
schroeder et al found no significant regional variation in perception of fracture severity in any of the morphologic or neurologic elements graded,13 which is highly relevant to the present investigation as it strongly suggests that there is no need to consider a point system with regional variation .
we expect the point system described previously to accurately reflect worldwide perceptions of injury severity , and we anticipate that there will be widespread adoption of the scoring system given the lack of variation in perception of injury severity .
future efforts will establish surgical thresholds based on questionnaires designed to distinguish which morphologic injury patterns and neurologic deficits are thought to require stabilization / decompression and sent to a large group of surgeons with an interest and experience in spinal trauma .
an inclusive approach will increase the generalizability of the resulting recommendations and allow for surgical thresholds to be evaluated on a regional basis to capture culture differences among surgeons and patients that influence treatment .
the ultimate goal is to use this classification and scoring system framework to perform randomized , controlled trials that would allow patient outcomes and not surgeon perception of patient outcomes to establish the surgical thresholds and algorithms .
although we used the results of a survey sent to surgeons thought to have particular expertise and experience in treating patients with spinal trauma , there was no practical metric to verify whether this belief was true .
this flaw is inherent to all studies that utilize expert opinion , although our intent in including a large number of surgeons was to mitigate the influence any single surgeon would hold over the study results .
the points assigned to each injury were arbitrary , as was the relative importance of morphology and neurologic injury .
we used the largely uniform stepwise perception of injury severity in the ascending injury subgroups to assign single - point increases with the few exceptions as described previously , but the magnitude of the point scale and relatively severity implied is somewhat arbitrary .
prospective application to injured patients with follow - up outcome measures along with the investigations to identify surgical thresholds will provide feedback about the point system described herein , which can be modified if necessary .
in this article , we have provided face validity for the aospine thoracolumbar classification system in that the consensus scoring system that has been developed aligns with the ordered classification itself .
furthermore , the hierarchy of neurologic and other modifiers was consistently rated and scored by surgeons and confirms the increasing severity of injury expressed within this classification system .
these results indicate that through utilizing the tl aosis , the aospine thoracolumbar classification system is an ideal system for the establishment of a globally accepted treatment algorithm for thoracolumbar trauma . | study design survey of 100 worldwide spine surgeons .
objective to develop a spine injury score for the aospine thoracolumbar spine injury classification system .
methods each respondent was asked to numerically grade the severity of each variable of the aospine thoracolumbar spine injury classification system . using the results , as well as limited input from the aospine trauma knowledge forum ,
the thoracolumbar aospine injury score was developed .
results beginning with 1 point for a1 , groups a , b , and c were consecutively awarded an additional point ( a1 , 1 point ; a2 , 2 points ; a3 , 3 points ) ; however , because of a significant increase in the severity between a3 and a4 and because the severity of a4 and b1 was similar , both a4 and b1 were awarded 5 points . an uneven stepwise increase in severity moving from n0 to n4 , with a substantial increase in severity between n2 ( nerve root injury with radicular symptoms ) and n3 ( incomplete spinal cord injury ) injuries
, was identified .
hence , each grade of neurologic injury was progressively given an additional point starting with 0 points for n0 , and the substantial difference in severity between n2 and n3 injuries was recognized by elevating n3 to 4 points .
finally , 1 point was awarded to the m1 modifier ( indeterminate posterolateral ligamentous complex injury ) .
conclusion the thoracolumbar aospine injury score is an easy - to - use , data - driven metric that will allow for the development of a surgical algorithm to accompany the aospine thoracolumbar spine injury classification system . |
autoimmune diseases ( ads ) are a family of more than 80 chronic , and often disabling , illnesses broadly characterized by immune system dysfunction leading to the loss of tolerance to self - antigens , presence of increased level of autoantibodies , inflammatory and mediatory cells and resulting chronic inflammation .
the family of ads is remarkable for its complexity and similar underlying mechanisms despite the involvement of multiple organ systems .
patients often endure lifelong debilitating symptoms , loss of organ function , reduced productivity at work and high medical expenses .
importantly here , as many ads present before or during a woman 's reproductive years , they can have effects on fetal and maternal outcomes , such as pregnancy loss in women with systemic lupus erythematosus , vasculitis and type 1 diabetes , and infertility in women with rheumatoid arthritis .
collectively , ads are common , affecting between five and nine percent of the population and are of considerable personal and public health burdens .
the reasons for their high prevalence , gender and ethnic disparities and rising incidence and prevalence remain unclear .
population - level phenomena such as mutation , migration , genetic drift and natural selection , have left an imprint on genetic variation that is likely to influence phenotypic expression in specific populations . given its role in driving genetic variation
, population genetics can help elucidate human genetic diversity and , consequently , disease etiology .
left untreated , most ads affect the ability to raise offspring that successfully reproduce and result in reduced reproductive fitness .
thus , alternative forces must exist that permit the relative high frequency of risk alleles . as immune and inflammatory responses can be highly sensitive to environmental change , evolutionary adaptation to specific environments
might have driven selection on immune - related genetic variants , impacting variant frequencies and leaving signatures of selection in the genome .
given that infectious organisms are strong agents of natural selection , it is plausible that alleles selected for protection against infection confer increased risk of autoimmune and allergic diseases , or their complications , as the hygiene hypothesis ' postulates . it is thought that the adaptation to pathogen pressure through functional variation in immune - related genes conferred a specific selective advantage for host survival , including protection from pathogens and tolerance to microbiota .
however , the emergence of such variation conferring resistance to pathogens is also influencing ad risk in specific populations . in the past decade , multiple genome scans for signatures of selection on common variation have identified many immune - related loci . similarly , over 130 genome - wide association studies have established ad - associated alleles .
this review summarizes the evidence for ad - associated loci under selection and the candidate selective pressures .
collectively , these results support the hypothesis that ad is , in part , a pleiotropic consequence of alleles conferring pathogen resistance and help explain the number of shared autoimmune variants conferring both disease risk and protection , as well as the health disparities in individuals and populations . given that genomic variation can have clinically important consequences ,
elucidating the patterns of variation and the functional role of the selective pressure might contribute to a better understanding of disease etiology and the development of new therapies for improved disease management .
the family of ads is remarkable for its complexity and similar underlying mechanisms despite the involvement of multiple organ systems .
they disproportionately afflict women and are among the leading causes of death for young and middle - aged women .
african americans are at higher risk than european americans for systemic lupus erythematosus and scleroderma ( systemic sclerosis ) , which they tend to develop earlier in life and experience more severe disease , but are at lower risk for type 1 diabetes , thyroiditis and multiple sclerosis . although most of these diseases are individually rare , in the aggregate they affect between five and nine percent of the population , and , for reasons that are poorly understood , their incidence and prevalence is rising . despite the variation in prevalence , incidence and disease severity that are known to vary among ethnic groups , little
is known about the genetic etiology of these diseases in the different populations and the reasons for the ethnic disparities remain elusive .
multiple lines of evidence suggest some degree of common genetic etiology in ads , including clustering of multiple ads in families and in individuals , and the number of confirmed genetic regions predisposing to several ads .
this genetic overlap is exemplified by the well - known associations of the human leukocyte antigen ( hla ) region with all ads , as well as other loci associated with multiple ad , such as il23r , tnfaip3 and il2ra .
nevertheless , the genetic heritability of ads is extremely variable , ranging from very high in crohn 's disease or ankylosing spondylitis to almost negligible in systemic sclerosis .
table 1 summarizes the ads with published genome - wide association studies and the number of disease - associated loci uncovered from these genome - wide association studies .
it is the general consensus that there is a common genetic background predisposing to autoimmunity , and that further combinations of more disease - specific variation at hla and non - hla genes , in interaction with epigenetic and environmental factors , contribute to disease and its clinical manifestations .
results from the most comprehensive evaluation of shared genetic autoimmune loci to date suggest that instead of resulting from common risk factors , autoimmunity may result from specific and multiple different pleiotropic effects .
this suggests that different population genetic factors ( for example , natural selection with coevolution with pathogens , random mutation , isolations , migrations and interbreeding ) in similar or distinct environments led to the establishment of the current plethora of loci that predispose to autoimmunity .
as the authors conclude , these results generate an appreciation for potential interplay between population genetic factors and environmental factors .
this appreciation has been renewed in a recent systematic analysis of loci shared among multiple ads , confirming that snp or haplotype sharing is complex , that often the most associated variant at a given locus differs and , even when shared , the same allele often has opposite associations .
population - level phenomena are the likely reason behind this complexity of gene effects in different ads .
human genome variation at the population level is typically thought to be shaped by five evolutionary processes : sexual reproduction , mutation , migration , random genetic drift and natural selection .
in addition , recombination is often an underappreciated driver of genomic diversity and genome evolution , as recombination events ( the exchange of genetic material between homologous chromosomes ) are accompanied by biased gene conversion , which causes allele frequency shifts that mimic the effects of natural selection , shapes patterns of diversity in human genomes and contributes to substantially increased risks of hereditary disease .
natural selection is the process by which a trait becomes either more or less common in a population depending on the differential reproductive success of those with the trait .
natural selection drives adaptation , the evolutionary process whereby over generations the members of a population become better suited to survive and reproduce in that environment .
negative ( or purifying ) selection decreases the prevalence of traits by purging deleterious alleles , and is the most common mechanism of selection . negatively selected variants are usually associated with rare mendelian disorders and have low population frequencies . as an example , endosomal toll - like receptors (
tlr3 , tlr7 , tlr8 and tlr9 ) , which are involved in the sensing of nucleic acids , mostly from viruses , have been subject to negative selection .
positive selection increases the prevalence of adaptive traits by increasing the frequency of favorable alleles .
positively selected variation is often associated with common complex traits and can achieve higher population frequencies .
a classic example of positive selection is the lactase ( lct ) gene , in which independent variants associated with lactase persistence raised to high frequency in different populations owing to the strong selective force of adult milk consumption .
the enrichment for signals of positive selection among genes associated with complex traits is well documented .
balancing selection favors genetic diversity by retaining variation in the population as a result of heterozygote advantage and frequency - dependent advantage .
one classical example is the hemoglobin- locus and the high prevalence of the hemoglobin- mutation that causes sickle cell anemia in regions where malaria is endemic : while individuals homozygous for the wild - type hemoglobin- are susceptible to malaria , and individuals homozygous for the hemoglobin- mutation have sickle cell anemia , heterozygous individuals are resistant to malaria and otherwise healthy .
another pertinent example of balancing selection is the hla ( also known as major histocompatibility complex ( mhc ) ) region , where highly polymorphic loci have a central role in the recognition and presentation of antigens to the immune system .
the heterozygote advantage against multiple pathogens contributes to the evolution of hla diversity , which confers resistance against multiple pathogens and explains the persistence of alleles conferring susceptibility to ad . nevertheless , there is also recent evidence that positive selection might be acting on specific hla alleles in a deme due to unique environmental pressures .
quintana - murci and clark provide a comprehensive description of popular statistical methods to detect the different types of selection .
there are three main classes of tests that detect regions under selection : ( i ) site frequency spectrum tests summarize the allele frequency distribution of polymorphisms , ( ii ) linkage disequilibrium tests uncover high - frequency haplotypes with extended linkage disequilibrium and ( iii ) population differentiation statistics detect local increases in the magnitude of population structure due to geographically restricted selection .
allele frequency spectrum tests ( for example , tajima 's d , fay and wu 's h - test ) can detect recent selective events ( < 250 000 years ) and are powerful for detecting completed or quasi - completed selective sweeps ; haplotype - based tests ( for example , integrated haplotype score ( ihs ) , cross population - extended haplotype homozygosity ( xp - ehh ) test ) are very powerful to detect very recent events of positive selection ( < 30 000 years ) ; population structure statistics ( for example , fst test ) are powerful for detecting selective differences among population , especially those that occurred after the out - of - africa dispersals ( < 60 000 years ) . as recently reviewed , allele frequency spectrum and haplotype - based tests are powered to detect classic selective sweeps , in which a novel adaptive variant arises de novo in a population and rapidly increases in frequency to ( or near ) fixation , purging variation at linked sites as they spread .
soft sweeps in which selection arises from variants already polymorphic in a population ( standing variation ) are more difficult to detect using these tests , especially when an adaptive trait is influenced by multiple loci . however , in a geographically restricted population being subjected to adaptive pressures , selection is likely to favor a weaker signature of adaptation , which may include subtle shifts in allele frequencies at multiple loci , resulting in an excess of allele frequency differentiation .
recent studies suggest that such soft sweeps may have been the most common mechanism of adaptation in recent human evolution .
interestingly , immune response pathways show evidence of polygenic adaptation , the process in which adaptation occurs by simultaneous selection on variants at many loci .
hence , methods based on population structure may be more useful in studies of recent human natural selection as they can detect more subtle changes in allele frequency .
as revised , it has been hypothesized that , in addition to genetic ( sequence ) variation , heritable epigenetic modifications can affect rates of fitness increase , as well as patterns of genotypic and phenotypic change during adaptation .
however , the role of epigenetic variation in the response to natural selection has not been formally assessed , as the methodology to test signatures of natural selection on epigenetic variation is just emerging .
given that , if untreated , ads can diminish reproductive potential and impair the ability to raise offspring that successfully reproduce , some evolutionary process must sustain the relative high frequency of risk alleles seen in current populations around the world . as the human genome is shaped by adaptation to environmental pressures at the population level
, one plausible reason for the higher frequency of disease risk alleles may be the direct effect of population - specific natural selection .
this hypothesis is supported by the experimental evidence for mhc heterozygote superiority against multiple pathogens , a mechanism that would contribute to the evolution of hla diversity and explain the persistence of alleles conferring susceptibility to disease .
there is compelling evidence that natural selection is acting on a significant fraction of the human genome .
immune function genes and pathways are consistently reported in tests for natural selection . as a result of several genome - wide scans , over 300 immune - related genes have been suggested as putative targets of positive selection .
although the challenge in validating the true signals remain , several genes involved in immune - related functions have been shown to be under selection . over 40 regions with evidence for selection and associated with at least one ad have been reported ( table 2 ) .
although slightly over half of these regions seem associated with a single disease , close to half are shared among ads , including the hla region , ptpn22 , tnfsf4 , arhgap31-cd80 , tnip1 and tyk2 .
a few ads have reported loci with signatures of positive selection : inflammatory bowel disease ( crohn 's disease and ulcerative colitis ) , celiac disease and systemic lupus erythematosus .
several of the genes in table 2 show patterns of genetic variation that are consistent with evidence for recent positive selection , such as the ptpn22 , itpr3 and blk regions , or snps in clec16a and uhrf1bp1 .
as discussed in the next section , resistance to protozoa and tuberculosis infection have been implicated as the selective pressures for ptpn22 and uhrf1bp1 , respectively .
interestingly , the systemic lupus erythematosus susceptibility allele in uhrf1bp1 is associated with decreased uhrf1bp1 rna expression in different cell subsets , suggesting that the disease risk allele under selection has a regulatory effect .
it is noteworthy that alleles under selective pressure can confer increased risk of ad manifestations , as shown by the evidence that variants within the apol1 gene associated with resistance to human african trypanosomiasis ( aka sleeping sickness ) in some african populations predispose to end - stage kidney disease in systemic lupus erythematosus .
the wide variety of environments inhabited by human populations is likely exerting different selective pressures that lead to adaptation through natural selection .
climatic factors such as altitude , latitude , ultra - violet radiation levels , temperature , as well as diet and pathogens have been reported as agents of selection driving adaptations to these environments and lifestyles .
for example , several correlations between temperature , body mass and metabolic rates have been reported , correlations between latitude with skin pigmentation are well - known , and latitude combined with specific genetic variation have been reported to increase susceptibility to hypertension .
signals of natural selection driven by annual photoperiod variation have been reported for schizophrenia , bipolar disorder and restless leg syndrome risk variants . in a comprehensive genome - wide test for adaptations to continuous climate variables ,
hanckock et al . found correlation between snps and climate variables , including an enrichment of individual snps involved in pigmentation and immune response , as well as for pathways related to uv radiation , infection and immunity , and cancer .
they note that their enrichment for pigmentation and immune response phenotypes contrasts with the metabolic traits with common evidence for adaptations to diet , subsistence and ecoregion . in a recent analysis of correlations between genetic risk of multiple diseases and worldwide migration trajectories ,
the authors report that variants associated with primary biliary cirrhosis , alopecia areata , inflammatory bowel disease , systemic lupus erythematosus , systemic sclerosis , ulcerative colitis and vitiligo , have undergone genetic risk differentiation associated with migration . in addition to these general correlations between climatic variables and particular traits , examples of selective pressures driving natural selection of specific loci include malaria resistance and -globin gene mutation , lactose tolerance and the lactase ( lct ) gene variation , skin pigmentation and the slc24a5 gene variation , high - altitude tolerance and the epas1 gene variation , and adaptation to cold resistance and the uncoupling protein ucp3 .
interestingly , expression qtls ( eqtls ) from immune function and metabolism genes are enriched in signals of environmental adaptation , which highlights the importance of regulatory variations in local adaptation .
nevertheless , the strongest effect of climate is in shaping the spatial pattern and species diversity of human pathogens , which is directly relevant to ad predisposition .
as recently reviewed , in the constant co - evolutionary battle between host and pathogen , pathogens that diminish reproductive potential , either through death or poor health , drive selection on genetic variants that affect pathogen resistance . according to the hygiene hypothesis ' first proposed by strachan in 1989
, the increased disease prevalence of autoimmune and allergic diseases in industrialized countries may be due to modern society 's limited pathogen exposure .
the hygiene hypothesis posits that humans have adapted to infectious exposures that were the norm in the past and that exposure was protective against ad . over
many generations environmental pressure may have favored alleles that affect resistance to pathogenic microorganisms , allowing humans to respond to immune system challenges differently but resulting in an increased risk of ads . as hancock et al
. suggested , it is likely that selection signals in immune - related loci may implicate variants evolving under a model of antagonistic pleiotropy , where the selective pressure was pathogen resistance , and the autoimmune disorder is a pleiotropic consequence of the resistance allele .
this could hence be a mechanism explaining the prevalence of ad risk alleles that are common in the population .
indeed , pathogens have been the main selective pressure through human evolution . in an analysis that included climate , diet regimes and pathogen loads , fumagalli et al . showed that the diversity of the local pathogenic environment is the predominant driver of local adaptation , and that climate conditions only had a relatively minor role .
in addition , they reported an enrichment of genes associated to ads , such as celiac disease , type 1 diabetes and multiple sclerosis , which supports the hypothesis that some susceptibility alleles for ads may be maintained in human population due to past selective processes .
the enrichment for signals of positive selection in inflammatory - disease susceptibility loci has been recently corroborated .
recent reviews of selection signatures left by pathogen - exerted pressure , including immune - related genes , can be found elsewhere .
genetic regions associated with susceptibility to different ads and evidence of selection that has been attributed to host pathogen coevolution are shown in table 2 . in slightly less than half of the regions with evidence for selection and ad - association ,
variation in the hla and sh2b3 has been reported as a protective factor against bacterial infection .
variants in the ifih1 gene , whose protein is a cytoplasmic helicase that recognizes rna of picornaviruses and mediates induction of interferon response to viral rna , have been shown to affect ifih1 function and host antiviral response . in the context of systemic lupus erythematosus ( sle )
has shown that fcgr2b is important in controlling the immune response to plasmodium falciparum , the parasite responsible for the most severe form of malaria , and suggests that the higher frequency of human fcgr2b polymorphisms predisposing to sle in asians and africans may be maintained because these variants reduce susceptibility to malaria .
machado et al . has suggested that helminth infection has driven positive selection of fcgrs variation .
grossman et al . implicated salmonella typhimurium and other exposures that directionally drive selection of the toll - like receptor 5 ( tlr5 ) gene , which is involved in recognition of flagellated bacteria . unlike endosomal tlrs , such as tlr7 and tlr8 , that have been subject to purifying selection , cell - surface tlrs involved in pathogen recognition ( tlr1 , tlr2 , tlr4 , tlr5 , tlr6 and tlr10 ) experienced more relaxed constraints .
uhrf1bp1 has been shown to be significantly differentially expressed in primary dendritic cells upon mycobacterium tuberculosis infection , suggesting that response to tuberculosis has shaped genetic variation at this locus .
nod2 shows complex signatures of selection , with some variation consistent with neutrality and other with balancing or recent positive selection , probably reflecting its association with multiple traits ( crohn 's disease , ulcerative colitis and mycobacterial infections ) .
another complex pattern is that observed for the nonsynonymous variant in ptpn22 that increases the risk of sle , rheumatoid arthritis , type 1 diabetes , vitiligo , autoimmune thyroid disease and ulcerative colitis , but is protective against crohn 's disease , despite ulcerative colitis and crohn 's disease being closely related phenotypes .
it is important to highlight examples where there is strong statistical evidence of selection plus an experimentally validated phenotypic effect likely to predispose to autoimmunity or its complications , as these examples provide the most convincing evidence of positive selection increasing ad susceptibility .
these include the sle - associated polymorphism of the inhibitory receptor fcgr2b , which is found at higher frequency in african and asian populations where malaria is endemic and enhances phagocytosis of plasmodium falciparum - infected erythrocytes , demonstrating that fcgr2b is important in controlling the immune response to malarial parasites . experimental characterization of a nonsynonymous variant in tlr5 , another sle - associated gene , showed that it leads to altered nf-b signaling in response to bacterial flagellin .
functional analysis revealed that cells from individuals carrying the sh2b3 risk allele display an increased proinflammatory cytokine production in response to muramyl dipeptide , a component in all bacterial cell walls , indicating that sh2b3 has a role in protection against bacteria infection .
as previously noted , it is noteworthy that variants within the apol1 gene known to be under strong selective pressure in some african populations predispose to a manifestation of sle , end - stage kidney disease .
apol1 has a role in innate immunity by protecting against trypanosoma brucei infection , the parasitic protozoa transmitted by the tsetse fly that causes human african trypanosomiasis ( a.k.a .
there is compelling evidence that the kidney disease risk alleles ' frequencies increased rapidly because of their potential protective effects against human african trypanosomiasis .
given that these disease risk adaptive alleles have been functionally validated , these examples support the hypothesis that the increased prevalence of ads may result , at least partially , from past events of selection that increased host resistance to infection .
this review summarizes the genetic regions associated with susceptibility to different ads and concomitant evidence for selection , including known agents of selection when known . uncovering these ad - associated loci under selection underscores the importance of population genetics and how the understanding of human genetic diversity is crucial to understanding disease etiology or treatment response at both the population and individual levels .
progress achieved in recent years is a direct consequence of large - scale projects such as the hapmap and hgdp .
however , the common variation analyzed by these projects does not capture the novel , deleterious or functional variants that , together with common variation , distinguish global human populations .
now , with genome sequence information available on a population scale from projects like the 1000 genomes , the role of selection in shaping ad risk can be assessed with unprecedented detail in an unbiased fashion .
recent studies using whole - genome sequence data helped identify the established functional snps in known loci and report novel candidate regions for positive selection . in the future
, analyses of heritable epigenetic variation will unveil signatures of natural selection on epigenetic variation .
the complexity of gene effects in different ads is remarkable , as shown , for example , by the complex signatures of selection seen at nod2 , and the plethora of shared variants that increase ad predisposition concordantly or discordantly , such as for ptpn22 .
these observations illustrate that the model whereby ad risk alleles are positively selected due to their protective effects against infections is naive .
a combination of population - level phenomena , including possibly bottlenecks , migration , admixture , natural selection and random genetic drift , are likely contributors to this complexity of gene effects . given the complex history of selective pressures acting on humans , unequal selective pressures and a diverse spectrum of plausible evolutionary models are expected to be exerted on susceptibility loci for ads .
it is likely that several pathogens have exerted pressure on the same loci and that selection can vary in form , intensity , time and space , which is consistent with the observation that both risk and protective alleles for ads increased in frequency due to selection . for most regions ,
the exact selective pressure leaving the signature of selection is unclear . clearly , these signatures are not necessarily the result of adaptation , but might be a consequence of random genetic drift . in any case ,
regardless of the population phenomenon shaping current human genetic diversity , this genetic variation is the basis of clinically relevant traits at both the individual and population levels .
an important next step to delineate the selective advantage conferred by these ad risk variants are functional studies using in vitro experiments and model organisms to identify the underlying functional variants and quantify the phenotypic consequences of the candidate adaptive alleles .
pathogen coevolution is ongoing and , despite the emergence of new pathogens ( for example , hiv ) , potential pathogens driving these host - specific adaptations are expected to have long - standing relationships with humans , including those that cause malaria , smallpox , cholera , tuberculosis and leprosy , as well as the human microbiome .
regardless of the agent of selection and the reasons for the emergence of both common and rare ad - causing alleles , incorporating population genetics to understand human genetic diversity will lead to a better understanding of the causes of health disparities , identification of functional variants and discovery of cellular mechanisms and contribute to the development of new therapies . | human genetic diversity is the result of population genetic forces .
this genetic variation influences disease risk and contributes to health disparities . autoimmune diseases ( ads )
are a family of complex heterogeneous disorders with similar underlying mechanisms characterized by immune responses against self .
collectively , ads are common , exhibit gender and ethnic disparities , and increasing incidence .
as natural selection is an important influence on human genetic variation , and immune function genes are enriched for signals of positive selection , it is thought that the prevalence of ad risk alleles seen in different population is partially the result of differing selective pressures ( for example , due to pathogens ) . with the advent of high - throughput technologies , new analytical methodologies and large - scale projects , evidence for
the role of natural selection in contributing to the heritable component of ads keeps growing .
this review summarizes the genetic regions associated with susceptibility to different ads and concomitant evidence for selection , including known agents of selection exerting selective pressure in these regions .
examples of specific adaptive variants with phenotypic effects are included as an evidence of natural selection increasing ad susceptibility .
many of the complexities of gene effects in different ads can be explained by population genetics phenomena . integrating ad susceptibility studies with population genetics to investigate how natural selection has contributed to genetic variation that influences disease risk will help to identify functional variants and elucidate biological mechanisms . as such , the study of population genetics in human population holds untapped potential for elucidating the genetic causes of human disease and more rapidly focusing to personalized medicine . |
diabetes mellitus is a syndrome characterized by chronic hyperglycemia and disturbances of carbohydrate , fat , and protein metabolism occurs due to deficiency of insulin ( type 1 ) or insensitivity of the target tissues to insulin ( type 2 ) . when untreated , it leads to multiorgan damage , specially cardiovascular , renal , neurological , and ocular .
currently , a wide variety of oral antidiabetic agents and insulin are available for the treatment of type 2 diabetes .
several oral antidiabetic agents like metformin , sulfonylureas , meglitinides , thiazolidinediones are currently available .
some of them increase the insulin secretion from pancreas while others increase peripheral utilization of glucose by increasing the sensitivity of peripheral tissues to utilize glucose . oral therapy for type 2 diabetes , when used appropriately , can safely assist patients to achieve glycemic target in the short term to medium term .
however , the progressive nature of type 2 diabetes usually requires a combination of two or more oral agents in long term .
issues of safety and tolerability , notably weight gain , often limit the optimal application on antidiabetic drugs such as sulfonylureas , meglitinides and thiazolidinediones .
therefore , there is a need for novel oral antidiabetic agents with different mechanism of action from existing oral antidiabetic agents .
bromocriptine mesylate quick release formulation has been approved by fda in may 2009 for the treatment of type 2 diabetes in adults as an adjunct to diet and exercise to improve glycemic control .
the concept of using this d2 receptor agonist for the treatment of type 2 diabetes came while studying the metabolism of migrating birds [ figure 1 ] .
the hibernation theory of circadian neuroendocrine rhythm which forms the basis for the use of bromocriptine use proposes that animals hibernating in the winter not only put on a lot of weight before they hibernate but also they become insulin resistant/ glucose intolerant which help them to conserve glucose and lipids for energy production .
extensive animal studies have shown that decreased hypothalamic dopaminergic tone may be involved in the pathogenesis of this insulin resistance .
the normal circadian cycle that results in leaner body in the summer and heavier body in winter is disturbed in humans because of abundant caloric intake round the year as in modern times human do not worry about food availability during summer resulting in the absence of lean phase .
stimulation of the hypothalamus promotes the release of several hormones that respond to traditional shift in caloric intake and storage .
quick release bromocriptine , given once in the morning , stimulates the hypothalamus to reset circadian rhythm if it was permanently stuck in a winter rhythm ( insulin resistant phase ) , thus improving insulin resistance and peripheral glucose disposal by central mechanism .
hence , there is a rationale for this drug , which attacks the problem of insulin resistance , type 2 diabetes and obesity ( which form a conglomere ) in a completely new way .
therefore , bromocriptine may prove to be a revolution in the field of treatment of type 2 diabetes .
very few studies have been carried out to clinically evaluate bromocriptine in type 2 diabetes mellitus and no such study was done in indian population .
so , the present study is undertaken to evaluate the efficacy and safety of bromocriptine monotherapy as well as its low dose combination with metformin in type 2 diabetes .
this was a prospective , randomized , double blind , parallel study carried out at the outpatient department of an endocrinology hospital over 12 weeks after obtaining approval by institutional ethics committee . newly diagnosed adult type 2 diabetes patients of either sex with fasting blood sugar in the range of 126 to 200 mg / dl
the following category of patients were excluded : type 1 diabetes mellitus , diabetic ketoacidosis , known hepersensitivity to ergot alkaloids or bromocriptine , nursing mothers , psychiatric disorder , syncopal migraine or patients taking antihypertensives .
sample size determined from previous studies was large but for feasibility a smaller sample was chosen .
105 patients were included in the study and randomized into 3 groups of 35 each .
simple randomization was done and allocation was concealed by employing different investigators for each step of random number generation , enrolloment , assignment of patients to treatment groups .
patients in group 1 received bromocriptine qr 2.4 mg once daily within 2 h of waking in the morning and with food to reduce the risk for gastrointestinal adverse effects such as nausea .
group 3 received bromocriptine qr 1.6 mg as in group 1 and metformin 500 mg two times daily .
as metformin is a standard first line drug for type 2 diabetes , it was chosen as the standard to compare against .
most patients eventually need two or more drugs in real life management of type 2 diabetes .
therefore a third group comprising the combination of both drugs was added with low dose bromocriptine .
after enrolment , detailed medical history and baseline values of fasting blood sugar ( fbs ) , post prandial sugar ( ppbs ) and hba1c and bmi were recorded .
efficacy was assessed by measuring fbs ( mg / dl ) , ppbs ( mg / dl ) at 6 and 12 weeks while hba1c ( % ) and bmi ( weight /height ) at 12 week .
safety was assessed in terms of systemic adverse effects by questioning patients at each visit and objective signs were assessed by clinical and biochemical examination .
routine hematological ( hb and tlc ) and biochemical investigations ( serum creatinine , sgpt , sgot ) were done at baseline and at the 12 week of study period .
data was checked for normality and then analyzed by z test for difference between two means , p value < 0.05 was taken as significant while p value > 0.05 was considered as insignificant .
baseline values of all three groups were comparable with respect to age , sex , habits , fbs , ppbs , hba1c , and bmi [ table 1 ] .
baseline data of group 1 , group 2 and group3 mean fbs reduction in group 1 compared with day 0 , at 6 weeks was not significant ( p<0.05 ) while that at 12 weeks was statistically significant ( p<0.05 ) [ table 2 ] . in groups 2 and 3 ,
reduction at 6 and 12 weeks was significant ( p<0.05 ) when compared to baseline .
12 weeks reduction of group 3 was significantly greater than that of group1 ( p<0.05 ) .
effect on mean fasting blood sugar ( mg / dl ) at 6 and 12 weeks group 1 mean ppbs reduction when compared with day 0 , at 6 weeks was not significant ( p<0.05 ) while that at 12 weeks was statistically significant ( p<0.05 ) [ table 3 ] . in groups 2 and 3 ,
reduction at 6 and 12 weeks was significant ( p<0.05 ) when compared to baseline .
12 weeks reduction of group 3 was significantly greater than that of group1 ( p<0.05 ) .
effect on mean postprandial blood sugar(mg / dl ) at 6 and 12 weeks mean reduction in glycosylated hemoglobin at 12 weeks when compared with respective baseline values was statistically significant ( p<0.05 ) in all three treatment groups [ table 4 ] .
effect on mean hba1c level ( % ) at 12 weeks all three treatment groups showed reduction in mean bmi which when compared with baseline was found to be statistically not significant ( p<0.05 ) [ table 5 ] .
effect on mean body mass index at 12 weeks in the present study , significant common adverse drug events observed were nausea , vomiting , and headache in all the three groups with comparable incidence .
none of the groups had adverse impact on hb level , total leucocyte count , serum creatinine , sgpt and sgot .
diabetes mellitus is a chronic metabolic syndrome of endocrine origin with multiorgan involvement and complications .
currently wide variety of oral hypoglycemic agents and insulin are available for the treatment of type 2 diabetes .
there is a need for novel antidiabetic agents with different mechanism of action from existing drugs.the traditional oral agents are often associated with an increased risk of adverse events ( hypoglycemia , weight gain ) and typically may become less effective over time as patients undergo progressive beta cell failure and often fail to achieve target glycemic control in patients , even when used as combination therapy .
bromocriptine , a dopamine receptor ( d2 ) agonist used for hyperprolactinemia and parkinsonism , has recently been approved for the treatment of type 2 diabetes in adjunct to diet and exercise to improve glycemic control .
studies carried out have shown that it can reduce fbs , ppbs and hba1c level significantly .
no study has been carried out to evaluate the effectiveness of bromocriptine in type 2 diabetes mellitus in indian population until now .
so , the present study was planned to evaluate the efficacy and safety of bromocriptine in type 2 diabetes as monotherapy and in combination with metformin . as depicted in graph 1 ,
mean fbs in group1 ( bromocriptine ) was reduced by 4.79 mg / dl at 6 weeks and by 16.09 mg / dl at 12 weeks . when compared to baseline reduction at 6 weeks ,
it was found to be statistically insignificant ( p>0.05 ) , whereas at 12 weeks it was significant ( p<0.05 ) . in group 2 ( metformin ) , it was decreased by 19.79 mg / dl at 6 weeks and by 37.36 mg / dl at 12 weeks . in group 3
( combination ) , there was a reduction in mean fbs by 26.26 mg / dl at 6 weeks and by 44.31 mg / dl at 12 weeks . in groups 2 and 3 ,
reduction in mean fbs at 6 and 12 weeks was found to be statistically significant when compared to baseline values ( p<0.05 ) .
previous study on bromocriptine by pijl et al . , found fbs reduction by 18 mg / ml after 16 weeks . in group 1 , reduction was significant at 12 weeks of treatment and this lag period could be due to its complex mechanism acting through neural circuits and providing a fine tuning of hypothalamic circadian rhythm . in the present study , combination of metformin with low dose bromocriptine
comparison of reduction in fasting blood sugar level ( mg / dl ) as shown in graph 2 , in group 1 reduction in mean ppbs at 6 weeks was 3.92 mg / dl and at 12 weeks it was 14.38 mg / dl .
when compared to baseline reduction at 6 weeks it was found to be statistically insignificant ( p>0.05 ) , whereas at 12 weeks it was significant ( p<0.05 ) .
similar findings were observed by kamath et al . , where the reduction in postprandial sugar was 16 mg / dl . in group 2
mean ppbs decreased by 19.94 mg / dl at 6 weeks and by 35.87 mg / dl at 12 weeks . in group 3
it decreased by 25.66 mg / dl at 6 weeks and by 43.71 mg / dl at 12 weeks . in groups 2 and 3 , the reduction in mean fasting blood sugar level at 6 and 12 weeks was found to be statistically significant when compared to baseline values ( p<0.05 ) . in the present study , combination of metformin with low dose bromocriptine significantly reduced ppbs as compared to bromocriptine or metformin alone .
comparison of reduction in postprandial blood sugar level ( mg / dl ) graph 3 shows the mean reduction in glycosylated hemoglobin at 12 weeks which was 0.46% in group 1 , 0.63% in group 2 and 0.74% in group 3 .
this reduction was statistically significant ( p<0.05 ) when compared with respective baseline values in all the groups .
comparison of reduction in glycosylated haemoglobin levels ( gm % ) thus it can be concluded from the findings of the present study that bromocriptine as monotharapy and in combination is effective in significantly reducing fbs , ppbs and hba1c level .
though there was a slight decrease in bmi at 12 weeks in all the groups , when compared to baseline values , reduction was statistically insignificant .
in present study there was no significant effect on bmi possibly due to short duration of study .
the cerebral mechanisms underlying the behaviours that lead to pathological overeating and obesity are postulated to involve dopamine .
dopamine modulates motivation and reward circuits and hence dopamine deficiency in obese individuals may perpetuate pathological eating as a means to compensate for decreased activation of these circuits .
strategies aimed at improving dopamine function may be beneficial in the treatment of obese individuals .
further extensive studies of long duration need to be carried out to evaluate the effectiveness of dopamine agonists including bromocriptine in obesity and in obese diabetic patients .
frequency of patients experiencing at least one adverse reaction in bromocriptine treated group 1 was 30.3 % , in metformin treated group 2 was 33.7% while in group 3 treated with bromocriptine and metformin it was 35.3% .
significant common adverse drug events observed were nausea , vomiting , headache in all three groups with incidence not significantly different from each other .
other adverse effects seen in bromocriptine treated groups are fatigue and dizziness . in previous studies ,
bromocriptine used in diabetes patients has demonstrated good safety and tolerability , most of the side effects were gastrointestinal and tend to be present on initiation or titration of dose and decreased on continuation of therapy .
thus from the results of present study it can be concluded that bromocriptine is a safe antidiabetic agent alone and in combination with metformin .
no drug had any adverse impact on the values of hemoglobin , total leukocyte count ( tlc ) , serum creatinine , sgpt , sgot .
apart from the efficacy parameters studied in the present study , various other favorable effects of bromocriptine in animal and human studies have been observed .
multiple animal studies have demonstrated metabolic improvements including significant weight loss , decreased levels of blood sugar and triglycerides , decreased insulin resistance and increased glucose tolerance .
human studies show similar effects , though the results are not as profound . from the safety point of view
, bromocriptine meets all the usfda cardiovascular safety guidelines and demonstrated 40% reduction in cardiovascular end points .
bromocriptines clinical utility should be maximal in obese , depressed patients with limited mobility and features of insulin resistance . despite well - established guidelines for glycemic control , many diabetes patients do not achieve targeted goals or have difficulty in maintaining it .
adherence to prescribed medicine , dietary and lifestyle regimens are all crucial factors in the management of type 2 diabetes .
bromocriptine has the advantage of acting through a completely different mechanism of action from currently available antidiabetic drugs , easy to use single daily dose which may boost compliance , can be combined with other antidiabetic drugs for synergistic effect and may help reduce their dose and consequent adverse reactions .
the dose approved for treatment of type 2 diabetes is 2 to 3 fold lower than usual doses in hyperprolactinemia and 10 to 20 fold lower than treatment for parkinson 's disease .
overall , bromocriptine demonstrates good safety and tolerability that most individuals are adherent to therapy .
india has the largest number of diabetic subjects in the world and therefore called as the diabetes capital of the world .
according to the diabetes atlas 2006 published by the international diabetes federation , the number of diabetics in india is around 40.9 million which is expected to rise to 69.9 million by 2025 unless urgent preventive steps are taken .
asian indian phenotype with certain unique clinical and biochemical abnormalities in indians which include increased insulin resistance , greater abdominal adiposity i.e. , higher waist circumference despite lower body mass index , lower adiponectin and higher high sensitive c - reactive protein levels makes asian indians more prone to diabetes .
so any modality of treatment including therapeutic lifestyle change ( tlc ) , pharmacotherapy can gradually but significantly improve the situation and can prevent , retard progression and complications of diabetes and reduce the financial burden on the individual patient and government for the awareness and treatment of the diabetes . a recent survey conducted on indian physicians about the knowledge and attitude about the use of bromopcriptine in diabetes management demonstrated the interest and high potential associated with bromocriptine use .
the present study is a small study both as regards to the number of patients included , the duration , also dose used was fixed . in india
more extensive studies including large number of patients with differing severity and comorbidities , more efficacy parameters and a flexible dosing pattern are required to determine the exact utility of this drug with novel mechanism .
results of the present study show that bromocriptine is effective in improving the markers of diabetes like fasting blood sugar , postprandial blood sugar , hba1c , even though the results are not as profound as that of metformin . combination with metformin in a low dose was found to be more effective in improving glycemic control than individual drugs alone . | context : diabetes mellitus is a chronic metabolic disorder of endocrinal origin with multiorgan involement . today 's physician has a lot many options to choose for treating type 2 diabetes , but does not always manages to achieve optimal glycemic control .
the newer drug bromocriptine acts by novel hypothalamic circadian rhythm resetting mechanism.objective:to evaluate the efficacy and safety of bromocriptine qr in type 2 diabetes.materials and methods:105 patients according to inclusion and exclusion criteria were randomized into three groups by simple randomization .
group 1 received bromocriprine 2.4 mg once daily , group 2 received metformin 500 mg twice daily while group 3 received bromocriprine 1.6 mg daily and metformin 500 mg twice daily .
baseline measurement of fasting and postprandial blood sugar , hba1c and bmi were followed up at 6th and 12th weeks .
safety evaluation was done by questioning the patient and also through routine hematological and biochemical parameters .
z test was used for analysis.results:group 1 showed significant reduction in fasting and postprandial sugar and hba1c at 12 weeks . while groups 2 and 3 showed even higher reduction in these parameters albeit with slightly more adverse drug events like nausea , vomiting compared to group 1.conclusion:bromocriptine qr is an effective and safe antidiabetic drug which can be employed as monotherapy or in conjuction with metformin to achieve and maintain optimal glycemic control . |
a 17-year - old male patient visited our institution with a complaint of a painful movable mass on the lateral distal aspect of the right femur that was not caused by trauma .
the mass had been slowly growing for 6 months and started to cause increasing pain from one month ago .
a firm and movable mass with a size of a quail egg was palpated in the lateral distal aspect of the right femur .
neither muscle weakness and numbness nor abnormal sensation was observed in the lower leg . in the absence of abnormal findings on radiography ,
ultrasound was performed , in which a cystic tumor approximately 4.12.5 cm in size was identified in the quadriceps femoris ( fig .
was considered unnecessary because it had a relatively distinct border and was well - encapsulated .
exploration and excision of the mass were performed under the assumption of a vascular lesion or a lipoma within the vastus lateralis of the quadriceps femoris .
noninvasive methods such as aspiration and steroid injection could be an option , but surgical excision was our treatment of choice in order to reduce the possibility of recurrence .
intraoperatively , spindle - shaped swelling was observed and a firm mass was palpated in the vastus lateralis of the quadriceps femoris .
the mass could be dissected with ease from the surrounding tissue and had a stalk as other ganglion cysts .
when the muscle sheath was removed , the mass was found to originate from the muscle , not from the muscle sheath . in the naked eye ,
the intramuscular cystic mass was 3.42.21.4 cm in size , weighed 4.7 g , and had some muscle tissue on its smooth surface ( fig .
2 ) . when dissected upon excision , clear jelly - like fluid was noted in the sac .
the biopsy results showed that the cystic wall was composed of fibrous tissue , lacked a synovial lining , and had muscular tissue on the surface , which led us to the diagnosis of an intramuscular ganglion cyst ( fig .
there has been neither recurrence nor complications for the 1 year and 6 months of follow - up period .
ganglion cysts are the most common soft tissue lesions of the hand that are composed of a fluid - filled sac connected to a synovial sheath , a tendon sheath , or a tendon .
some studies associate them with mucoid degeneration of soft tissues , outpouchings of tissues through defects in the joint capsule or tendon sheath , or a trauma3 ) .
the most common area of occurrence is the radial side of the extensor digiti communis tendon on the dorsum , followed by the area between the flexor carpi radialis and the abductor pollicis longus tendons on the volar side of the wrist4 ) .
ganglions cysts arise from joints in most cases and rarely from the tendon sheath , cartilage , cruciate ligaments , nerves , and muscles5 - 7 ) .
although most of the ganglia around the knee joint are located in the tendon sheath or joint capsule , they can form in the meniscus , anterior cruciate ligament , posterior cruciate ligament , and common peroneal nerve9 ) .
intraosseous ganglion cysts that are characteristically located in the epiphysis of long bones are mostly solitary and involve the distal end of the tibia . currently , there are only 2 reported cases of ganglion cysts in the quadriceps femoris that were diagnosed using plain radiography , ultrasound , and computed tomography . in our patient ,
the majority of ganglion cysts are palpable tender lumps without any accompanying symptoms . however ,
when a ganglion cyst exerts pressure on the nerves or blood vessels , pain , numbness , and muscle weakness may occur or tenderness and discomfort during joint motion may present from the initial stage .
although ultrasound is a valuable diagnostic tool , it is limited in its ability to evaluate the relationship between a cyst and adjacent soft tissues .
mri is essential for the differential diagnosis from other masses such as vascular lesions and lipomas . in our patient ,
mri was considered unnecessary because the mass was seen well - encapsulated with a distinct border on ultrasound .
it is important to identify during surgery whether a lesion is a mass , has a connection with a joint , or is associated with intraarticular disease and to perform thorough excision in order to reduce the likelihood of recurrence .
ganglia rarely recur with proper curative measures , but inappropriate treatment methods can result in a recurrence rate of 50%3 ) .
the available treatment options range from non - surgical methods such as rupture by direct pressure , aspiration with or without a steroid injection , and draining using threads to surgical excision with or without arthroscopy .
the cure rate of ganglion cysts has no association with gender , age , and size and location .
nelson et al.4 ) reported that the cure rate was 94% after excision under general anesthesia or axillary nerve anesthesia , 84% after excision under local anesthesia with a tourniquet in place , and 65% after rupture by direct pressure or aspiration with a steroid injection . in the study by aydin et al.10 ) on 40 cases of excision of ganglion cysts of the hand , the recurrence rate was 22% , damage to the median palmar cutaneous nerve was observed in 10% , radial nerve damage was present in 5% , and joint stiffness occurred in 5% .
they cautioned that the possibility of recurrence and other complications should be taken into consideration in determining a surgical option for the treatment of a ganglion cyst .
intramuscular ganglion cysts are rare with only a few cases involving the hand present in the literature .
, we described a case of an intramuscular ganglion cyst in the vastus lateralis of the quadriceps femoris that was successfully treated with excision without complications . | ganglion cysts are common lesions that are most often found around the joints of the hands and feet .
ganglia around the distal femur usually occur within the synovial membrane or tendon sheath , but rarely within muscles .
several cases of intramuscular ganglions in the hand and wrist have been reported , but a ganglion cyst in the quadriceps muscle has rarely been addressed in studies . in this report , we present a 17-year - old patient with a painful movable mass in the intramuscular area of the quadriceps femoris that was diagnosed by ultrasound and treated by excision and biopsy . |
measuring efficiency has been common in a large range of activities , including health care ( 1 , 2 ) , insurance services ( 3 ) , universities ( 4 , 5 ) , and research activities ( 6 ) . analyzing efficiency through dea ( data envelopment analysis ) is an assessment of output - input ratio in order to find relative efficiencies of decision - making units ( dmus ) ( 7 ) .
dea is well known as a powerful and also easily understandable analytical tool ( 9 , 10 ) .
further , its superiority compared with other techniques has been proven by several studies ( 11 , 12 ) . during the last few decades
, the health sector has experienced a dramatic growth in cost in developed and developing countries , and inefficiency of health sectors , at least in part , has contributed to this growth ( 13 , 14 ) . in response to this situation ,
extensive empirical studies have focused on the measurement of efficiency of health institutions around the world . while hospitals are the subject of most of these studies , there are few studies about health research center efficiency .
nowadays , due to the rapid progress of research systems , the regular collection and reporting information about the performance of research centers are common practices .
research center information and reports can provide a general overview about their performance ( 15 , 16 ) , and the next logical step is to begin comparison of the efficiency of research centers . through such comparisons ,
the most efficient research units can be identified and considered as benchmarks for improving the efficiency and effectiveness of others ( 17 ) .
iran also has experienced an impressive advance in knowledge production during the past decades ( 18 , 19 ) .
the numbers of health research centers increased from 53 in 2001 to 359 in 2010 during the last decade .
research centers are growing rapidly in iran and will continue to provide excellent opportunities in knowledge production ( 15 ) .
also iran s government provides great financial support for health research centers and research projects in order to advance knowledge production more progressively ( 15 , 20 ) .
our study is the first step in analyzing efficiency of knowledge production in health areas and can be a good model for analyzing efficiency of knowledge production , especially for developing and in - transition countries .
the aim of our study is to measure efficiency of knowledge production by a sample of health research centers using data envelopment analysis .
this paper used a comprehensive methodology of data envelopment analysis ( dea ) to measure health research center efficiency in iran .
this retrospective and applied study conducted in 2015 used input and output data of 16 health research centers affiliated with a health sciences university during 20102014 .
the 16 research centers included virology , nephrology and urology , nano biotechnology , microbiology , gastroenterology and hepatology , behavioral sciences , neurosciences , human genetics , sports physiology , trauma , molecular biology , biotechnology , nutrition and hygiene , chemical injuries , medicine and religion and health management .
the input and output data of the health research centers for years 20102014 were collected from the iran ministry of health and medical education ( mohe ) profile .
the input data included the average amount of annual budget , faculty researchers , and staff researchers employed in each health research center during five years ( 20102014 ) .
these inputs could well represent labor and capital used in the knowledge production process and have been widely considered in previous studies as the main factors affecting knowledge production in research centers ( 20 , 21 ) .
the output data included the average number of published papers indexed in isi web of science , paper citations according to scopus website report , and the textbooks written by each research center during 20102014 .
these outputs introduced and reported by the iran ministry of health as the main indicators of measuring knowledge production of health research centers .
these outputs also have been used in previous studies for measuring knowledge production of research centers ( 15 , 16 ) . using the average five years ( 20102014 ) of inputs and outputs data was because the result of some research activities appear in the long - run ( five year ) .
the three main models of dea , included input - oriented model , output - oriented model , and hyperbolic - oriented model in the vrs ( variable returns to scale ) approach , were used to study the relative efficiency of health research centers .
the input - oriented model focused on minimizing the resources for a certain level of production , while the output - oriented model focused on maximizing production in a certain level of resources .
the hyperbolic - oriented model focused on both resource minimizing and production maximizing simultaneously ( 22 ) . in dea models , the efficiency scores 1 means complete efficiency and the efficiency scores smaller than 1 means relatively not complete efficacy .
the input and output data of each research center were entered into r software , and the numerical score of each research center s efficiency were calculated and reported .
the numerical values of each research center s efficiency in three main models , including input - oriented , output - oriented , and hyperbolic , are reported in table 1 .
eight research centers have complete efficiency in all the three models , including chemical injuries , nutrition and hygiene , trauma , human genetic , medicine and religion , gastroenterology and herpetology , microbiology , and virology .
dispersion indexes of efficiency scores in each of the three models are shown in table 2 .
the minimum efficiency score in input- , output- , and hyperbolic - oriented models was 0.043 , 0.048 , and 0.11 , respectively .
the applied biotechnology research center have the minimum efficiency level in the input - oriented model with a score of 0.043 and the nano biotechnology research center has a minimum efficiency level in both output - oriented and hyperbolic - oriented models with 0.048 and 0.11 efficiency score , respectively . the mean efficiency score in input - oriented , output - oriented , and hyperbolic - oriented models was 0.781 , 0.671 , and 0.798 , respectively . in table 3 ,
research centers with less than the mean scores reported as an under - average efficiency research center . according to table 3 , seven research centers were below the average efficiency in the input - oriented model , and six research centers were below the average efficiency in output - and hyperbolic - oriented models .
five research centers ( including applied biotechnology , sports physiology , behavioral sciences , health management , and nano biotechnology ) were under the average efficiency in all the three models .
the considerable advances in health science in the twentieth century have become obvious , and it is really necessary to maintain this capacity for continued scientific advance in the twenty - first century .
this goal is not achievable without a greater focus on efficiency of health science researches .
therefore , focusing on the efficiency of a health research center seems to be critical .
improving the efficiency of health research centers can provide a suitable foundation for these crucial health scientific advances . in recent years
, the number of health research centers has expanded , which have had a significant role in health knowledge production .
assessing the efficiency of research centers is vital for effective performance and utilization of knowledge production activities , and our study is a first step is this area .
the application of dea can enable health research centers to identify deficiencies and taking appropriate actions for improvement . in this study ,
the efficiency of health research centers was evaluated based on three basic data envelopment analysis ( dea ) models : input - oriented , output - oriented , and hyperbolic - oriented . in the input - oriented model , the mean of efficiency score was 0.781 .
it means that , among 16 research centers , seven research centers ( 44% ) have been working below average efficiency level and should reduce their inputs for producing the same level of outputs . in the output - oriented model ,
this means that six research centers ( 37% ) have been working under the average efficiency level and should increase their outputs by the same level of outputs , and the average performance can be 0.33 percent better . based on the hyperbolic - oriented model , the mean of efficiency score was 0.798 .
so , according to the results of the model among 16 research centers , six research centers ( 37% ) have been working under the average - efficiency level .
based on the findings of this study , only 50% percent of health research centers ( eight from 16 ) were completely efficient .
in all the studied models , 31% of research centers ( including applied biotechnology , sports physiology , behavioral sciences , health management , and nano biotechnology ) were identified as under - average efficiency research centers .
so it is necessary for health research centers to improve their efficiency through better management of available resources .
the application of dea to assess randd efficiency of 29 universities in china indicated that universities that have improved their management achieved a high efficiency value regardless of whether their original randd strengths were strong or weak .
they expressed that such a measure is proved to be helpful for motivating universities to keep on improving their randd management ( 23 ) . also agasisti and haelerman in their study analyzed the performance of public universities in italy and the netherlands . according to their findings , the relative efficiency of italian and dutch universities is largely dependent on policy adoption . when italian universities were much more efficient in cost minimizing for a given level of activity , dutch universities were more efficient in realizing european goals and minimizing resources to obtain a high number of graduates ( 4 ) . while many studies have measured the efficiency of hospitals and health service institutions , there are only a few studies on measuring the efficiency of health research centers . in a similar study by dabagh
( 24 ) , efficiency and productivity of the research sectors of 31 capital universities of iran have been investigated using data envelopment analysis ( dea ) ; results showed that most universities ( 74% ) were technically inefficient in research filed in 2007 . in another study by afonso and santos ( 25 ) on relative efficiency of portuguese public universities by dea , output efficiency scores were 0.77 , which means that , with the same inputs , the average faculty seems to be obtaining 23-percent - less performance than it should . also in a study by kuah and wong ( 26 ) in malaysia
, the efficiency of 30 universities was assessed through dea in teaching and research activities separately .
the results showed that three universities were efficient in their teaching activities and 11 universities were efficient in their research activities .
out of these 30 universities , only one university was efficient in all the criteria . according to the results of our study , a large gap exists between maximum and minimum efficiency scores of different research centers .
for example , taylor and harris ( 27 ) showed considerable and time progressive differences in relative efficiency among south african universities .
also in a study by dabagh ( 24 ) , the result of five years following efficiency suggests that , despite large differences in relative efficiency of universities in iran , there has been no significant improvement in research efficiency in these five years .
a study of australian public university efficiency suggests that australian universities are operating at a fairly high level of efficiency relative to each other ( 28 ) .
however , an empirical study by zhigang et al . on total factor productivity in china showed that the country s eastern regions were significantly and constantly more efficient than the western regions during the years 19782003 ( 29 ) . due to the undesirable efficiency level in a large number of health research centers and also due to the large gap between efficiency of health research centers , our study suggests future studies should focus on identify factors affecting efficiency in health research centers .
a balanced efficiency improvement is necessary in order to make health science progress more desirable . in order to reach this goal ,
it is necessary that future studies focus on the institutional and environmental factors affecting health research center efficiency .
also some factors include manager s experience and skills mentioned previously by some studies ( 30 ) ; still there are many unknown factors affecting efficiency of health research centers .
the present study has limitations that we would like to note as follows : first , in our study , we compare research centers together without considering their field of activity . comparing efficiency of research centers in different field of sciences separately such as clinical and none clinical research centers can provide more accurate comparisons .
second , we use only a nonparametric method to measure efficiency of research centers , but using parametric methods also can be useful for such comparisons .
our study was just a first step in analyzing efficiency of knowledge production in health areas , and future studies should be done without such limitations .
despite increasing the number of health research centers in recent years and their role in producing knowledge and health promotion in society , based on the results of this study , one - half of the studied research centers are operating below full efficiency .
also , a significant number of research centers ( one - third ) are operating under an average efficiency level , and still there is a large gap between research center efficiency relative to each other .
thus , the higher level of efficiency in a significant number of the health research centers will occur through more efficient management of human resources and capital .
further researches are needed for measuring and following the efficiency of knowledge production by health research centers around the world and over a period of time using dea techniques . because many health research centers regard themselves in global competition , international comparisons are also necessary in future studies . | introductionefficiency analysis is necessary in order to avoid waste of materials , energy , effort , money , and time during scientific research .
therefore , analyzing efficiency of knowledge production in health areas is necessary , especially for developing and in - transition countries . as the first step in this field , the aim of this study was the analysis of selected health research center efficiency using data envelopment analysis ( dea).methodsthis retrospective and applied study
was conducted in 2015 using input and output data of 16 health research centers affiliated with a health sciences university in iran during 20102014 .
the technical efficiency of health research centers was evaluated based on three basic data envelopment analysis ( dea ) models : input - oriented , output - oriented , and hyperbolic - oriented .
the input and output data of each health research center for years 20102014 were collected from the iran ministry of health and medical education ( mohe ) profile and analyzed by r software.resultsthe mean efficiency score in input - oriented , output - oriented , and hyperbolic - oriented models was 0.781 , 0.671 , and 0.798 , respectively .
based on results of the study , half of the health research centers are operating below full efficiency , and about one - third of them are operating under the average efficiency level .
there is also a large gap between health research center efficiency relative to each other.conclusionit is necessary for health research centers to improve their efficiency in knowledge production through better management of available resources .
the higher level of efficiency in a significant number of health research centers is achievable through more efficient management of human resources and capital .
further research is needed to measure and follow the efficiency of knowledge production by health research centers around the world and over a period of time . |
lymphoscintigraphies performed during sentinel lymph node ( sln ) mapping and biopsies in breast cancer have discovered lymphatic extra - axillary drainage in 20 - 27% of cases .
extra - axillary nodes most often located are : ipsilateral internal mammary chain ( imc ) ( 17% ) , intra - mammary ( 3% ) , supraclavicular ( 2% ) and interpectoral ( 2% ) nodes .
contralateral axillary drainage is an unexpected finding that is a source of controversies in the management of these patients.[310 ] this situation is more exceptional when there is not prior breast surgery,[710 ] especially when internal mammary chain drainage is associated .
a 48-years - old woman with no prior surgery of breast or axilla was diagnosed of breast ductal carcinoma in retroareolar region of the right breast with size of 2 cm .
lumpectomy and sentinel lymphatic node ( sln ) biopsy was suggested . the day prior to surgery , a lymphoscintigraphy was performed with peritumoral injection of two doses of 74 mbq of technetium-99m - nanocolloid ( volume of 0.1 ml ) each one .
planar images were acquired at 120 min of injection , including chest in anterior and lateral projections .
we acquired images of 3 min in a 256 256 matrix using a planar source of cobalt-57 in anterior projection and an anatomical point source of tc99 m in order to delineate the body shape and locate the nodes .
the images showed drainage to ipsilateral axillary nodes , ipsilateral imc and contralateral axillary nodes [ figure 1 ] .
in the left we show anterior image of lymphoscintigraphy after 2 peritumoral injection of tc-99m - nanocolloid in the retroareolar region of right breast .
bilateral axilla and ipsilateral ( internal mammary chain ) imc drainage are depicted with at least , 3 nodes in right axilla , 2 in imc and only one in left axilla . in the right upper quadrant ,
in the left lateral projection we can see tracer injection activity in the breast and the only contralateral axillary node . in the right
lower quadrant , right lateral projection shows activity in the breast and in the main axillary node in the operating room , lumpectomy and sln biopsy of the three territories were performed .
frozen section and subsequent 2 mm step sections of the paraffin embedded nodes did not reveal metastases in any of the lymph nodes .
the patient was treated with chemotherapy , radiotherapy and hormone therapy and no significant complications were observed .
there are several works , which show patients with contralateral axillary drainage but with prior history of surgery due to breast cancer , mammoplasty , axillary dissection due to melanoma or excissional biopsy , etc .
, these abnormal drainages probably would be caused by surgical disruption of the physiological lymphatic pathway , and the clinical implications of them are different to these cases with no prior surgery procedures in the breast or axilla . we have found only four articles with contralateral axillary drainage and no prior surgery in breast or ipsilateral axilla.[710 ] simultaneous imc nodes were not found in any of them . in two cases , the lymphoscintigraphy showed bilateral drainage and all nodes were selected as sentinel and excised .
histological studies did not reveal tumor spread in all nodes and did not impact in the management of the patients .
tumor spread of a contralateral axillary lymph node is considered to be due to distant metastasis ( m1 ) by mean of conventional diagnosis procedures , or due to the presence of other occult primary cancer in contralateral breast .
differentiation between a systemic metastatic disease and an atypical lymphatic spread would be of great interest in the prognosis and management of these patients .
lymphoscintigraphy is the only procedure capable of separating these clinical situations , because extra - axillary lymph nodes are not detectable using blue dye alone.[79 ] metastases to a contralateral axillary lymph node reflect the lymphatic drainage of the tumor as seen on lymphoscintigraphy .
so , the patient should be considered to have a node positive and not metastatic ( m1 ) disease .
most lymph passes from the breast along interlobular vessels to the subareolar plexus and follows the veins of the breast to the axilla ( 75% ) .
lymph from the medial part of the breast , in addition to the axilla , drains into the parasternal lymph nodes .
some authors think that lymph from the skin of the breast may pass to the abdominal wall and opposite breast through the subdermal plexus as it is seen in melanoma , but retrosternal crossover to the contralateral side could be the pathway if there is internal mammary involvement .
we think that in our patient , the lymph could have passed through the retrosternal pathway to the contralateral side because the tumor was located deep in the retroareolar region as we can see in the figure 1 .
krause and colleagues described a case , which showed that the only metastatic lymph node was the contralateral axillary sentinel node , a finding , which may impact on adjuvant therapy , if this situation were ignored .
, showed a patient with bilateral axillary drainage in which both axillary nodes were positive ; ipsilateral lymphadenectomy was performed and four more lymph nodes were removed from the left ( contralateral ) axilla .
all nodes were free of metastases . a complete level i and level ii axillary lymph node dissection was not performed on the contralateral side because of concerns about bilateral lymphedema .
probably , this would be the right choice and could be implemented selecting the contralateral nodes by mean of manual exploration removing all nodes enlarged and/or hardened.[1113 ] we think , as the same of carmon , et al .
, that all ectopic nodes should be considered as sentinel nodes and must be biopsied to reach the most complete staging as possible .
the knowledge of extra - axillary lymph nodes containing metastases may lead to better selection of patients for various adjuvant treatments such as radiotherapy to extra - axillary sites of metastatic sentinel lymph nodes or alteration of chemotherapy modalities if an extra - axillary sentinel node is the only metastatic lymph node .
in our opinion , lymphoscintigraphy should be always used in addition to or instead of dye methods for lymphatic mapping .
the only way to find a metastasis in a contralateral axillary lymph node arose from the primary carcinoma in the opposite breast is to use lymphoscintigraphy and perform sentinel node biopsy ( snb ) of contralateral nodes if founded .
the distinction between node - positive and systemic metastatic disease is important considering the different treatment objectives and modalities for node positive versus metastatic breast cancer . | lymphatic drainage outside the ipsilateral axilla in breast cancer is discovered in as many as 25% of all sentinel lymph node procedures and it is evidenced by lymphoscintigraphy .
contralateral axillary drainage is a rare clinical situation , mostly if there is not prior breast surgery , but this situation is extremely exceptional if we also found internal mammary chain drainage .
we have not found such a case published in literature .
this atypical finding is source of doubts because it could have clinical implications in the management of patients .
we present a woman just diagnosed of breast carcinoma with hot nodes in bilateral axillary and ipsilateral internal mammary chain lymph nodes , shown by preoperative lymphoscintigraphy .
we review published cases with contralateral axillary drainage , which enhance the role of scintigraphic procedure in such cases and show induced controversies in selecting the most adequate management . |
in the daily lives of most modern people , the time spent working with the neck bent
forward , working on a computer , riding in cars , and engaging in leisure activities while in
a sitting positions has been increasing .
this sitting causes overloads in the soft tissues
that support the neck , and repeated damage adversely affects neck stability .
therefore ,
understanding neck - stabilizing exercises is necessary not only to prevent neck diseases but
also to reduce chronic neck pain .
recently , among therapeutic exercises , the importance of
stabilizing exercises that strengthen deep muscles , such as the longus colli muscle and the
longus capitis muscle , which play leading roles in maintaining the stability of the spine in
order to keep the cervical spine in the neutral position , has been noticed .
many previous studies have reported that stabilizing exercises are quite effective at
reducing chronic neck pain and improving neck function1 .
previous studies have also reported that neck - stabilizing exercises
greatly affected the strength and endurance of deep muscles . among these stabilizing
exercise methods , a method using slings2
and a method using cervical flexion exercises3 and a pbu in the supine position4 have been reported .
recently , neck - stabilizing exercises that can be
performed in standing positions have been developed , so that neck - stabilizing exercises can
be performed anytime , anywhere5 .
. reports in which
massage , a passive method , was directly applied to the longus colli muscle and its effect on
the strength and endurance of the dnf are rare .
patient with acute pain can not perform
stabilizing exercises , and elderly persons with low cognitive abilities can not improve their
neck stabilization abilities through stabilizing exercises that require active
participation .
the present study directly applied massage to the longus colli muscle , which is an
important element in neck stabilization , and examined its effect of the massage on the
strength and endurance of the deep muscles , important elements in neck stabilization .
longus colli massage process ( the throat and bronchial tubes were pushed to one side
using a finger .
ischemic compression applied to the longus colli ) the subjects who participated in the present study were 60 students of n university in
korea .
they were divided into an experimental group of 30 subjects ( 15 males and 15 females )
and a control group of 30 subjects ( 15 males and 15 females ) .
the experimental group s age
was 23.13.2 years , its height was 166.47.9 cm , and its weight was 62.711.7 kg .
the
control group s age was 23.14.3 years , its height was 166.37.2 cm , and its weight was
61.610.3 kg .
there were no statistically significant difference between the two groups in
age , height or weight(p>0.05 ) .
those
who had undergone surgery , those who were receiving treatment at a hospital , and those who
had ruptured cervical disks were excluded from the study .
the purpose of the study and the
entire process of the experiment were explained to all the subjects , and voluntary written
agreements were received from all of them before they participated in the experiment .
the experimental group was given massage in order to relax the longus colli muscle , while
the control group was given general neck massage .
after applying the massage once , the
immediate effect was tested . in the experimental group , for massage of the longus colli
muscle , the subjects first lay down on massage table in the supine position and with c7
placed on the table , and c6 off the table in order to induce cervical spine extension . at
this time ,
the subject s head was supported by the hands of the therapist in order to induce
slow extension .
thereafter , in order to find out the tonus of the longus colli muscle ,
extension was induced to the left and to the right , while 15 of cervical rotation was
maintained .
then , massage was applied to the longus colli muscle on the side that was
thought to have high muscle tone , between the left and right longus colli muscles , in order
to relax this muscle . in this technique , the practitioner first checked the flexibility of
the subject s throat and bronchial tubes by pushing the respective parts from left to right
using the index and middle fingers of both hands .
then , in order to relax a particular side
of the longus colli , the practitioner pushed the throat and bronchial tubes in the opposite
direction , while applying a horizontal force so as to have the longus colli exposed .
after
this , the practitioner fixed his / her index and middle fingers on the subject s throat and
bronchial tubes in a hook shape , and pressed the tense area of the longus colli with the tip
of the thumb of the opposite hand in the dorsal direction ; then gently pressed again , by
placing the opposite thumb over the first thumb on the area . at this time , the therapist
compressed the muscle at strength that would not cause tonus of the superficial muscles
while sensing the relaxation of the longus colli muscle with the fingertips ( fig .
the longus colli massage was mainly applied
to segments around c5 , where shortening occurs .
each compression was maintained for
approximately 57 seconds , and the compression was repeated 56 times or until muscle
relaxation was felt by the therapist s fingertips . after massage , relaxation was checked
through retests6 . for the control group ,
massage was applied to the sternocleidomastoid muscle , the superior trapezius muscle , the
musculus levator scapulae , and the posterior muscles of the neck7 .
the strength and endurance of the dnf were measured via revised cranio - cervical flexion
tests ( ccft ) using a pbu ( chattanooga group , australia ) .
strength was measured as the time
which the maximum voluntary contractile strength ( mvcs ) , the pressure of the maximum push
over a base pressure of 80 mmhg , could be maintained , and endurance was measured as the time
during which the intermediate pressure between the base pressure and mvcs could be
maintained5 .
the experimental results were statistically analyzed using spss 12.0 ko ( spss , chicago , il ,
usa ) .
after the general characteristics of the subjects were determined , the paired t - test
was used to compare the changes in strength and endurance that occurred between pre - test and
post - test in each group .
the differences between the two groups were tested using the
independent t - test .
difference : difference of pre - test and post - test * p<0.05 the strength and endurance of the experimental group and the control group before and after
the experiment were compared .
the strength of the experimental group showed a statistically
significant difference between before and after the intervention , and the endurance of the
experimental group showed an increase , although the increase was not statistically
significant ( table 1 ) .
the independent sample
t - test was conducted to test the significance of the differences in the pre - test , the
post - test , and the differences between the pre - test and post - test values of the strength and
endurance between the two groups ; none of the differences were statisticaly significant
( table 2 ) .
in the present study , massage was directly applied to the longus colli muscle , which plays
an important role in neck stabilization , and the effects of the massage on the strength and
endurance of the dnf were examined . among previous studies ,
there are reports of massage
directly applied to the longus colli muscle resulting in increases in the range of motion of
the cervical spine6
. however , no previous
studies in which massage was directly applied to the longus colli muscle , as was done in the
present study , could be found . among the studies that reported improvement in the strength
and endurance of the longus colli muscle after exercises , jeon et al .
reported improvement
in the strength and endurance of subjects who performed cervical stabilizing exercises in
either the standing or supine positions5 .
conducted craniocervical flexion exercises and cervical flexion exercises for
chronic neck pain patients for seven weeks , aiming to improve the strength and endurance of
the cervical flexor muscle , and reported , the strength of the cervical flexor muscle was
improved8 . in the present study ,
massage was directly applied to the longus colli muscle , and a
statistically significant , increase in muscle strength after the experiment was found in the
experimental group .
this result is considered have been the result of muscle relaxation
induced by direct ischemic compression of the longus colli muscle after the bronchus and the
esophagus had been pushed to one side . however , although endurance showed an increase in the
average value , the increase was not statistically significant .
the independent t - test did
not find statisticaly significant differences between the grops , indicating that exercises
are more effective than massage at improving the strength and endurance of the longus colli
muscle .
however , because patients with acute pain or elderly persons with low cognitive
abilities can not actively participate in neck - stabilizing exercises , massage could be
applied to the longus colli muscle , which is the deep neck flexor muscle , as an alternative
method to improve strength and endurance , which are measures of neck stabilization .
therefore , longus colli muscle massage can be applied to patients who can not perform
neck - stabilizing exercises or before other neck - stabilizing exercises are performed . | [ purpose ] the purpose of the present study was to examine the effects of longus colli
muscle massage on the strength and endurance of the deep neck flexor muscle in adults .
[ subjects ] a total of 60 subjects were divided into an experimental group of 30 subjects
and a control group of 30 subjects .
[ methods ] the experimental group received massage of
the longus colli muscle , which is the deep neck flexor muscle , and the control group
received superficial neck muscle massage . the strength and endurance of both the
experimental group and the control group were measured before and after the intervention
using a pressure biofeedback unit ( pbu ) .
[ results ] after the experiment , the strength of
dnf of the experimental group showed a statistically significant increase , and the
endurance of dnf of the experimental group showed an increase in its average value .
the
independent sample t - test revealed no statistically significant differences in the groups .
[ conclusion ] massage of the longus colli muscle , which is the deep neck flexor , was shown
to improve its strength and endurance , which are measures of neck stabilization .
therefore , longus colli muscle massage can be performed for patients who can not perform
neck - stabilizing exercises or before performing other neck - stabilizing exercises . |
bacterial strains , growth conditions , and the peptide : b. abortus biotype 1 isolate from korean cattle was used for the
construction of a vaccine by means of gi24 .
b. abortus strain rb51 served as the comparative vaccine ( versus the vaccine candidate ) .
b.
abortus strain 544 ( atcc 23448)smooth , virulent bacteria of the b. abortus biovar 1 strain served as the virulent challenge strain
( table 1table 1.bacterial strains and plasmids used in this studystrain / plasmiddescriptionsource or referencestrainsb .
abortusbiotype 1isolate from korean cattlelab stockstrain rb51commercial vaccine strainlab stockstrain 544atcc23448 , a smooth virulent b. abortus biovar 1 strain ) .
these strains were grown in brucella broth and on brucella agar ( becton dickinson , sparks , md , u.s.a . )
at 37c .
the gi24 ( grfrrlrkktrkrlkkigkvlkwi - nh2 ) peptide was chemically synthesized by peptron ( daejeon , south korea ) .
the b.
abortus biotype 1 isolate was kindly supplied by the national veterinary research and quarantine service ( nvrqs ) , anyang , korea , after
identification of the phenotypic characteristics of the b.abortus biotype 1 isolate by typical biochemical tests and
brucella biotyping using the co2 requirement for primary isolation , h2s production , urease activity , growth in the
presence of dyes , agglutination with monospecific sera , phage typing and oxidative metabolic rates .
construction of a b. abortus vaccine candidate : a single colony ( round with smooth margins and approximately 2 mm in diameter )
of b. abortus biotype 1 isolate was individually inoculated into 200 ml of brucella broth , and the cultures were incubated at
37c with slow agitation to obtain optical density ( od ) of 0.3 at 600 nm .
gi24 was added into the culture broth at 40 g / ml and
was incubated at 37c to lyse the isolate . after 24 hr , lysis induction
was determined by counting the number of viable cells after incubation on brucella agar
for 72 hr at 37c .
after the lysis process , the cell lysates were harvested by centrifugation at 4,000 g for 10 min . finally , the harvested
cells were washed three times with sterile phosphate - buffered saline ( pbs ) , then resuspended in pbs at the concentration of approximately 3 10
cells / ml and stored at 20c .
preparation of a b. abortus strain rb51 formulation : strain rb51 was cultured in brucella broth for 48 hr at 37c .
the bacteria
were washed with sterile pbs three times and resuspended in sterile pbs at approximately 3 10 colony - forming units ( cfus)/ml .
transmission electron microscopy ( tem ) : the lysate samples for tem to examine the intracellular alterations in the b. abortus
cells before and after treatment with peptide gi24 were prepared in the same manner as the b. abortus lysates .
immunization and sample collection : balb / c mice were subdivided into four groups , each group containing 10 mice .
all the mice were inoculated at
6 weeks of age [ 0 week postinoculation ( wpi ) ] .
ten mice of group a were inoculated ip with sterile pbs , as the control group was .
mice of groups b and c were also
vaccinated ip with approximately 3 10 cfus of b. abortus strain rb51 and approximately
3 10 cells of the brucella vaccine candidate .
group d was orally immunized with approximately 3 10 cells of the
vaccine candidate .
blood and vaginal washing samples were collected 0 , 2 , 4 and 6 wpi for evaluation of the immune response according to the method described in
another study .
the animal experiments described in this study were conducted with approval ( cbu 2015 - 052 ) of the
chonbuk national university animal ethics committee in accordance with the guidelines of the korean council on animal care .
immune responses evaluated by an enzyme - linked immunosorbent assay ( elisa ) : a modified elisa was performed to assess the
brucella lipopolysaccharide ( lps)-specific igg and iga titers in serum and vaginal washing samples , using the b. brucella ab elisa 2.0 kit
( bionote , hwaseongsi , gyeonggi - do , republic of korea ) . briefly
, serum samples and vaginal washing samples were diluted 1:50 and 1:3 with pbs , respectively .
the
plates were incubated with a horseradish peroxidase - conjugated goat anti - mouse igg or iga antibody ( southern biotechnology associates , birmingham , al , u.s.a . ) .
enzymatic reactions were carried out by addition of a substrate solution containing o - phenylenediamine ( sigma - aldrich , st .
louis , mo , u.s.a . ) and
were analyzed on an automated elisa spectrophotometer ( thermo scientific multiskan go , thermo fisher scientific oy , ratastie , vantaa , finland ) at 492 nm .
results
of the elisa are expressed as the mean optical density ( od ) standard deviation .
quantitation of cytokines from splenocytes : from each group , five mice were euthanized , and spleens were removed aseptically 4 wpi .
the splenocytes were prepared according to the method described elsewhere [ 1 , 38 ] .
the prepared spleen cells were seeded in 24-well tissue culture plates at 2 10/well [ 1 , 38 ] .
the splenocytes were stimulated in vitro with heat - inactivated bacteria of
b. abortus strain 544 ( 10 cells / well ) , concanavalin a ( 0.5 g / well ) as a positive control or the medium as an
unstimulated control , and incubated at 37c , 5% co2 and 95% humidity [ 1 , 52 ] .
culture supernatants were collected after 72 hr of restimulation and stored at 70c until use for cytokine quantification [ 1 , 52 ] .
quantification of cytokines by an elisa : elisa was used to measure the concentration of interleukin ( il)-4 , il-10 , interferon - gamma ( ifn- ) , and
tumor necrosis factor alpha ( tnf- ) in the supernatants using the mouse cytokine elisa ready - set - go reagent kit ( ebioscience inc . , san diego , ca , u.s.a . ) [ 6 , 7 , 42 ] .
challenge experiments : for challenge experiments , the challenge strain , strain 544 was prepared .
briefly , the strain was grown in brucella broth
at 37c for 24 hr and was resuspended at approximately 10 cfus / ml . all mice were challenged ip 6 wpi with 100 l of
the challenge strain .
two weeks after the challenge , the spleen weights of all mice were measured , and each spleen was diluted 1:100 using brucella broth . a total
of 100 l of the diluted media was spread on blood agar to count the number of viable strain 544 cells from the spleens at 2 weeks after the
challenge . if no colony was detected on the blood agar , then the number of viable cells of challenge strains from the spleen of the mice corresponded to
< 10 cfus . briefly
, blended spleen samples were plated on brucella agar with or without rifampicin ( 50 g / ml ) ,
because strain rb51 is resistant to rifampicin .
the vaccine and challenge strains were confirmed by pcr using a
b. abortus - specific primer ( 5-gac gaa cgg aat ttt tcc aat ccc ) , rb51/2308 primer ( 5-ccc cgg aag ata tgc ttc gat cc ) and
is711-specific primer ( 5-tgc cga tca ctt aag ggc ctt cat ) with enhanced brucella amos pcr primers [ 10 , 11 ] .
statistical analysis : to evaluate the differences among various vaccinated groups , absorbance data from elisas were subjected to analysis of
variance with post hoc tukey s test for pairwise comparison in the spss software , version 16.0 ( spss inc . ,
chicago , il , u.s.a . ) . the
kruskal - wallis test followed by dunn s procedure for multiple comparisons between groups
was carried out to compare the log - transformed cfu values in organs
obtained from each vaccinated group of mice with the values in the respective control group in the bacterial challenge experiments .
the bacterial cells were incubated with 40 g / ml of gi24 for 30 hr at 37c .
, in the untreated b. abortus cells , full intracellular contents and an intact cell membrane were observed ( fig .
1b ) , the bacteria treated with gi24 showed clear cytoplasmic
spaces , and a disrupted membrane of b. abortus ( with visible pores ) was observed .
the bacterial cells were incubated with 40 g / ml of gi24 for 30 hr at 37c . the humoral immune response in immunized mice : serum igg titers against b. abortus lps in groups
c and d were 0.07 0.009 and
0.08 0.025 before immunization and increased gradually to 1.78 0.484 and 1.66 0.407 at 6 wpi ( fig .
2.serum igg ( g / ml ) and vaginal iga ( g / ml ) titers against brucella
abortus 1119 - 3 lps in mice intraperitoneally or orally immunized with each vaccine candidate .
group a mice were immunized with sterile pbs ; group
b mice were intraperitoneally immunized with 3 10 cfus of b. abortus strain rb51 ; group c mice were intraperitoneally
immunized with 3 10 cells of the b. abortus lysates ; group d mice were orally immunized with 3 10 cells of the
b. abortus lysates .
data shown are the means of all mice in each group , and error bars show the standard deviations ( sd ) .
asterisks
indicate a significant difference between the values of the groups immunized with the vaccine candidate ( * p0.05 ) and those of the control
group . ;
p<0.01 ) . in addition , the vaginal iga titers against the b. abortus lps in groups b , c and d were 0.13 0.037 , 0.14
0.033 and 0.11 0.025 , respectively , before immunization and 0.2 0.061 , 0.7 0.239 and 0.36 0.185 , respectively , 2 wpi .
four wpi , the iga titers of groups
b , c and d were 0.44 0.093 , 0.66 0.195 and 0.45 0.108 , respectively ( p<0.01 ) .
the iga titers of groups b , c and d were 0.23 0.036 ,
0.87 0.089 and 0.36 0.103 , respectively , 6 wpi ( fig . 2 ; p<0.01 ) .
serum igg ( g / ml ) and vaginal iga ( g / ml ) titers against brucella
abortus 1119 - 3 lps in mice intraperitoneally or orally immunized with each vaccine candidate .
group a mice were immunized with sterile pbs ; group
b mice were intraperitoneally immunized with 3 10 cfus of b. abortus strain rb51 ; group c mice were intraperitoneally
immunized with 3 10 cells of the b. abortus lysates ; group d mice were orally immunized with 3 10 cells of the
b. abortus lysates .
data shown are the means of all mice in each group , and error bars show the standard deviations ( sd ) . asterisks
indicate a significant difference between the values of the groups immunized with the vaccine candidate ( * p0.05 ) and those of the control
group .
cytokine analysis : the mean concentrations of il-4 in response to heat - inactivated b. abortus cells from splenocytes of mice
from groups a , b , c and d were 25 8.477 pg / ml , 112.1 21.129 pg / ml , 168.7 36.53
pg / ml and 199 63.116 pg / ml , respectively ( p<0.05 ) ( fig . 3fig .
3.il-4 ( pg / ml ) , il-10 ( pg / ml ) , tnf - a ( pg / ml ) and ifn-
( ng / ml ) concentrations in the supernatants of the splenocytes stimulated with heat - inactivated brucella
abortus strain 544 at 4 wpi .
group a mice were immunized with sterile pbs ; group b mice were intraperitoneally immunized with 3 10
cfus of b. abortus strain rb51 ; group c mice were intraperitoneally immunized with 3 10 cells of the b.
abortus lysates ; group d mice were orally immunized with 3 10 cells of the b. abortus lysates .
data are presented
as the mean of all mice in each group ; error bars show sd .
asterisks indicate a significant difference between the values of mouse groups b , c and d
( * p<0.05 ) and those of the control group . ) .
the il-10 concentrations of the mice from groups a , b , c and d were 17.4 10.644 pg / ml , 72.1 21.829
pg / ml , 101.5 39.049 pg / ml and 92.5 48.855 pg / ml ,
respectively ( p<0.05 ) .
the tnf- concentration of mouse groups a , b , c and d were 162.5 18.208 pg / ml ,
375.8 76.996
pg / ml , 276.3 61.266 pg / ml and 316 87.429
pg / ml , respectively ( p<0.05 ) .
in addition , the levels of ifn- in groups a , b , c and d were 1 0.385
ng / ml , 3.7 1.29 ng / ml , 2.4 0.274 ng / ml and 1.6 0.091
ng / ml , respectively ( p<0.01 ) .
il-4 ( pg / ml ) , il-10 ( pg / ml ) , tnf - a ( pg / ml ) and ifn-
( ng / ml ) concentrations in the supernatants of the splenocytes stimulated with heat - inactivated brucella
abortus strain 544 at 4 wpi .
group a mice were immunized with sterile pbs ; group b mice were intraperitoneally immunized with 3 10
cfus of b. abortus strain rb51 ; group c mice were intraperitoneally immunized with 3 10 cells of the b.
abortus lysates ; group d mice were orally immunized with 3 10 cells of the b. abortus lysates .
data are presented
as the mean of all mice in each group ; error bars show sd .
asterisks indicate a significant difference between the values of mouse groups b , c and d
( * p<0.05 ) and those of the control group .
protection of mice against a virulent challenge : all mice were challenged ip 6 wpi with approximately 4 10 cfus of the challenge
strain . among the five mice of group
a , the challenge strain was isolated from all mice , and the number of isolates was 115,296 15,446 cfus / spleen .
the
challenge strain was isolated from all mice of group b , and the number was 19,243 9,229 cfus / spleen .
in contrast , in group c , the challenge strain was isolated
from three out of five mice , and the number was only 4,267 2,512 cfus / spleen . among four of five mice in group d
, the wild - type b. abortus
strain was isolated , and the number was 13,118 1,005 cfus / spleen ( fig .
4.protection against b. abortus strain 544 in mice immunized with b. abortus strain rb51 or b. abortus
ghost cells .
group a mice were immunized with sterile pbs ; group b mice were intraperitoneally immunized with 3 10 cfus of b.
abortus strain rb51 ; group c mice were intraperitoneally immunized with 3 10 cells of the b. abortus lysates ; and
group d mice were orally immunized with 3 10 cells of the b. abortus lysates .
six weeks postinoculation ( wpi ) , the mice were
challenged with b. abortus strain 544 .
two weeks later , the number of viable brucella cells recovered from the spleen was
determined .
data represent the mean of all mice in each group ; error bars show sd .
asterisks indicate a significant difference between the values of mouse
groups b , c and d ( * p<0.05 ) and those of the control group . ) .
protection against b. abortus strain 544 in mice immunized with b. abortus strain rb51 or b. abortus
ghost cells .
group a mice were immunized with sterile pbs ; group b mice were intraperitoneally immunized with 3 10 cfus of b.
abortus strain rb51 ; group c mice were intraperitoneally immunized with 3 10 cells of the b. abortus lysates ; and
group d mice were orally immunized with 3 10 cells of the b. abortus lysates .
six weeks postinoculation ( wpi ) , the mice were
challenged with b. abortus strain 544 .
two weeks later , the number of viable brucella cells recovered from the spleen was
determined .
data represent the mean of all mice in each group ; error bars show sd .
asterisks indicate a significant difference between the values of mouse
groups b , c and d ( * p<0.05 ) and those of the control group .
a novel method for production of an inactivated b. abortus vaccine candidate is based on b. abortus lysis using one of the amps
leading to formation of empty cell envelops , termed brucella lysate vaccine .
the peptides bearing a net positive charge get attached to the
negatively charged phospholipids in the cell membranes of gram - negative and gram - positive bacteria [ 23 , 48 ] , followed by insertion of the peptide into to the bacterial cell membrane ,
resulting in formation of a transmembrane pore [ 20 , 37 ] . as soon as pores form
,
they disrupt and destabilize the bacterial cell membrane , and then all cytoplasmic content comes out leading to bacterial lysis [ 20 , 37 ] , with intact natural outer membrane lps structures that have highly immunogenic properties .
vaccination via the mucosal route is a feasible method to induce acquired immune responses against infectious agents in humans and animals . in most animals ,
live
attenuated brucella strains were used as mucosal vaccine candidates in several studies [ 14 , 21 , 36 ] .
however , the inherent safety risks , such as bacterial replication , may prevent
the use of live attenuated brucella strains as vaccines .
other studies showed that salmonella gallinarum cell lysates can
generate protective antibody - mediated as well as cell - mediated immunity ( cmi ) when used as a vaccine via various routes in chickens [ 13 , 22 ] .
bacterial lysates represent a relatively new concept for improvement of the vaccine technology ,
but this novel approach has been seldom tested on brucella . as reported in this study , we constructed a brucella vaccine
candidate using gi24 , one of amps . in this study , we tested whether the brucella vaccine candidate can be administered orally as well as ip to
protect mice from a challenge infection with wild - type b. abortus .
we investigated the efficacy of the brucella vaccine
candidate , constructed using gi24 , for protection against brucellosis caused by strain rb51 in murine models .
we first examined the effect of the
brucella vaccine candidate ( constructed using gi24 ) on induction of cmi and protection . in line with our initial hypothesis , ip or oral
immunization with the brucella vaccine candidate significantly increased antibody titers and cmi responses in comparison with the unimmunized
group . in the present study
, we evaluated humoral immunity as well as cmi induced by immunization with the brucella vaccine candidate and its
protective efficacy against challenge infection with b. abortus strain 544 .
the route of immunization alters the course of the immune response
being induced by the vaccine candidate .
therefore , we evaluated two routes of immunization , ip and oral , to find the optimal immunization route required for an
effective immune response .
approximately 3 10 and 3 10 cells of the brucella vaccine candidate were used as the dose
for immunization via ip and oral routes , respectively . a vaccine inducing protective immune responses , such as secretory iga , at the mucosal surface
would be an
ideal vaccine , because it would prevent the entry of ( and colonization by ) pathogens and consequently would prevent the disease . in the present study , immune responses , such as serum igg and mucosal iga ,
the serum igg and vaginal iga titers in the vaccine candidate - inoculated groups irrespective of the immunization route were significantly increased as
compared to the unimmunized group .
furthermore , induction of cytokines in splenocytes collected from all mice immunized with the vaccine candidate or strain rb51
and restimulated in vitro with heat - inactivated bacteria of b. abortus strain 544showed powerful th2 type immunity ( il-4 levels
represent the th2 type of immunity ) . generally , induction of mucosal iga is strongly dependent on a type th2 immune response .
major cytokines that enhance iga
responses are il-5 , il-6 and il-10 [ 33 , 44 ] . in this study ,
il-10 levels were
significantly higher in all the mice immunized the brucella vaccine candidate than in control mice .
these results show that the production of
cytokines that are associated with enhancement of an igg and iga response was also significantly increased by the brucella vaccine candidate .
it is well known that brucella is a facultative intracellular pathogen . for clearance of an intracellular pathogen ,
therefore , in this study , we evaluated cellular immune responses using elisa kits
for cytokines .
the results revealed strong secretion of tnf- and ifn- into the culture supernatant when splenocytes were restimulated in vitro ,
giving an indication of the th1 type of immune response ; this result is in agreement with the previously shown protective response .
for efficient macrophage
functioning , such as killing and clearance of an intracellular pathogen , ifn- is an essential effector cytokine [ 32 , 43 ] . in the present study ,
a strong th1 response ( tnf- and ifn- ) was induced by ip immunization with strain rb51 or by ip or
oral vaccination with the brucella vaccine candidate .
these results showed that immunization with the brucella vaccine candidate
irrespective of the immunization route effectively induces the cytokines that are related to cmi as well as humoral immunity . in mouse
brucellosis models , the vaccine - induced protective immune responses are analyzed by comparing a reduction in a bacterial load in the spleen , liver or
both between the vaccinated mice and unvaccinated animals .
on the basis of that study , mice vaccinated with the
vaccine candidate and strain rb51 showed a decrease in the splenic counts of brucella after an ip challenge with wild - type b.
abortus strain 544 .
nevertheless , the notable difference between mice immunized with the vaccine candidate and with strain rb51 was the number of
bacterial cells isolated from the mice after the challenge with virulent b. abortus strain 544 . among the 5 mice immunized ip with the
brucella vaccine candidate , the challenge strains were isolated from the spleens of only 3 mice .
the challenge strains were isolated from the
spleens of only 4 mice among the 5 mice orally immunized with the vaccine candidate .
in contrast , the strains were detected in spleens of all mice immunized ip
with strain rb51 .
furthermore , in this study , mice immunized ip with the vaccine candidate showed the best protection against infection with virulent b.
abortus strain 544 .
this observation suggests that ip immunization with the brucella vaccine candidate involves intact lps , which is a
potent natural immunogen .
therefore , the brucella vaccine candidate elicits antibodies to lps , which are effective at protecting the animals in
mouse models of brucellosis . | brucella abortus cells were lysed by the n - terminal 24-amino acid fragment ( gi24 ) of the 36-amino acid peptide pmap-36 ( porcine myeloid
antimicrobial peptide 36 ) .
next , the protection efficacy of the lysed fragment as a vaccine candidate was evaluated .
group a mice were immunized with sterile
pbs , group b mice were intraperitoneally ( ip ) immunized with 3 108 colony - forming units ( cfus ) of b. abortus strain rb51 , group c
mice were immunized ip with 3 108 cells of the b. abortus vaccine candidate , and group d mice were orally immunized with 3
109 cells of the b. abortus vaccine candidate .
brucella lipopolysaccharide ( lps)-specific serum igg titers were
considerably higher in groups c and d than in group a. the levels of interleukin ( il)-4 , il-10 , tumor necrosis factor alpha ( tnf- ) and interferon gamma ( ifn- )
were significantly higher in groups b
d than in group a. after an ip challenge with b. abortus 544 , only group c mice showed a significant
level of protection as compared to group a. overall , these results show that ip immunization with a vaccine candidate lysed by gi24 can effectively protect mice
from systemic infection with virulent b. abortus . |
we studied 2 outbreaks of orally transmitted chagas disease ( 120 cases , 5 deaths ) .
the first occurred in chichiriviche , vargas state , a coastal community ( population 800 persons ) 50 km northwest of caracas , venezuela .
the outbreak occurred at a primary school where food was prepared on site . in early april 2009 ,
a total of 71 children ( 613 years of age ) who attended the morning school shift and 14 adults became ill .
exposure of these persons to t. cruzi was established by use of igm and igg elisa .
parasitemia was observed in 33 of the patients with serologic results positive for t. cruzi infection ( 9,10 ) .
the second outbreak occurred in antimano , a peri - urban slum southwest of central caracas ( technical appendix figure 1 ) . in may 2010 ,
35 patients with suspected t. cruzi infection were examined at hospital miguel perez carreno in caracas .
patients reported that they regularly ate at the same local communal canteen . among the patients tested , 15 were positive for t. cruzi igm and igg ( 9 ) .
parasitemia in 14 patients was confirmed indirectly by hemoculture . of the 35 patients , 21 ( 2 adults , 19 children )
, we undertook intensive additional sampling of contemporary , nonhuman sources local to each outbreak and of human and nonhuman sources from more distant localities throughout venezuela . in total , 246 t. cruzi strains and clones were typed for 23 microsatellite markers ( technical appendix 1 table ) ( 8) .
a list of the samples and their sites of origin is in technical appendix 2 ) .
individual level sample clustering was defined first by constructing a neighbor joining tree based on pairwise distances between multilocus genotypes ( figure 1 ) . a second analysis used k - means clustering and discriminant analysis of principal components ( figure 2 ) ( 11 ) . to assess connectivity between human and nonhuman outbreak cases ,
population - level genetic diversity was assessed first by calculating allelic richness then private allele frequency over loci between each human nonhuman population pair ( table 2 ) .
unrooted neighbor joining tree showing genetic clustering among trypanosoma cruzi isolates from 2 outbreaks of oral disease in northern venezuela .
based on pairwise genetic distances ( 1 proportion of shared alleles ) between multilocus microsatellite profiles ( 23 loci ) generated from 246 isolates and clones .
indicate isolates associated with the outbreaks . discriminant analysis of principal components showing genetic clustering among trypanosoma cruzi isolates from 2 outbreaks of oral disease in northern venezuela .
ellipses correspond to the optimal ( as defined by the bayesian information criterion minimum ) number of population clusters among the genotypes analyzed .
images indicate sample host origin ( human , rodent , marsupial , or triatomine ) , while colors correspond to the key in figure 1 .
a full list of samples and population assignments ( numbered boxes ) is included in technical appendix 2 .
lower left shows linearized fst ( genetic differentiation ) values ; upper right shows associated p values .
clustering results determined by discriminant analysis of principal components and neighbor joining were broadly congruent . in the former ,
substantial overlap existed between some clusters , especially those from capital and vargas states ( e.g. , those labeled
x in figure 2 ) , while others were highly distinct ( e.g. , cluster 8 in figure 2 ) .
human isolates from both oral outbreaks are extremely distinct from non - orally transmitted isolates collected from humans throughout venezuela .
almost all these presumably vector - transmitted strains are closely related to one another , despite their geographic dispersal ( cluster 8 in figure 2 ) . by comparison ,
oral outbreak strains that were isolated a mere 50 km apart ( clusters 2 , 5 , 7 , 9 , 1517 ) are far more globally diverse . unlike most human isolates in venezuela , which are distinct from nonhuman strains , oral outbreak isolates are interspersed among nonhuman strains from venezuela .
furthermore , samples from both outbreaks clustered among nonhuman strains local to that outbreak , clearly indicating a local origin .
oral samples from each outbreak are polyphyletic with respect to strains from their immediate environment , a finding consistent with multiple contamination events or multiclonal infection sources .
fst values further support connectivity between outbreak and local environmental samples in both antimano and chichiriviche ( table 1 ) .
a lack of private alleles between human and nonhuman isolates also supports a local source for the chichiriviche outbreak ( table 2 ) .
fst values in the 4-way comparison between outbreak and local environmental strains are , however , somewhat equivocal with respect to the entire dataset ( table 1 ) .
cluster analysis showed that the human and nonhuman strains from chichiriviche interspersed with strains from other states in venezuela ( figure 2 ) .
thus , we can not confirm a uniquely local origin for the chichiriviche outbreak , despite a low value for fst , and it is possible that some contaminating strains originated elsewhere .
this study demonstrates the value of rigorous molecular epidemiologic analysis of orally transmitted t. cruzi outbreaks , including the importance of appropriate sampling to identify the origin of the infecting strains .
the foodstuff that propagated the peri - urban outbreak in antimano was certainly contaminated locally .
an active nonhuman transmission cycle in the slums of caracas , maintained by rattus rattus rodents and panstrongylus geniculatus triatomines , is the likely source .
the chichiriviche outbreak , however , has potential sources both in and outside the immediate area .
as found in chagas disease outbreaks linked to aa palm fruit in brazil ( 12 ) , the t. cruzi parasite can survive for several days in some foodstuffs ( 13 ) .
also , triatomines can survive for months in harvested crops ; thus , multiple hygiene interventions are potentially necessary along the food production line ( 14 ) .
nonetheless , if the foodstuff implicated was prepared locally , local contamination represents the most likely source of the outbreak .
crucial to understanding parasite transmission in general , we believe , are genetic differences between strains from orally and non - orally transmitted human cases .
all tci strains appear to be infective to humans and adapted to long - term carriage ( 8) .
however , the presence of a common , reduced - diversity tci genotype cluster ( tcidom ) among a high proportion of human chagas disease cases in south america is also well established ( 7,8 ) .
we originally hypothesized that tcidom was maintained , despite the presence of sympatric and infective sylvatic strains , because of low parasite transmission efficiency by invasive sylvatic vectors ( 8) .
thus , unlike tcidom strains , those from orally transmitted t. cruzi cases demonstrated high genetic diversity and clearly originated from local nonhuman t. cruzi populations .
however , it is also true that all tcidom strains we isolated originated from patients with chronic infection , and all orally transmitted cases were in the acute phase .
we can not , therefore , rule out a role for immune selection in driving the frequency of tcidom infections among humans ; such selection represents an intriguing topic for future enquiry .
molecular tools and reference datasets are now available to determine the source of acute chagas disease outbreaks within days of their occurrence .
the plummeting cost of such analyses means it is time to apply population genetic techniques and markers developed for trypanosomes as genuine epidemiologic tools .
photo of antimano , a suburban slum of caracas , map showing the geographic distribution of trypanosoma cruzi i isolates analyzed , and primers and chromosomal positions for microsatellite loci used in a study of the molecular epidemiologic source tracking of 2 outbreaks of oral chagas disease , venezuela .
trypanosoma cruzi isolates and clones analyzed in a study of the molecular epidemiologic source tracking of 2 outbreaks of oral chagas disease , venezuela . | oral outbreaks of chagas disease are increasingly reported in latin america .
the transitory presence of trypanosoma cruzi parasites within contaminated foods , and the rapid consumption of those foods , precludes precise identification of outbreak origin .
we report source attribution for 2 peri - urban oral outbreaks of chagas disease in venezuela via high resolution microsatellite typing . |
alzheimer s disease ( ad ) is a complex neurodegenerative disease , which can be divided into two major forms : early - onset ad ( eoad ) and late - onset ad ( load ) with dividing age of 65 years , respectively .
three dominant causative genes for eoad are amyloid precursor protein ( app ) , presenilin-1 ( psen1 ) , and presenlin-2 ( psen2 ) .
mutations in these genes could increase the production of amyloid beta ( abeta , a ) peptide , resulting in their aggregation and deposition , leading to neuronal toxicity and loss .
five to ten percent of all ad cases under 65 years of age can be categorized as eoad with an autosomal dominant inheritance pattern ( familial eoad ) .
however , family history of the disease could also be absent in several eoad patients , in which it could be described as occurring due to de novo mutations.1 even though majority of pathogenic eoad mutations ( almost 200 mutations ) were found in psen1 , only limited numbers of pathogenic mutations were reported in app ( 25 cases ) and psen2 ( 14 cases ; http://www.alzforum.org/mutations ) .
mutations in psen2 ( ch1 227,058,272227,083,804 ) are rare , and majority of the reported cases were from patients of european ancestry.2 only a few psen2 mutations were previously reported in asia , but emerging recent studies found several novel mutations in korean and chinese patients.25 phenotypes in ad patients with psen2 mutations were milder than those in with psen1 mutations .
additional interesting symptoms were also associated with psen2-mutation carriers , including atypical dementia , dementia with lewy bodies , and frontotemporal dementia ( http://www.alzforum.org/mutations ) .
age of disease onset could range between 45 and 88 years.6 here , we present the detailed case report of a first korean eoad patient with a psen2 r62c mutation in asia . in the two main databases , ad and frontotemporal dementia ( ftd ) mutation database ( http://www.molgen.ua.ac.be/admutations/ ) and alzgene database http://www.alzforum.org/mutations ) database , this mutation was previously reported in patients from europe with load and pathogenic nature unclear.7
a 49-year - old , right - handed man visited our memory clinic in april 2013 with memory complaints , which were noticed in 2011 by the patient and his family members .
his memory disturbances were gradually increasing , frequently making him lose his belongings without noticing and exhibiting severe memory loss .
other personality changes appeared , such as forgetting important promises to his friends , easily confusing the detailed must do - lists or difficulties in the calculations .
sometimes , he asked other people to repeat their conversations due to his failures in comprehending the talks .
the patient was working as a physical education teacher in a middle school , and showed significant impairments and carried out inappropriate decision making and social functions .
he could not manage his work as a teacher , for example , creating tests and making decisions at school .
impairments appeared in his daily life activities , such as mistakes in transferring money through telebanking .
he presented abnormal behavioral symptoms with an obsessive personality , for example , being stingy with money and also did not allow anyone to spend his money without prior discussions and permission .
patient did not have any medical disease prior to his cognitive decline , and family history was unremarkable since neither his parents nor his siblings showed any cognitive impairment or dementia so far .
routine laboratory tests , including blood chemistry , electrolyte , and urine analyses , were normal .
serum venereal disease research laboratory ( vdrl ) results , including those for treponema pallidum particle agglutination assay ( tpha ) , were negative .
this patient was diagnosed according to the diagnostic criteria for probable ad by the national institute of neurological and communicative diseases and stroke / alzheimer s disease and related disorder association , which were ascertained by clinical features , neuropsycho - logical test , and brain magnetic resonance imaging ( mri ) imaging with single - photon emission computed tomography ( spect ) .
brain mri revealed mild cortical atrophy without any ischemic change or other lesions ( figure 2a ) .
brain spect showed moderate hypoperfusion in the frontal , limbic , and temporal areas ( figure 2b ) .
the reduced cerebral blood flow from the images of brain spect also supported the ad diagnosis .
the patient underwent comprehensive neuropsychological tests , and he scored 23 out of 30 ( 2 percentile ) on the mini - mental state examination .
the subscores for time orientation and seven - serial calculations were 4 out of 5 and 1 out of 5 , respectively .
the delayed word recall score was 1 out of 3 , and he scored 1 out of 3 in the task of clock drawing ( figure 3 ) .
his score fell in the abnormal range on the attention task of backward digit span ( 2.5 percentile ) .
performances in copying the intersecting pentagon test and rey osterrieth complex figure test were normal , but his performances on clock drawing were severely impaired ( 5 percentile ) . on the seoul verbal learning test , he was able to recall 13 items ( 0.02 percentile ) in three consecutive immediate recall trials , but only 3 items ( 0.3 percentile ) in the 20-minute delayed recall .
poor visual memory score of 10 out of 36 ( 1 percentile ) was obtained in the delayed recall of the rey osterrieth complex figure test .
he showed abnormal performances in several tasks of frontal and executive functions , especially in the controlled oral word association test , stroop test , and trail making test .
patient was diagnosed as probable eoad , based on clinical assessment , detailed neuropsychological tests , and neuroimaging studies including brain mri and spect .
we were unable to make segregation analysis , since all family members and relatives refused the genetic test .
none of his family members presented any cognitive decline or dementia phenotypes , but they did not agree in releasing other family information .
this patient was included in the clinical research center for dementia of south korea study , which screened potential causative mutations in 100 patients with eoad.8 details of the clinical and scientific analyses of eoad mutations in the clinical research center for dementia of south korea study have not been included previously . a detailed clinical investigation of this patient with detailed personal interview , mri , spect imaging data , and bioinformatic analyses are presented .
venous blood samples ( 5 ml ) were collected from the patient with an ethylenediaminetetraacetic acid blood drawing tube .
blood sample was centrifuged at 1,500 g for 5 min , followed by separation of white blood cells ( buffy coat ) for its storage at 80c .
dna was purified using geneall blood protocol kit ( geneall biotechnology , seoul , korea ) , and purified dna was kept at 20c until the analysis . a polymerase chain reaction ( pcr)-based analysis was performed using primers , as previously described.2 targeted potential eoad genes were as following : app exons 16 and 17 , psen1 , and psen2.912 since the diagnosis of eoad is complicated due to the pathologic overlaps within diverse neurodegenerative disorders , this patient was also screened for progranulin ( pgrn ) , microtubule - associated protein tau ( mapt ) , and prion ( prnp ) genes.1315 the pcr products were purified using geneall expin pcr kit ( geneall biotechnology ) .
single - strand conformation polymorphism was performed.16 samples were mixed with formamide and incubated for 10 minutes at 95c to generate single - stranded dna bands .
for separation , native polyacrylamide electrophoresis ( biorad , seoul , korea ) was performed for 21 hours .
sybr gold ( thermo fisher scientific , waltham , ma , usa ) staining was used to visualize the dna bands . to confirm the mutation ,
all pcr products were sequenced with the same primer sets in both directions by bioneer inc .
( dajeon , korea ) . before sequencing , we purified the pcr products by using geneall pcr kit ( geneall biotechnology ) .
big dye terminator cyclic sequencing was performed with the abi 3730xl dna analyzer ( http://eng.bioneer.com/home.aspx ; bioneer inc . ) .
sequence variants were identified by ncbi ( http://www.ncbi.nlm.nih.gov/gene ) and uniprot ( http://www.uniprot.org ) databases .
mutations were analyzed by polyphen-2 ( http://genetics.bwh.harvard.edu/pph2/ ) online in silico prediction program , which predicted the pathogenic nature of missense mutations .
polyphen-2 used homology search , multiple sequence alignment , profiling , identity - based scores , and structure - based search parameters ( such as accessible surface area and hydrophobicity features ) .
these predictions could define the putative role of missense variants and could predict whether they were probably / possibly damaging or benign .
polyphen-2 also provided the multiple sequence alignment , which compared the homologous sequences from different organisms .
humdiv was used for testing the pathogenicty of rare alleles , where mildly damaging variants must be treated as possibly damaging .
humvar was used in case of mendelian disorders , where more damaging alleles should be distinguished from the less damaging ones.17 we also analyzed the mutation with sift algorithm ( http://sift.jcvi.org/ ) , which used several databases such as swiss - prot , swiss - prot / trembl , and ncbi protein database .
the software analyzed the possibility on the pathogenic properties of mutations by comparing the mutant and normal variants .
sift scores of point mutation were calculated for weighting the mutations as possibly damaging allele or tolerated ( nondamaging ) , with a dividing score of 0.05 , respectively.18 protein structure prediction analysis of psen2 was performed by raptor x program at the university of chicago ( http://raptorx.uchicago.edu ) .
normal psen2 was composed of 488 amino acids , starting at 61 residues and continuing till residue 448 .
the prediction was performed at raptor x web server for both normal psen2 and r62c mutation .
based on psen2 information from the uniprot database , r62c mutation belonged to the topological domain , positions 187 , including the mutation site .
a 49-year - old , right - handed man visited our memory clinic in april 2013 with memory complaints , which were noticed in 2011 by the patient and his family members .
his memory disturbances were gradually increasing , frequently making him lose his belongings without noticing and exhibiting severe memory loss .
other personality changes appeared , such as forgetting important promises to his friends , easily confusing the detailed must do - lists or difficulties in the calculations .
sometimes , he asked other people to repeat their conversations due to his failures in comprehending the talks .
the patient was working as a physical education teacher in a middle school , and showed significant impairments and carried out inappropriate decision making and social functions .
he could not manage his work as a teacher , for example , creating tests and making decisions at school .
impairments appeared in his daily life activities , such as mistakes in transferring money through telebanking .
he presented abnormal behavioral symptoms with an obsessive personality , for example , being stingy with money and also did not allow anyone to spend his money without prior discussions and permission .
patient did not have any medical disease prior to his cognitive decline , and family history was unremarkable since neither his parents nor his siblings showed any cognitive impairment or dementia so far .
routine laboratory tests , including blood chemistry , electrolyte , and urine analyses , were normal .
serum venereal disease research laboratory ( vdrl ) results , including those for treponema pallidum particle agglutination assay ( tpha ) , were negative .
this patient was diagnosed according to the diagnostic criteria for probable ad by the national institute of neurological and communicative diseases and stroke / alzheimer s disease and related disorder association , which were ascertained by clinical features , neuropsycho - logical test , and brain magnetic resonance imaging ( mri ) imaging with single - photon emission computed tomography ( spect ) .
brain mri revealed mild cortical atrophy without any ischemic change or other lesions ( figure 2a ) .
brain spect showed moderate hypoperfusion in the frontal , limbic , and temporal areas ( figure 2b ) .
the reduced cerebral blood flow from the images of brain spect also supported the ad diagnosis .
the patient underwent comprehensive neuropsychological tests , and he scored 23 out of 30 ( 2 percentile ) on the mini - mental state examination .
the subscores for time orientation and seven - serial calculations were 4 out of 5 and 1 out of 5 , respectively .
the delayed word recall score was 1 out of 3 , and he scored 1 out of 3 in the task of clock drawing ( figure 3 ) .
his score fell in the abnormal range on the attention task of backward digit span ( 2.5 percentile ) .
performances in copying the intersecting pentagon test and rey osterrieth complex figure test were normal , but his performances on clock drawing were severely impaired ( 5 percentile ) . on the seoul verbal learning test , he was able to recall 13 items ( 0.02 percentile ) in three consecutive immediate recall trials , but only 3 items ( 0.3 percentile ) in the 20-minute delayed recall .
poor visual memory score of 10 out of 36 ( 1 percentile ) was obtained in the delayed recall of the rey osterrieth complex figure test .
he showed abnormal performances in several tasks of frontal and executive functions , especially in the controlled oral word association test , stroop test , and trail making test .
patient was diagnosed as probable eoad , based on clinical assessment , detailed neuropsychological tests , and neuroimaging studies including brain mri and spect .
genetic tests were performed in the patient to evaluate possible mutations . we were unable to make segregation analysis , since all family members and relatives refused the genetic test .
none of his family members presented any cognitive decline or dementia phenotypes , but they did not agree in releasing other family information .
this patient was included in the clinical research center for dementia of south korea study , which screened potential causative mutations in 100 patients with eoad.8 details of the clinical and scientific analyses of eoad mutations in the clinical research center for dementia of south korea study have not been included previously .
a detailed clinical investigation of this patient with detailed personal interview , mri , spect imaging data , and bioinformatic analyses are presented .
venous blood samples ( 5 ml ) were collected from the patient with an ethylenediaminetetraacetic acid blood drawing tube .
blood sample was centrifuged at 1,500 g for 5 min , followed by separation of white blood cells ( buffy coat ) for its storage at 80c .
dna was purified using geneall blood protocol kit ( geneall biotechnology , seoul , korea ) , and purified dna was kept at 20c until the analysis . a polymerase chain reaction ( pcr)-based analysis was performed using primers , as previously described.2 targeted potential eoad genes were as following : app exons 16 and 17 , psen1 , and psen2.912 since the diagnosis of eoad is complicated due to the pathologic overlaps within diverse neurodegenerative disorders , this patient was also screened for progranulin ( pgrn ) , microtubule - associated protein tau ( mapt ) , and prion ( prnp ) genes.1315 the pcr products were purified using geneall expin pcr kit ( geneall biotechnology ) .
single - strand conformation polymorphism was performed.16 samples were mixed with formamide and incubated for 10 minutes at 95c to generate single - stranded dna bands . for separation , native polyacrylamide electrophoresis ( biorad , seoul , korea ) was performed for 21 hours .
sybr gold ( thermo fisher scientific , waltham , ma , usa ) staining was used to visualize the dna bands . to confirm the mutation ,
all pcr products were sequenced with the same primer sets in both directions by bioneer inc .
( dajeon , korea ) . before sequencing , we purified the pcr products by using geneall pcr kit ( geneall biotechnology ) .
big dye terminator cyclic sequencing was performed with the abi 3730xl dna analyzer ( http://eng.bioneer.com/home.aspx ; bioneer inc . ) .
sequence variants were identified by ncbi ( http://www.ncbi.nlm.nih.gov/gene ) and uniprot ( http://www.uniprot.org ) databases .
mutations were analyzed by polyphen-2 ( http://genetics.bwh.harvard.edu/pph2/ ) online in silico prediction program , which predicted the pathogenic nature of missense mutations .
polyphen-2 used homology search , multiple sequence alignment , profiling , identity - based scores , and structure - based search parameters ( such as accessible surface area and hydrophobicity features ) .
these predictions could define the putative role of missense variants and could predict whether they were probably / possibly damaging or benign .
polyphen-2 also provided the multiple sequence alignment , which compared the homologous sequences from different organisms .
humdiv was used for testing the pathogenicty of rare alleles , where mildly damaging variants must be treated as possibly damaging .
humvar was used in case of mendelian disorders , where more damaging alleles should be distinguished from the less damaging ones.17 we also analyzed the mutation with sift algorithm ( http://sift.jcvi.org/ ) , which used several databases such as swiss - prot , swiss - prot / trembl , and ncbi protein database .
the software analyzed the possibility on the pathogenic properties of mutations by comparing the mutant and normal variants .
sift scores of point mutation were calculated for weighting the mutations as possibly damaging allele or tolerated ( nondamaging ) , with a dividing score of 0.05 , respectively.18 protein structure prediction analysis of psen2 was performed by raptor x program at the university of chicago ( http://raptorx.uchicago.edu ) .
normal psen2 was composed of 488 amino acids , starting at 61 residues and continuing till residue 448 .
the prediction was performed at raptor x web server for both normal psen2 and r62c mutation .
based on psen2 information from the uniprot database , r62c mutation belonged to the topological domain , positions 187 , including the mutation site .
pcr - single - strand conformation polymorphism showed different mobility patterns for this patient s sample , which suggested the presence of mutation exon 4 of psen2 ( figure 4a ) .
sanger sequencing revealed a heterozygosity of arginine ( cgc , arg , r ) to cysteine ( tgc , cys , c ) exchange at codon 62 , which was previously reported in europe as possibly a pathogenic variant ( figure 4b and c).7,2024 we did not observe any pathogenic mutation in app , psen1 , prnp , pgrn , or mapt genes .
psen2 r62c mutation was checked against the korean center for diseases and controls database ( http://www.cdc.go.kr/cdc/contents/ ) , a database of whole genome sequences from 622 healthy control individuals in korea , but this mutation was not found .
mutation was also checked in the exome aggregation consortium ( exac ) ( http://exac.broadinstitute.org/ ) browser for reporting all variants from 60,706 unrelated individuals .
the previous association studies revealed that this mutation was found in both ad patients and in a few healthy controls.7,2124 polyphen-2 prediction supported the data of sala frigerio et al , suggesting psen2 r62c as a possibly damaging variant with humdiv and humvar scores of 0.877 and 0.513 , respectively.22 polyphen-2 multiple sequence alignment revealed that r62 might not be conserved among vertebrates .
multiple sequence alignment suggested that psen2 sequences of african elephants and giant pandas were similar to human psen2 , with arg at the same position .
however , in psen - like genes of some vertebrate , different amino acids took place of arg : glycine in tasmanian devil and short - tailed opossum , threonine in while duckbill platypus , and asparagine in turkey , respectively .
three - dimensional structure modeling data revealed that psen2 187 topological domain had an n - terminal loop structure up to residue 71 ( figure 5 ) , suggesting divergent structures between normal psen2 and its mutant r62c .
the mutation may not have strong defined structure , but reveals dynamic motion in the loop structure .
the highly distinct properties of arg and cys could have dramatic impact on the structure and functions of psen2 .
arg is positively charged with high hydrophilicity and bulk volume , while cys presents a smaller volume and has a sulfhydryl ( -sh ) group with negative charge , which can form disulfide bond with other cys residues inside the ps2 protein or with other proteins .
this new sh - bonding potential could result in altered protein functions , which may influence the altered a production .
previously , psen2 r62c mutation was reported in load patients and one healthy individual from the netherlands in europe.2124
psen2 r62c was also reported in a caucasian belgian ad patient with age of onset at 70 years without a detailed clinical report.20,21,23 sala frigerio et al detected psen2 r62c in one ad patient , whose brother was also diagnosed with ad .
again , no detailed report on the clinical symptoms or disease progression was available , including their age of onset , phenotypes , or genetic testing of their relatives .
hence , the previous reports on psen2 r62c did not segregate the relatives of the patients with the disease status.7,2124 here , we discovered the same mutation in a korean male patient with probable eoad for the first time in asia .
even though his family did not present with any previous neurologic diseases , this mutation could not be categorized as a probable de novo case , because his family did not agree in revealing any information about their ages nor allowed further genetic testing of siblings and descendants .
exchange from arg to cys could allow psen2 to form a disulfide bond with another cys inside the psen2 protein or with other proteins near psen2 at cytoplasmic ph ( ph 7.2 ) level .
these new sh bonds could have the potential to create novel intermolecular or intramolecular structures that could be involved in pathogenic mechanisms . at the same locus of psen2 r62 , another mutation to histidine ( his , h )
psen2 r62h appeared as both homozygous and heterozygous stages , suggesting for the potential of higher number of unaffected individuals and less or no pathogenicity . for h62r , the exchange of arg to his might not have a dramatic effect on psen2 , since his and arg are both positively charged and large amino acids . on the other hand , exchange with cys
nevertheless , polyphen-2 and sift analyses suggested that psen2 r62c could be more pathogenic than r62h .
both algorithms revealed psen2 r62c as possibly damaging , but the r62h was predicted as benign.25 in addition , the exac database showed much lower frequency for r62c in unaffected individuals than r62h .
sleegers et al revealed that since both r62c and r62h mutations were rare variants , it would be difficult to perform association study with them ; however , they may occur more frequently in patients than in unaffected individuals.21 guerreiro et al designed an algorithm for classifying psen1 and psen2 mutations as definitely pathogenic , probably pathogenic , possibly pathogenic , or nonpathogenic groups . if the mutation was found in larger number of patients than controls , it still could be categorized as a risk factor or possibly pathogenic
mutations.26
psen2 r62c and r62h are found in higher frequency of patients with dementia than in controls .
the measured a levels in the cellular in vitro or in vivo studies of many of the mutations may help in verifying the effect of mutations on increasing a production .
psen2 r62h mutation did not affect the a production or a 42/40 ratio.27,28 ertekin - taner et al examined the correlation of plasma a42 levels between r62c and r62h.7 in this study , a significant association was found between psen2 polymorphisms and plasma a42 levels in first - degree relatives with load at a level of 0.03.4 this study was repeated in a large community - based load setting , but no significant association was detected .
these findings suggested that even though these psen2 mutations might increase the levels of a42 in ad patients , they might not be prominent genetic factors in disease onset .
since the analyses for psen2 r62c were not performed thoroughly , the mutational effects on a levels , its metabolism , or altered functions of psen2 function can not be conclusive .
in addition , psen2 r62c and r62h could be also affected potentially from the gene
environment interactions.7 besides r62c and r62h , seven missense mutations in the n - terminal region of psen2 protein are described in table 1 .
majority of them in the n - terminus loop region are categorized as mutations with unclear pathogenicity or nonpathogenic variants .
psen2 t18 m was discovered in a german patient with parkinson s disease at 62 years of age , and ad symptoms did not appear until the age of 70 years .
the functional effect r29h variant remained unknown , but polyphen-2 suggested that a29h might have some pathogenic effect.26
psen2 g34s was discovered in a caucasian load patient from the netherlands , but this mutation did not segregate the family members completely with the disease .
psen2 g34s did not show clear association with increased a42/a40 ratio.21
psen2 p69a was found in a load patient where segregation also could not be assessed , and dobricic et al suggested that p69a might be a nonpathogenic variant.30
psen2 r71w was detected in a load patient without segregation of relatives with the disorder .
previously , polyphen-2 predictions suggested that this mutation might be involved in eoad , but we could not confirm it.21,24,26,31
psen2 k82r was found in a chinese eoad patient , who presented additional symptoms such as depression and language impairment .
she developed memory loss at the age of 53 , and family history remained unclear.26
psen2 a85v from a sardinian family was the only pathogenic mutation in the n - terminal region of psen2 , which was verified from affected family members with ad or dementia with lewy bodies .
we discovered a psen2 r62c in a male korean patient with eoad for the first time in asia . from the analyses against two major ad databases , ad and ftd mutation databases and the alzgene databases ,
psen2 r62c was verified as a rare mutation with unclear pathogenic nature , which was similar to the his variant at residue 62 , r62h .
however , r62c might be involved in pathogenicity , since in silico analyses from polyphen-2 , sift , and three - dimensional modeling suggested pathogenic effects without clear mechanisms .
the free sh group in cys could associate by forming novel disulfide bridge in the loop with nearby proteins or with other sulfurs or metals , leading to altered protein function .
limitation of our study was that we could not make segregation analysis from other family members , since all relatives and family members declined the genetic test , nor revealing other information .
since both psen2 r62c and r62h are relatively rare variants in asia , the association studies may be difficult .
lastly , the functional / cellular studies of these mutations in future could be helpful in verifying their possible roles in ad pathogenicity . | a 49-year - old korean male patient with dementia was diagnosed with probable early - onset alzheimer s disease ( ad ) .
he presented with memory problems , personality changes , and disorientation .
his family history of dementia was probably negative , since no family member with dementia was found or mentioned .
mild cortical atrophy was observed upon magnetic resonance imaging analyses of his brain , and the single - photon emission computed tomography analysis revealed hypoperfusion in the frontal , temporal , and limbic lobes .
the patient was tested for mutations in app , psen1 , psen2 , pgrn , mapt , and prnp genes .
genetic analysis revealed r62c mutation in psen2 gene .
psen2 r62c mutation was previously reported in european populations , including dutch and belgian families with ad . herein , we present the first case report of psen2 r62c mutation in asia .
polyphen-2 and sift software analyses predicted this mutation as possibly damaging , suggesting its potential involvement with ad . in silico
protein structural prediction analyses of psen2 r62 and c62 revealed two divergent structures , suggesting that large perturbations of r62c mutation might cause dysfunctions of psen2 , which may alter the normal amyloid production . |
the vitreous humor ( volume of ~4 ml in an adult ) comprises the majority of the eye s volume1 and is responsible for several visually debilitating conditions such as retinal tear and detachment , macular hole , macular pucker , and vitreomacular traction ( vmt ) .
the vitreous has a uniform gel - like consistency at birth , but liquefaction begins soon thereafter , with pockets of aqueous fluid appearing by the age of 4 years.2 as aging continues , the loss of type ix collagen increases the surface area on sticky type ii collagen fibrils and allows them to fuse when they come into contact.3,4 collagen aggregation promotes vitreous liquefaction , resulting in pockets of aqueous fluid , most of which are situated within the central vitreous , while others lie adjacent to the posterior hyaloid membrane .
the collagen fibers that attach the posterior hyaloid surface to the internal limiting membrane ( ilm ) also slowly degenerate with advancing age.5 twenty percent of the vitreous is liquefied by the age of 18 years and 50% by the age of 8090 years.6 vitreous liquefaction , together with weakening of the adhesion between the posterior hyaloid and the ilm , promotes posterior vitreous detachment ( pvd).5 investigators using funduscopic evaluations reported that the prevalence of pvd was 50% in patients over the age of 50 years and 65% in patients over the age of 65 years.7 subsequent histologic analyses challenged the validity of these findings8 and called into question the accuracy of funduscopic examination because many eyes diagnosed with pvd actually had vitreous schisis with preretinal bursae that had been mistaken for posterior hyaloid separation.9 the introduction of optical coherence tomography ( oct ) has given us a deeper understanding of vitreoretinal interface ( vri ) abnormalities .
spectral domain optical coherence tomography ( sd - oct ) accurately visualizes the vri with an axial resolution of 36 m .
high - resolution , cross - sectional images show that vitreous detachment is a gradual , stepwise process that begins in the peripheral macula .
this slowly extends posteriorly to involve the fovea and often culminates with sudden , complete release from the fovea and disc , thereby clinically presenting as a pvd.2 the extent of shallow detachment between the posterior hyaloid and the central 6 mm of the retina s ilm can be accurately measured with sd - oct .
the limitations of oct imaging become apparent , however , when we attempt to distinguish complete pvd , where the separated posterior hyaloid often lies > 2 mm above the ilm and is outside the range of the oct image , from no detachment.10 in these eyes , clinical examination of the vitreous together with b - scan ultrasound is necessary to diagnose a complete pvd.11 the prevalence of pvd increases with age , but the extent of partial vitreous separation in different age groups is still under investigation.12,13 this issue becomes clinically important because a partially detached vitreous can exert traction on the ilm and cause ilm damage that leads to macular pucker and macular hole .
vmt alters the severity of chorioretinal vascular conditions such as diabetic macular edema14,15 and neovascular age - related macular degeneration1619 and limits the effectiveness of pharmacologic treatment with drugs that inhibit vascular endothelial growth factor.20 pharmacotherapy to hasten vitreomacular separation had been developed to treat symptomatic vmt .
the intravitreal injection of ocriplasmin ( jetrea ; thrombogenics , leuven , belgium ) cleaves remaining vitreoretinal adhesions and restores macular anatomy and function in some symptomatic eyes.21 the present study was conducted to determine the prevalence of vitreomacular adhesion ( vma ) in a large clinic population .
this retrospective chart review was approved by the mayo clinic institutional review board . for this retrospective study ,
all patients examined by one of the authors ( mws ) between january 1 , 2013 and june 30 , 2013 , were identified from the electronic scheduling calendar , and data from the electronic medical record were evaluated for the study .
exclusion criteria included previous pars plana vitrectomy , high myopia ( > 6 d ) , history of uveitis , and unavailability of evaluable oct images .
patients who were evaluated more than once during the study period were counted only once for the overall vma .
demographic information on each patient was obtained from the medical record and tabulated for analysis .
identified clinical data included the following : best - corrected visual acuity ; intraocular pressure ; slit - lamp examination of the anterior segment ; lens status ( phakic , aphakic , pseudophakic ) including the location of any intraocular lens ( anterior or posterior chamber ) ; biomicroscopic examination of the vitreous optic disc , macula , and retinal vessels ; and indirect ophthalmoscopic examination of the peripheral retina .
the vitreous was examined with slit - lamp biomicroscopy using a 78 or 90 d handheld condensing lens .
the vitreous was characterized as either completely detached or attached , based on the presence or absence of a partial or complete weiss ring .
the biomicroscopic status of the vitreous was correlated with the results of the oct scan , and inconsistencies , though rare , were reconciled by the investigators . throughout the study period , all patients
not previously seen in the department of ophthalmology by one of the authors ( mws ) within the previous 6 months were routinely evaluated with sd - oct ( cirrus ; carl zeiss meditec ag , jena , germany ) . for patients seen more than once during the study period ,
usually at least one set of scans were performed with additional scans performed at the discretion of the investigator , based upon the patients diagnoses and need for treatments .
the state of the posterior hyaloid membrane on oct was characterized by the presence or absence of separation from the ilm in any part of the horizontal foveal scan or vertical construct . any observed separation of the vitreous from the ilm , either partial or complete , resulted in a characterization of partial vitreous separation or incomplete pvd .
if the vitreous appeared to be completely separated from the ilm throughout the scan , an observation of weiss ring on the biomicroscopic exam was required before the eye could be characterized as having a complete pvd .
eyes with no vitreoretinal separation observed on oct were characterized as having either complete pvd or completely attached vitreous , depending upon the biomicroscopic examination .
the length of vitreoretinal separation on oct in each eye was measured with electronic calipers .
the lengths of vitreomacular separation on the horizontal scan and vertical construct were averaged to determine a single number .
because the strength of the vma varies among common retinal conditions , and the degree of adhesion can affect disease severity and response to therapy , separate calculations were performed on eyes with age - related macular degeneration ( which did not undergo intravitreal injections ) and diabetic retinopathy ( without previous laser photocoagulation ) , and eyes without retinal abnormalities .
descriptive statistics , including mean and standard deviation , were calculated with excel microsoft office 2013 software .
demographic information on each patient was obtained from the medical record and tabulated for analysis . identified clinical data included the following : best - corrected visual acuity ; intraocular pressure ; slit - lamp examination of the anterior segment ; lens status ( phakic , aphakic , pseudophakic ) including the location of any intraocular lens ( anterior or posterior chamber ) ; biomicroscopic examination of the vitreous optic disc , macula , and retinal vessels ; and indirect ophthalmoscopic examination of the peripheral retina .
the vitreous was examined with slit - lamp biomicroscopy using a 78 or 90 d handheld condensing lens .
the vitreous was characterized as either completely detached or attached , based on the presence or absence of a partial or complete weiss ring .
the biomicroscopic status of the vitreous was correlated with the results of the oct scan , and inconsistencies , though rare , were reconciled by the investigators .
throughout the study period , all patients not previously seen in the department of ophthalmology by one of the authors ( mws ) within the previous 6 months were routinely evaluated with sd - oct ( cirrus ; carl zeiss meditec ag , jena , germany ) . for patients seen more than once during the study period , usually at least one set of scans
were performed with additional scans performed at the discretion of the investigator , based upon the patients diagnoses and need for treatments .
the state of the posterior hyaloid membrane on oct was characterized by the presence or absence of separation from the ilm in any part of the horizontal foveal scan or vertical construct .
any observed separation of the vitreous from the ilm , either partial or complete , resulted in a characterization of partial vitreous separation or incomplete pvd .
if the vitreous appeared to be completely separated from the ilm throughout the scan , an observation of weiss ring on the biomicroscopic exam was required before the eye could be characterized as having a complete pvd .
eyes with no vitreoretinal separation observed on oct were characterized as having either complete pvd or completely attached vitreous , depending upon the biomicroscopic examination .
the length of vitreoretinal separation on oct in each eye was measured with electronic calipers .
the lengths of vitreomacular separation on the horizontal scan and vertical construct were averaged to determine a single number .
because the strength of the vma varies among common retinal conditions , and the degree of adhesion can affect disease severity and response to therapy , separate calculations were performed on eyes with age - related macular degeneration ( which did not undergo intravitreal injections ) and diabetic retinopathy ( without previous laser photocoagulation ) , and eyes without retinal abnormalities .
descriptive statistics , including mean and standard deviation , were calculated with excel microsoft office 2013 software .
the mean age of the cohort was 69.9 years ( standard deviation : 14.7 ) with 29.8% of patients under the age of 65 years . mean intraocular pressures in the right and left eyes were 14.95 and 14.99 mmhg , respectively .
lens status reflected the age distribution of the population with 62.0% of eyes being phakic , 37.4% pseudophakic , and 0.6% aphakic .
the prevalence of partial vitreous separation below the age of 50 years was 61.7% , and it peaked at 71.2% in the first analyzed 5-year interval ( 5054 years ) .
the prevalence of partial pvd then decreased steadily through each 5-year interval until reaching 5.6% in the 95- to 99-year group .
the prevalence of no vitreous separation , calculated by the formula : ( all eyes partial separation
pvd by biomicroscopy)/all eyes , was 36.7% in the < 50-year group , dropped to 20.3% in the 60- to 64-year group , and then steadily rose to 65.6% in the 85- to 89-year group , after which it remained at or > 50% .
the prevalence of complete pvd ( determined by biomicroscopy ) rose from a low of 1.7% in the < 50-year group , to a high of 29.2% in the 75- to 79-year group , after which it decreased but remained > 22% .
for all eyes with partial separation of the vitreous , the average length of vitreous attachment to the macula is listed by age groups in figures 1 and 2 .
the length of vitreous attachment decreased from a high of 4.6 mm in the < 50-year age group to 2.4 mm in the 95- to 99-age group . a trend - line ( calculated by linear regression )
this shows that the average length of vma decreases by an average of 0.045 mm per year after the age of 50 years .
patients with normal retinas , macular degeneration , and diabetic retinopathy were evaluated for partial vitreoretinal separation ( figure 3 ) .
patients with macular degeneration resembled those with normal retinas , but there was a nonstatistically significant trend for greater vitreoretinal adhesion in patients with diabetic retinopathy .
the mean age of the cohort was 69.9 years ( standard deviation : 14.7 ) with 29.8% of patients under the age of 65 years . mean intraocular pressures in the right and left eyes were 14.95 and 14.99 mmhg , respectively .
lens status reflected the age distribution of the population with 62.0% of eyes being phakic , 37.4% pseudophakic , and 0.6% aphakic .
the status of the vitreous attached , partial separation , and complete detachment is summarized by 5-year increments in table 2 .
the prevalence of partial vitreous separation below the age of 50 years was 61.7% , and it peaked at 71.2% in the first analyzed 5-year interval ( 5054 years ) .
the prevalence of partial pvd then decreased steadily through each 5-year interval until reaching 5.6% in the 95- to 99-year group .
the prevalence of no vitreous separation , calculated by the formula : ( all eyes partial separation
pvd by biomicroscopy)/all eyes , was 36.7% in the < 50-year group , dropped to 20.3% in the 60- to 64-year group , and then steadily rose to 65.6% in the 85- to 89-year group , after which it remained at or > 50% .
the prevalence of complete pvd ( determined by biomicroscopy ) rose from a low of 1.7% in the < 50-year group , to a high of 29.2% in the 75- to 79-year group , after which it decreased but remained > 22% .
for all eyes with partial separation of the vitreous , the average length of vitreous attachment to the macula is listed by age groups in figures 1 and 2 .
the length of vitreous attachment decreased from a high of 4.6 mm in the < 50-year age group to 2.4 mm in the 95- to 99-age group . a trend - line ( calculated by linear regression )
this shows that the average length of vma decreases by an average of 0.045 mm per year after the age of 50 years .
patients with normal retinas , macular degeneration , and diabetic retinopathy were evaluated for partial vitreoretinal separation ( figure 3 ) .
patients with macular degeneration resembled those with normal retinas , but there was a nonstatistically significant trend for greater vitreoretinal adhesion in patients with diabetic retinopathy .
few studies have documented the age - dependent separation of the posterior hyaloid from the ilm of the retina.12,13 our study evaluated a large group of patients , but it was neither randomized nor does it represent the general population .
our cohort excluded patients below the age of 14 years ( because of the practice composition ) , so the pediatric population , a demographic in whom the vitreous is usually completely attached , is underrepresented .
the population served by the author s practice has a large number of retirees , so even though ophthalmology clinics generally see more elderly than young patients , this cohort probably includes a disproportionate number of patients over the age of 65 years .
finally , the author s subspecialty practice includes patients with vitreoretinal conditions such as age - related macular degeneration , diabetic retinopathy , and epiretinal membranes .
our study showed a remarkably high ( 71.2% ) prevalence of partial vitreous separation in the 50- to 54-year group .
this decreased steadily through successive age groups , consistent with the notion that the vitreous completely separates at an increasing rate in populations over the age of 50 years .
the proportion of patients in our study with partial vitreous separation differs from that reported by itakura and kishi.13 for patients in each of the 50- to 59- , 60- to 69- , 70- to 79- , and 80- to 89-year age groups , our proportions with partial vitreous separation were considerably higher than theirs ( 64.4% vs 40% , 51.1% vs 30% , 32.5% vs 10% , 18.0% vs 2% ) .
the reasons for these differences are unclear but may include the racial compositions of the cohorts , the health of the cohorts ( clinic population vs healthy volunteers , general ophthalmology / vitreoretinal practice vs healthy volunteers ) , and the sizes of the cohorts ( 1,436 vs 368 ) .
itakura and kishi did not describe the phakic status of their cohort , but 38% of our patients had previously undergone cataract extraction . since their cohort was composed of volunteers and ours was a clinic population ( many of whom had already undergone cataract surgery ) , this may have contributed to the higher separation rates seen in our study .
they reported a high incidence of complete vitreous detachment ( 80% ) in patients over the age of 80 years but did not use b - scan ultrasonography to verify this number . nonetheless , this is probably closer to the actual prevalence of complete pvd than we determined in our retrospective series .
our data prevent us from accurately determining the prevalence of pvd in the older age groups .
but if we compare our 80- to 84-year group to the 50- to 54-year - old group and assume that the differences in partial vitreomacular separation represent progression to complete pvd , we can estimate that > 70% of our 80- to 84-year - old patients had complete pvds .
this number is very close to that reported by itakura and kishi.13 a large prospective study of 2,198 subjects detected higher rates of partial vitreous separation.12 in the 50- to 59- , 60- to 69- , 70- to 79- , and 80- to 89-year age groups , ~85% , 61% , 30% , and 29% of patients had partial vitreous separation .
common to all three studies is the detection of higher rates of partial separation in the younger group ( 5059 years ) , which decreases with advancing age .
the decreasing prevalence of partial separation with advancing age in our study was initially accompanied by an increase in the prevalence of complete pvd , from 1.7% in the < 50-year group to 29.2% in the 70- to 74-year group .
counterintuitively , the prevalence of pvd then dropped to 22.6% in the 90- to 94-year group .
prospective studies of pvd have used both clinical examination and b - scan ultrasound to make this diagnosis .
many authors contend that ultrasound is necessary to correctly diagnose a pvd , since the predominantly transparent posterior hyaloid can be easily missed by clinical examination , thereby underestimating the true prevalence.11,22 confirmatory ultrasound was not used in our study , and this probably affected the pvd prevalence rates we discovered , particularly for the 75 + -year age groups .
younger patients generally have minimal cataracts and clear media , which makes for more accurate biomicroscopic evaluation of the vitreous . in advancing age groups ,
cataracts develop and vitreous syneresis progresses , thereby making accurate assessment of the posterior hyaloid more difficult .
additionally , the increasing prevalence of previous cataract surgery with accompanying capsular opacification and retained intracapsular lens material makes sensitive visualization of the posterior segment more difficult .
we believe that these factors contributed to our underestimating the prevalence of pvd in the advancing age groups and may explain the decreasing prevalence of pvd in our study after the age of 74 years .
our study was conducted over a 6-month period and is more representative of a snapshot of the posterior hyaloid in a clinic population rather than a longitudinal study .
a trend - line showed a decrease of 0.045 mm per year in the length of vma throughout the cohort .
unfortunately , this is not a longitudinal study , and we can not use this trend to accurately extrapolate the progression rate of posterior hyaloid separation . however , we can use the data from the different age groups to create a rough estimation for the fastest possible progression of posterior hyaloid separation . if we assume that the initial pvd prevalence in the 50- to 54-year age group is accurate and that all patients in the 85- to 89-year group have either partial or complete pvd , then 87% of patients would have developed pvds during the 35-year aging period . equally distributed , this means that a maximum of 2.5% of patients over the age of 50 years could develop a pvd each year .
the widespread adoption of sd - oct together with the introduction of pharmacologic vitreolysis with ocriplasmin has increased interest in vri abnormalities such as symptomatic vmt .
the average age of patients treated with ocriplasmin is 68 years.23 we found a partial vitreomacular separation prevalence of 44.7% in the 65- to 69-year group , which is well above that of the 80 + -year age groups but significantly below the rate of the 50- to 54-year group .
we believe that in patients who develop vitreoretinal separation at a younger age , adhesions are weaker between the posterior hyaloid and ilm , and therefore , they separate completely rather than developing symptomatic persistent traction .
we propose that symptomatic vmt peaks in the seventh decade because a relatively high ( though dwindling ) number of patients have still not developed pvd and the remaining patients without pvd possess an increased adhesive strength .
our data also suggest a trend toward later vitreomacular separation in patients with diabetic retinopathy , consistent with the widely held belief that the posterior hyaloid in diabetic patients adheres more strongly to the ilm .
we found that the average length of attachment between the posterior hyaloid and ilm decreases with age .
this is consistent with the observation that vitreous detachment begins in the peripheral macula and spreads slowly over a period of years.24 more elderly segments of the population have higher prevalences of complete pvds , and those without pvds have probably been experiencing progressive vitreoretinal separation for longer periods of time .
therefore , the smaller number of patients without pvd have shorter average lengths of persistent adhesion .
this study provides a cross - sectional view of vitreoretinal separation in a clinic population and suffers from several weaknesses .
data regarding axial lengths are not available . though we are confident about the accuracy of partial vitreoretinal separation as shown by sd - oct , the prevalences of no vitreous separation and complete vitreous detachment in the older population groups are not accurate .
b - scan ultrasonography would have improved the accuracy of the complete pvd data , but ultrasound is rarely used to evaluate vitreous detachment outside the confines of a prospective study .
in summary , we have found that the prevalence of partial vitreous separation peaks no later than the sixth decade and the average length of residual vitreoretinal adhesion follows a similar pattern .
we suspect that patients with diabetic retinopathy may have stronger vitreoretinal adhesions , but larger studies are needed to confirm this .
we believe that future prospective , population studies will better characterize the epidemiology and progression rates of vitreoretinal separation . | backgroundvitreous degeneration begins soon after birth and accelerates throughout life .
vitreous liquefaction with a slowly progressive separation of the posterior hyaloid from the peripheral macula usually leads to complete posterior vitreous detachment .
the purpose of this study is to measure the age - related prevalence of partial vitreous separation and the length of residual vitreous adhesion in an ophthalmology clinic population.methodspatients examined by the senior author ( mws ) during a 6-month period were included in a retrospective chart review .
demographic data and spectral domain optical coherence tomography scan results were gathered .
data analysis with descriptive statistics focused on the prevalence and extent of partial vitreous separation.resultsthe mean age of the study patients was 69.9 years , and 62% were phakic .
the highest prevalence of partial posterior hyaloid separation from the internal limiting membrane ( 71.2% ) was seen in the 50- to 54-year age group .
this prevalence rate steadily decreased to 5.6% in the 95- to 99-year age group .
the prevalence of complete vitreous detachment as determined by slit - lamp biomicroscopy increased from 1.7% in the < 50-year age group to a maximum of 29.2% in the 75- to 79-year group .
the length of vitreomacular adhesion averaged 4.6 mm in the 50- to 54-year age group and steadily decreased to 2.1 mm in the 90- to 95-year group.conclusionvitreomacular separation affects the majority of eyes in the sixth decade of life .
the prevalence of partial vitreous separation decreases with advancing age , probably because an increasing number of these patients progress to complete posterior vitreous detachment . |
a 56-year - old man presented with a 12-month history of tingling ulcers on both feet ( figure 1a ) .
he was otherwise well . on examination there were ulcers on both feet , 11.5 cm in diameter with a macerated edge .
skin - colored to red - brown papules were noted on both knees ( figure 1b ) as well as irregular red circles on both calves .
skin biopsies taken from knee papules and red circles on the lower leg showed extravascular eosinophilic deposits in the dermis ( figure 1c ) , which stained positive with pas , igm ( figure 1d ) and kappa stains , but negative with congo red .
investigations are summarised here : serum kappa free chains , 85.9 mg / l ( normal 3.319.4 mg /
l ) ; serum lambda free chains , 17 mg / l ( normal 7.726.3 mg / l ) ; serum free kappa chains /free - lambda chains ratio , 5.05 ( normal 0.261.65 ) ; serum electrophoresis , elevated ig m gradient ; serum immunofixation , monoclonal ig m / kappa - type immunoglobulin ; urine immunofixation , monoclonal kappa - free chain ; bone marrow microscopy , infiltrate of lymphoplasmocytic cells ( 15% ) ( most cd20 and cd138 positive ) with preferential expression for igm .
the cutaneous manifestations of waldenstrm macroglobulinaemia have been classified as either infiltration by neoplastic cells or those related to the paraproteinaemia , such as immunoglobulin deposition , cryoglobulinaemia or blood hyperviscosity .
approximately 5% of patients with waldenstrm macroglobulinaemia develop cutaneous manifestations , with neoplastic infiltration being the least common .
prognosis does not appear to be influenced by the presence or absence of cutaneous disease .
the patient presented with a burning red face which was initially diagnosed as rosacea but with time the more typical purple infiltration developed .
macroglobulinaemia the neuropathy is typically sensorimotor , initially with paraesthesias and numbness in the feet and hands .
the possible mechanisms in waldenstrm macroglobulinaemia include microangiopathy , direct infiltration of nerves , endoneural deposition of igm protein or amyloidosis , hyperviscosity or immunoglobulin deposit in blood vessels .
the tingling ulcers in our patient are probably an example of this and , although biopsies were not taken from the ulcers , may have been due to igm deposition given he also had macroglobulinaemia cutis .
there has been only one other case reported , to our knowledge , of ulcers on the sides of the feet .
these developed in areas of hyperkeratosis and were associated with dysaesthesia and a burning sensation . however , the biopsy showed a leukocytoclastic vasculitis with a neutrophilic infiltrate .
another report described igm dermal deposits that presented as painful hyperkeratotic papules on the soles of the feet that were not ulcerated .
storage papules , also known as macroglobulinaemia cutis , are rare but well described and are usually found on the knees or other extensor surfaces .
they are usually skin - coloured , red - brown or translucent , and may be umbilicated , crusted or haemorrhagic .
after five cycles the lower limb ulceration and neurological symptoms had resolved , although the blood igm level did not change .
waldenstrm macroglobulinaemia is a low - grade lymphoplasmacytoid lymphoma that usually presents with nonspecific systemic symptoms , but cutaneous changes may be the initial manifestation and hence may present first to a dermatologist . although our patient presented with tingling ulcers , the diagnosis was reached after skin biopsy from knee papules demonstrated dermal immunoglobulin deposition . | a 56-year - old man presented with tingling ulcers on the feet . on further skin examination , papules on the knees were observed .
biopsies revealed extravascular eosinophilic deposits of igm , and waldenstrm macroglobulinaemia was diagnosed .
the skin manifestations have resolved with chemotherapy .
peripheral neuropathy and storage papules are rare manifestations of waldenstrm s macroglobulinaemia . |
taehwa , danyang - gun , chungcheongbuk - do , korea ( n 3707'01.42 '' , e 12829'05.11 '' ) .
healthy leaves were collected from conifer species growing at the site , including 3 juniper trees ( juniperus rigida siebold et zucc . ) , 4 japanese larch trees [ larix kaempferi ( lamb . ) carr . ] , and 5 pine trees ( pinus densiflora siebold et zucc . ) .
each tree was growing at a distance of at least 100 m from the other sampled trees between 400~800 m in altitude , and all trees were sampled using a gps , an aluminium tag , and a tagging tape : april ( spring ) , july ( summer ) , and november ( autumn ) .
all the 2-yr - old leaves were washed with tap water and then placed for 3 min in 1% naocl solution , 2 min in 70% ethanol , and finally washed twice with distilled water .
these surface - sterilized leaves were cut into 4 segments that were 5 mm in length and then placed into 3 types of culture medium : potato dextrose agar ( pda ) , malt extract agar , and water agar .
any hyphae that extended from the leaf fragments were used in a successive culture with pda .
all isolates were grouped into morphotypes on the colony shape , height , and color of the aerial hyphae , in addition to the base color , growth rate , margin characteristics , surface texture , and depth of growth into the medium .
dna was extracted according to the protocol of the dneasy plant mini kit ( qiagen , hilden , germany ) , and pcr was performed to amplify the internal transcribed spacer ( its ) region , including 5.8s rdna , by using the primers its1f and its4 under the following conditions : 94 for 5 min , followed by 30 cycles of 94 for 30 sec , 50 for 30 sec , 72 for 1 min , and a final extension at 72 for 5 min .
the pcr products were sequenced and compared with reference sequences on ncbi by using blast .
taehwa , danyang - gun , chungcheongbuk - do , korea ( n 3707'01.42 '' , e 12829'05.11 '' ) .
healthy leaves were collected from conifer species growing at the site , including 3 juniper trees ( juniperus rigida siebold et zucc . ) , 4 japanese larch trees [ larix kaempferi ( lamb . ) carr . ] , and 5 pine trees ( pinus densiflora siebold et zucc . ) .
each tree was growing at a distance of at least 100 m from the other sampled trees between 400~800 m in altitude , and all trees were sampled using a gps , an aluminium tag , and a tagging tape : april ( spring ) , july ( summer ) , and november ( autumn ) .
all the 2-yr - old leaves were washed with tap water and then placed for 3 min in 1% naocl solution , 2 min in 70% ethanol , and finally washed twice with distilled water .
these surface - sterilized leaves were cut into 4 segments that were 5 mm in length and then placed into 3 types of culture medium : potato dextrose agar ( pda ) , malt extract agar , and water agar . finally , the samples were cultivated in an incubator at 25 and in darkness .
any hyphae that extended from the leaf fragments were used in a successive culture with pda .
all isolates were grouped into morphotypes on the colony shape , height , and color of the aerial hyphae , in addition to the base color , growth rate , margin characteristics , surface texture , and depth of growth into the medium .
dna was extracted according to the protocol of the dneasy plant mini kit ( qiagen , hilden , germany ) , and pcr was performed to amplify the internal transcribed spacer ( its ) region , including 5.8s rdna , by using the primers its1f and its4 under the following conditions : 94 for 5 min , followed by 30 cycles of 94 for 30 sec , 50 for 30 sec , 72 for 1 min , and a final extension at 72 for 5 min .
the pcr products were sequenced and compared with reference sequences on ncbi by using blast .
a total of 59 morphotypes were isolated from the host plants ( table 1 ) . only morphotypes with a 97% similarity value were used for analysis ( fig .
depending on host plants , 6 species were identified among the 12 morphotypes from the juniper trees , 8 species among 21 morphotypes from the japanese larch , and 11 species among 26 morphotypes from the pine trees .
one species was isolated from both the juniper tree and the japanese larch , 3 species were isolated from both the japanese larch and the pine tree , and 2 species were isolated from both the pine tree and the juniper tree .
in addition , n. diffusa was the most abundant species in the juniper tree ; p. papaya , the japanese larch ; and l. pinastri , the pine tree ( table 1 ) .
the shannon index ( h ' ) was used to assess the species diversity of the endophytic fungi ( table 1 ) . in the juniper tree ,
the total h ' was 1.47 , and the highest h ' ( 1.00 ) was observed in april . in the japanese larch ,
the h ' was 1.74 , and the highest h ' ( 1.33 ) was observed in november . in the pine tree ,
h ' was 1.58 , and the highest h ' ( 1.43 ) was observed in november .
depending on sampling season , 7 species were identified among the 9 morphotypes in april , 8 species among 21 morphotypes in july , and 9 species among 29 morphotypes in november .
one species was isolated during both april and july , 4 species were isolated during both july and november , and 1 species was isolated during both april and november ( table 1 ) .
more than 600,000 species of endophytic fungi are theorized to exist worldwide , and various scientific approaches have been used to detect endophytic fungi to date . in the present study ,
h ' was the highest in the japanese larch , with the greatest number of endophytes being isolated during july and november .
these results indicate that endophytes do not only exist in the leaf and/or do not use horizontal transfer ; instead , endophytes might extend from the plant tissue like a branchlet . during july and august ,
the climate in korea is warm and humid ; hence , climate probably affects endophytic dispersal .
these results also indicate that the number of morphotypes belonging to endophytic fungi increases across the season .
therefore , it is likely that a combination of these factors enhanced the species diversity of the japanese larch .
j. rigida is mainly distributed in lower altitude forests , while l. kaempferi is primarily distributed in middle altitude forests .
however , p. densiflora is found at higher altitudes , where the forest conditions are cooler and drier in the korean peninsula .
thus , most endophytic fungi were obtained from lower to middle altitudes , with only one species of endophytic fungi being discovered at an altitude above 800 m. this observation indicates that endophyte distribution is influenced by the distribution of host plants .
surveys for endophytic fungi have been conducted at all altitudes , with specimens being found at all sites ; however , endophytic fungi have an ability to adapt to variations in abiotic and biotic conditions along the altitudinal gradient .
thus , standpoints of host specificity and adaptation ability are required , which is only possible through the collection and study of endophytes as ecological components and biological resources . | the needled leaves of three conifer species were collected in mt .
taehwa during different seasons of the year .
total 59 isolates and 19 species of endophytic fungi were isolated from the leaves and identified using morphological and molecular characteristics . as a result ,
shannon index was different in its host plant ; larix kaempferi had a highest value of species diversity .
according to the sampling season , 9 species of 19 species were isolated during fall season .
the results suggest that the existing of host plant and sampling season are major factors of distribution of endophytic fungi . |
convincing evidence continues to emerge from clinical trials that gene therapy is
yielding therapeutic effects in patients suffering from a wide range of
diseases .
in particular , liver - directed gene therapy is
becoming a promising modality to obtain sustained hepatocyte - specific expression
of secreted factors into the circulation .
this has implications for other
liver - borne genetic and complex diseases . despite these successes ,
there have
been concerns regarding the efficacy and safety of some gene delivery
approaches .
the major limiting factors are insufficient and/or transient
transgene expression levels and inappropriate expression of the transgene in
unwanted cell types .
higher vector doses are typically used in gene therapy
clinical trials to improve therapeutic efficacy . however
, this often triggers
t - cell mediated immune responses against the vector capsid antigens
displayed by transduced cells , particularly hepatocytes , in the context of mhc
class i. this contributes to
the elimination of the gene - modified cells and liver toxicity , resulting in
short - term gene expression . moreover , inadvertent transgene expression in
antigen - presenting cells ( apcs ) , increases the risk of untoward immune responses
against the gene - modified hepatocytes and/or the therapeutic transgene
product . hence , there is a need to generate improved gene
therapy vectors allowing the use of lower and safer vector doses that enable
sustained hepatocyte - specific expression of the therapeutic gene .
typically , conventional methods of vector design rely on haphazard
trial - and - error approaches whereby transcriptional enhancers are combined with
promoters to boost expression levels .
though this can sometimes be
effective , it often results in non - productive combinations that
result in either modest or no increased expression levels of the gene of
interest in hepatocytes and/or loss of liver specificity .
an intrinsic bias associated with the
design of gene therapy vectors is that it frequently relies on the in
vitro characteristics of its regulatory elements in cell lines , which
is usually not predictive of their in vivo performance .
moreover ,
these conventional approaches in vector design do not take into account the
importance of including evolutionary conserved regulatory motifs into the
expression modules , which is particularly relevant for clinical translation . in silico design
offers unique opportunities to generate robust
liver - specific gene therapy vectors and overcome some of the limitations of
conventional gene therapy vector development .
though data - mining has been used
to identify cis - regulatory modules ( crms ) and analyze
expression patterns in vivo , surprisingly
however , this area of research remains largely unexplored in the context of
vector development and gene therapy . to address this caveat and overcome some of
the limitations of conventional vector design ,
we have developed and validated
an alternative strategy to generate efficient , liver - specific gene therapy
vectors using computational methods . in this study , we relied on a genome - wide
in silico identification of crms that contained clusters
of evolutionarily conserved transcription factor binding site ( tfbs )
motifs associated with robust hepatocyte - specific expression .
we employed a
comprehensive computational strategy that takes into account not only the
over - representation of a given tfbs but also its context - dependent
co - occurrence with other tfbs .
such an approach has not yet been used to improve the
performance of gene therapy vectors , which underscores the novelty of this
study .
this computational approach has broad implications for liver - directed
gene therapy and allows for the use of lower and thus safer vector doses , while
maximizing therapeutic efficacy in pre - clinical mouse models and in non - human
primates .
furthermore , the validation of these liver - specific crms
provides new insights into the molecular determinants underlying transcriptional
control in hepatocytes .
rational de novo design of robust liver - specific gene therapy
vectors relies on the identification of tissue - specific crms
associated with high hepatocyte - specific expression . to identify these
critical hepatocyte - specific crms ( i.e. ,
hs - crms )
this
bio - informatics approach was originally used to identify crms
associated with differential gene expression in response to in
vitro stimuli . in this
study
, we extended this computational approach to identify evolutionary
conserved crms associated with highly expressed liver - specific
promoters .
one of the unique features of this computational strategy is that
it takes into account the over - representation of a given tfbs and
its context - dependent co - occurrence with other tfbs on a
genome - wide scale .
a total of 14 different hs - crms were identified , ranging in size
from 41 bp to 551 bp ( figure
1 and table 1 and
supplementary figure s1 and table s2 ) .
the hs - crm
comprised putative binding sites for eight different tfs : hnf1 ,
c / ebp , lef1 , fox , irf , lef1/tcf , tal1/e47 , and myod .
these
hs - crms contain a molecular signature that
represents a hallmark of highly expressed genes in the liver .
we observed
that most hs - crms contain multiple tfbs that are similar
but the uniqueness of each hs - crm is dictated by its specific
arrangement of the tfbs . since all hs - crms exhibited a high degree
of phylogenetic conservation among 44 divergent species , this suggests a
strong evolutionary selective pressure to maintain these particular
tfbs combinations for high tissue - specific expression .
phylogenetic conservation also increases the likelihood that the performance
of the hs - crms is preserved across species in the process of
clinical translation .
we subsequently assessed the efficiency and specificity of these novel
hs - crms in liver .
we first expressed human clotting factor ix
( hfix ) from a chimeric promoter , composed of a potent liver - specific minimal
transthyretin promoter ( ttr ) in conjunction with the
different hs - crm ( figure 2a ) .
we
choose hydrodynamic delivery to screen the 14 different hepatocyte - specific
cis - regulatory modules ( hs - crms ) since it allows for a rapid and
reliable assessment of their in vivo performance , without having to
produce aav vectors for each of them .
only the best hs - crm was then
selected for a subsequent comprehensive characterization in an aav vector
context ( see below ) .
semi - high throughput hydrodynamic transfection of these
expression plasmids into c57bl/6 mice revealed that the large majority
( i.e. , 85% ) of the de novo designed hs - crms
resulted in a significant increase in hfix expression ( figure 3a ) . in particular , the most potent
hs - crm8 resulted in a robust improvement in hfix expression ,
yielding up to 2.5 g / ml .
we subsequently validated the
hs - crm8 element in aav9-based vectors in which the
hfix gene was driven from either a moderately active
( palm ) ( figure 3b ) or potent
liver promoter ( ttr ) ( figure
3c ) .
the de novo designed hs - crm8 element in
the aav vector resulted in a robust and sustained 100-fold increase in hfix
expression from the moderately active palm promoter ( p
< 0.0001 ) ( 5 10 vg , figure 3b ) .
moreover , this hs - crm8
element significantly improved hfix expression seven- to tenfold ( p
<
0.00005 ) , even when a potent liver - specific promoter was used like
ttr ( figure 3c ) , consistent
with the increased hfix expression after hydrodynamic transfection
( figure 3a ) .
this resulted in
stable therapeutic hfix expression levels of about 100% , even at relatively
low vector doses ( 5 10 vg ) .
the
hs - crm8 element increased transcription from both ttr
and palm promoters ( supplementary figure s2a , b ) .
the
increased hfix antigen levels correlated strongly with the fix mrna
levels ( supplementary figure s2c ) .
there was no significant
difference in vector copy number in mice injected with the vectors with or
without the hs - crm8 element ( supplementary figure s2d ) . the
vector copy number correlated with the vector doses .
the potency of the hs - crm8 element was subsequently evaluated
through assessment of phenotypic correction in hemophilic fix - deficient mice
following a tail - clip injury .
the treated mice survived the injury in
contrast to naive controls , consistent with a significant dose - dependent
reduction in blood loss ( naive mice : 1,167 215
l ; 5 10 vg / mouse :
596 92 l ( p
vg / mouse :
495 82 l ( p < 0.00005 ) . hfix
mrna expression was restricted to the liver ( figure 3d , e ) , even
upon administration of extremely high vector doses
( 3 10 vg ) , despite the detection of
viral genomes in non - hepatic tissues ( i.e. , heart , spleen , muscle ,
etc . )
( data not shown ) . to further enhance hfix expression from the hs - crm8-ttr - fix
construct , we subsequently replaced hfix intron a with an mvm
intron upstream of the hfix transgene ( figure 2c ) or its codon - optimized equivalent
( hfixco ) ( figure 2d ) .
each of these
changes led to a significant increase in hfix expression ( figure 4a , b ) .
this optimized hs - crm8-ttr - mvm - fixco
construct was then packaged into scaav for subsequent validation in a
non - human primate model .
the scaav9-hs - crm8-ttr - mvm - hfixco vector was injected into two cynomolgus
macaques ( oug6 and obev7 ) at a dose of
9 10 vg / kg .
there was a steady
increase in the circulating hfix protein in both animals that reached a peak
by week 4 , corresponding to 1,718 ng / ml and 885 ng / ml for oug6
and obev7 , respectively ( figure 5a ) .
these levels correspond to therapeutic fix levels in the 2035% range
of normal fix levels .
after a few weeks , the hfix transgene expression began
to decline ( figure 5a ) , which could be
ascribed to the induction of anti - hfix antibodies ( figure 5c , d ) , often
seen in non - human primates following aav liver gene transfer ,
despite the use of liver - specific promoters .
the aav9 vector administration
also triggered anti - aav9 neutralizing antibodies ( figure 5b ) .
both animals had consistent coagulation
parameters despite the occurrence of hfix inhibitory antibodies
( supplementary figure s3 ) .
this indicates that the anti - hfix
antibodies did not cross - react with the macaque fix .
the animals did not
develop any adverse events or toxicity ( supplementary figures s4 and
s5 ) .
human fix mrna expression was restricted to the liver
whereas only background levels were apparent in any other organ or tissue
tested ( figure 6a ) .
this confirms the
liver - specificity of the synthetic in silico designed
hs - crm8-ttr promoter , consistent with the mouse data
( figure 3d , e ) .
the aav9 vector resulted predominately in liver
transduction though significant gene transfer was also apparent in heart ,
lungs , spleen , kidney and brain ( figure
6b ) .
one of our animals ( oug6 ) was euthanized due to unexpected complications
unrelated to the gene therapy , which occurred after a surgical procedure to
obtain a liver biopsy .
we subsequently tried to eliminate the anti - hfix
inhibitors in the remaining animal ( obev7 ) following , transient
immunosuppression with cyclosporin ( csa ) and rituximab ( rtx ) .
this led to a
modest increase in circulating fix levels ( figure
5a ) , consistent with a decrease in anti - hfix antibodies
( figure 5c , d ) .
this was consistent with the profound decrease in
cd20 b cells ( figure 5e ) ,
whereas the percentage of cd3 t cells remained unaffected
( figure 5f ) .
unfortunately , only
partial and transient immunosuppression could be achieved , since the animal
refused to ingest csa after the initial treatment .
transient
immunosuppression also led to transient and partial decline in anti - aav9
neutralizing antibodies ( figure 5b ) .
rational de novo design of robust liver - specific gene therapy
vectors relies on the identification of tissue - specific crms
associated with high hepatocyte - specific expression . to identify these
critical hepatocyte - specific crms ( i.e. ,
hs - crms )
this
bio - informatics approach was originally used to identify crms
associated with differential gene expression in response to in
vitro stimuli . in this
study
, we extended this computational approach to identify evolutionary
conserved crms associated with highly expressed liver - specific
promoters .
one of the unique features of this computational strategy is that
it takes into account the over - representation of a given tfbs and
its context - dependent co - occurrence with other tfbs on a
genome - wide scale .
a total of 14 different hs - crms were identified , ranging in size
from 41 bp to 551 bp ( figure
1 and table 1 and
supplementary figure s1 and table s2 ) .
the hs - crm
comprised putative binding sites for eight different tfs : hnf1 ,
c / ebp , lef1 , fox , irf , lef1/tcf , tal1/e47 , and myod .
these
hs - crms contain a molecular signature that
represents a hallmark of highly expressed genes in the liver .
we observed
that most hs - crms contain multiple tfbs that are similar
but the uniqueness of each hs - crm is dictated by its specific
arrangement of the tfbs . since all hs - crms exhibited a high degree
of phylogenetic conservation among 44 divergent species , this suggests a
strong evolutionary selective pressure to maintain these particular
tfbs combinations for high tissue - specific expression .
phylogenetic conservation also increases the likelihood that the performance
of the hs - crms is preserved across species in the process of
clinical translation .
we subsequently assessed the efficiency and specificity of these novel
hs - crms in liver .
we first expressed human clotting factor ix
( hfix ) from a chimeric promoter , composed of a potent liver - specific minimal
transthyretin promoter ( ttr ) in conjunction with the
different hs - crm ( figure 2a ) .
we
choose hydrodynamic delivery to screen the 14 different hepatocyte - specific
cis - regulatory modules ( hs - crms ) since it allows for a rapid and
reliable assessment of their in vivo performance , without having to
produce aav vectors for each of them .
only the best hs - crm was then
selected for a subsequent comprehensive characterization in an aav vector
context ( see below ) .
semi - high throughput hydrodynamic transfection of these
expression plasmids into c57bl/6 mice revealed that the large majority
( i.e. , 85% ) of the de novo designed hs - crms
resulted in a significant increase in hfix expression ( figure 3a ) . in particular , the most potent
hs - crm8 resulted in a robust improvement in hfix expression ,
yielding up to 2.5 g / ml .
we subsequently validated the
hs - crm8 element in aav9-based vectors in which the
hfix gene was driven from either a moderately active
( palm ) ( figure 3b ) or potent
liver promoter ( ttr ) ( figure
3c ) .
the de novo designed hs - crm8 element in
the aav vector resulted in a robust and sustained 100-fold increase in hfix
expression from the moderately active palm promoter ( p
< 0.0001 ) ( 5 10 vg , figure 3b ) .
moreover , this hs - crm8
element significantly improved hfix expression seven- to tenfold ( p
< 0.00005 ) , even when a potent liver - specific promoter was used like
ttr ( figure 3c ) , consistent
with the increased hfix expression after hydrodynamic transfection
( figure 3a ) .
this resulted in
stable therapeutic hfix expression levels of about 100% , even at relatively
low vector doses ( 5 10 vg ) .
the
hs - crm8 element increased transcription from both ttr
and palm promoters ( supplementary figure s2a , b ) .
the
increased hfix antigen levels correlated strongly with the fix mrna
levels ( supplementary figure s2c ) .
there was no significant
difference in vector copy number in mice injected with the vectors with or
without the hs - crm8 element ( supplementary figure s2d ) .
the potency of the hs - crm8 element was subsequently evaluated
through assessment of phenotypic correction in hemophilic fix - deficient mice
following a tail - clip injury .
the treated mice survived the injury in
contrast to naive controls , consistent with a significant dose - dependent
reduction in blood loss ( naive mice : 1,167 215
l ; 5 10 vg / mouse :
596 92 l ( p < 0.0005 ) ;
2 10 vg / mouse :
495 82 l ( p < 0.00005 ) .
hfix
mrna expression was restricted to the liver ( figure 3d , e ) , even
upon administration of extremely high vector doses
( 3 10 vg ) , despite the detection of
viral genomes in non - hepatic tissues ( i.e. , heart , spleen , muscle ,
etc . )
( data not shown ) . to further enhance hfix expression from the hs - crm8-ttr - fix
construct , we subsequently replaced hfix intron a with an mvm
intron upstream of the hfix transgene ( figure 2c ) or its codon - optimized equivalent
( hfixco ) ( figure 2d ) .
each of these
changes led to a significant increase in hfix expression ( figure 4a , b ) .
this optimized hs - crm8-ttr - mvm - fixco
construct was then packaged into scaav for subsequent validation in a
non - human primate model .
the scaav9-hs - crm8-ttr - mvm - hfixco vector was injected into two cynomolgus
macaques ( oug6 and obev7 ) at a dose of
9 10 vg / kg .
there was a steady
increase in the circulating hfix protein in both animals that reached a peak
by week 4 , corresponding to 1,718 ng / ml and 885 ng / ml for oug6
and obev7 , respectively ( figure 5a ) .
these levels correspond to therapeutic fix levels in the 2035% range
of normal fix levels .
after a few weeks , the hfix transgene expression began
to decline ( figure 5a ) , which could be
ascribed to the induction of anti - hfix antibodies ( figure 5c , d ) , often
seen in non - human primates following aav liver gene transfer ,
despite the use of liver - specific promoters .
the aav9 vector administration
also triggered anti - aav9 neutralizing antibodies ( figure 5b ) .
both animals had consistent coagulation
parameters despite the occurrence of hfix inhibitory antibodies
( supplementary figure s3 ) .
this indicates that the anti - hfix
antibodies did not cross - react with the macaque fix .
the animals did not
develop any adverse events or toxicity ( supplementary figures s4 and
s5 ) .
human fix mrna expression was restricted to the liver
whereas only background levels were apparent in any other organ or tissue
tested ( figure 6a ) .
this confirms the
liver - specificity of the synthetic in silico designed
hs - crm8-ttr promoter , consistent with the mouse data
( figure 3d , e ) .
the aav9 vector resulted predominately in liver
transduction though significant gene transfer was also apparent in heart ,
lungs , spleen , kidney and brain ( figure
6b ) .
one of our animals ( oug6 ) was euthanized due to unexpected complications
unrelated to the gene therapy , which occurred after a surgical procedure to
obtain a liver biopsy .
we subsequently tried to eliminate the anti - hfix
inhibitors in the remaining animal ( obev7 ) following , transient
immunosuppression with cyclosporin ( csa ) and rituximab ( rtx ) .
this led to a
modest increase in circulating fix levels ( figure
5a ) , consistent with a decrease in anti - hfix antibodies
( figure 5c , d ) .
this was consistent with the profound decrease in
cd20 b cells ( figure 5e ) ,
whereas the percentage of cd3 t cells remained unaffected
( figure 5f ) .
unfortunately , only
partial and transient immunosuppression could be achieved , since the animal
refused to ingest csa after the initial treatment .
transient
immunosuppression also led to transient and partial decline in anti - aav9
neutralizing antibodies ( figure 5b ) .
in this study , we have validated a computational approach to improve the
performance of current gene therapy vectors .
our data provide evidence that
evolutionary conserved clusters of tfbs motifs corresponding to
hepatocyte - specific crms could be identified in silico that
are strongly associated with robust liver - specific expression in vivo .
we discovered that the selected hs - crms identified contain a
molecular signature composed of tfbs clusters that
represent a hallmark of highly expressed genes in the liver .
we demonstrated
that these hs - crms resulted in a substantial increase in transgene
expression in the liver after gene therapy , while maintaining a high degree of
tissue - selectivity .
in particular , a significant 10- to 100-fold increase in
gene expression could be obtained depending on the type of hs - crm and
promoter used .
we subsequently validated the potential of
these hepatocyte - specific hs - crms in a clinically relevant therapeutic
setting and demonstrated robust and sustained supra - physiologic expression
levels of coagulation fix following hepatic transduction with aav vectors .
the
use of these potent hs - crms therefore allows for the use of lower and
thus safer vector doses in gene therapy clinical trials , that may in turn reduce
the risk of developing t - cell mediated immune responses against the vector
antigens displayed on the transduced target cells .
in silico vector design may therefore pave the way towards a more
rational and comprehensive strategy for vector optimization with broad
implications to improve the efficacy and safety of gene therapy
indeed , we have
confirmed the superiority of the hs - crm8 expression module for
liver - directed gene therapy using other transgenes ( i.e. , factor viii ,
phenylalanine hydroxylase ; ref .
15 and data
not shown ) another advantage of these crms is their small size so that
they can be readily accommodated into viral vectors in conjunction with
relatively large therapeutic transgenes . the use of hyper - active fix transgenes
can further enhance vector performance .
we subsequently validated the efficacy and safety of the aav - fix vectors
containing the most potent hs - crm8 expression module in a non - human
primate model .
systemic vector administration resulted in robust therapeutic fix
levels in the 2035% range at clinically relevant vector doses
( 9 10
the performance of our vector compared
favorably with that of one of the most potent aav vectors that was used in a
clinical trial in patients suffering from severe hemophila b. subjects injected with
6 10
vg / kg , comparable to the dose used
in our primate study , resulted in ~13% fix . in those subjects injected
with higher vectors doses ( i.e. ,
2 10
previously , we had
assessed the performance of the same clinical trial vector in cynomolgus
macaques .
when calibrating
the vector doses , it would appear that our current vector design is about
tenfold more efficient .
since a human fix transgene was employed , it is known
that this could potentially evoke neutralizing and non - neutralizing anti - hfix
antibodies in non - human primate models .
this is consistent with our observations that anti - hfix
antibodies were detected in the recipients which accounted for the subsequent
decline in fix expression .
this provided us with an opportunity to test whether
we could restore fix by transient immunosuppression using rituximab and csa .
fix
could be detected after immune - suppression , even up to nearly 2 years after gene
transfer .
this suggests that the fix - transduced hepatocytes persisted long - term
and that the short - term fix expression was primarily due to a humoral immune
response .
our results caution that rituximab by itself may not suffice to treat
patients that develop inhibitory antibodies after gene therapy and that more
complex combination therapies may be required to achieve optimal immune
suppression . in this study , we choose to employ aav9 instead of aav8 .
previously , gao et
al . evaluated the relationship of phylogenetic relatedness of distinct
aav serotypes and serologic activity .
polyclonal antisera generated against aav2 cross - reacted
to a lesser extent with aav9 than with aav8 .
conversely , polyclonal antisera
generated against aav9 cross - reacted to a lesser extent with aav2 than when the
polyclonal antisera were generated against aav8 .
these experiments suggest that
it may be desirable to select aav9 over aav8 to overcome neutralization by
pre - existing anti - aav2 antibodies in human subjects , which represents an
important bottleneck in clinical trials .
moreover , we choose to explore aav9
since the bio - distribution had not systematically been analyzed in non - human
primates .
finally , we had previously conducted a side - by - side comparison between
aav8 and aav9 and found that aav9 transduced the liver just as efficiently as
aav8 in mice .
the advantage of this particular computational approach used to identify the
tissue - specific crms compared to other in silico approaches
was discussed previously .
one
particularly distinctive feature is that it identifies not only the
tfbs that are over - represented in liver - specific crms but
also allows for the identification of frequently co - occurring tfbs .
consequently , this in silico analysis is more comprehensive and allows
for the identification of tfbs elements that tend to cluster together
in crms of genes that are highly expressed in a given target tissue
( in casu liver ) .
it therefore takes into account the actual
context - dependent tfbs interactions from a broad genome - wide
perspective , instead of just relying on the over - representation of a single tfbs
element .
our previous
studies had shown that the present computational approach was more reliable ,
compared to other data - mining strategies that typically rely solely on the
over - representation of a given tfbs , regardless of its context .
the current
in silico strategy that defines the hs - crms has been further
improved by taking into account cross - species evolutionary conservation of the
tfbs clusters .
this approach is more comprehensive and increases
the likelihood that the superior performance of the hs - crms is
maintained in patients undergoing gene therapy , consistent with the robust fix
levels obtained in the non - human primate model .
in addition , this study goes
beyond the initial description and validation of the algorithm and establishes a bona fide
causal relationship between the presence of these evolutionary conserved
tfbs clusters and high liver - specific expression .
in contrast , the
previously published bioinformatics approach was applied in a different context
and merely established correlations between tfbs clusters and differentially
expressed genes following a given physiologic stimulus . however , that study did
not provide direct proof of the impact of such tfbs clusters on gene expression
levels .
this study addresses this caveat by direct in vivo gene
transfer , hereby providing experimental in vivo validation of the
bioinformatics algorithm .
though the majority of hs - crms resulted in a
significant increase in gene expression , a few elements had only limited impact .
this suggests that other unknown factors may influence gene expression , such as
the spacing between a given hs - crm and the promoter / tata box
or epigenetic factors and chromatin remodeling .
this study sets the stage to
further refine the in silico analysis to improve gene therapy vectors .
moreover , the most potent hs - crm8 could be used in conjunction with
other liver - specific promoters , beyond those characterized in this study .
this study has broad implications for the design of better gene therapy vectors
that target different diseases . the same principle could be applied to other
vectors or to other organs ,
tissues or promoters , which we have validating experimentally in heart and
skeletal muscle ( rincon my , sarcar s , danso - abeam d , keyaerts m , matrai j ,
samara - kuko e , et al . unpublished data ) .
this may ultimately impact not
only on the clinical translation of gene therapy but also on fundamental
biological and transgenic studies that rely on the use of robust tissue - specific
gene expression .
. the accession
numbers of the genes from which the hepatocyte - specific crms
( i.e. , hs - crm1 to 14 ) are derived are listed in table 1 .
identification of hepatocyte - specific crms . to identify the
hepatocyte - specific ( hs)-crms ( figure
1 and table 1 and
supplementary figure s1 and table s1 )
, we used a step - wise in
silico strategy : ( i ) we first identified liver - specific genes that are
highly expressed in the liver based on statistical analysis of micro - array
expression data of normal human tissues ; ( ii ) we then
extracted the corresponding promoter sequences up to 800 bp upstream of
the transcriptional start site ( tss ) from public databases ( ncbi36/hg18
genome assembly ) ; ( iii ) using the transfac database we subsequently mapped
tfbs elements to these promoters .
we then identified the tissue - specific crm
using a multidimensional scaling ( mds)/ differential distance matrix ( ddm )
approach , as described ; ( iv )
finally , the cross - species conservation was taken into consideration to identify
highly conserved hs - crms .
the different hs - crms
( table 1 and supplementary table
s1 ) were synthesized by conventional oligonucleotide synthesis and
cloned upstream of the liver - specific minimal transthyretin ( ttr )
( figure 2a ) or paralemmin
( palm ) promoters ( figure 2b ) ,
driving the expression of the factor ix ( fix )
mini - gene . alternatively ,
the ttr promoter coupled to the hs - crm8 element was used to
drive wild - type fix ( figure 2c ) or
its codon - optimized counterpart ( figure 2d )
in the context of a self - complementary ( sc ) aav backbone .
these vectors also contained a minute
virus of mice ( mvm ) mini - intron .
vector titers were determined by quantitative ( q )
pcr ( see supplementary methods ) .
wild - type or hemophilic mice were transfected hydrodynamically with
plasmids or injected intravenously with purified aav9 vectors .
hfix antigen
levels were assayed in citrated mouse plasma using a hfix - specific enzyme - linked
immunosorbent assay ( elisa ) .
the scaav9-hs - crm8-ttr - mvm - hfixco vector ( figure 2d )
( 9 10 vg / kg ) was intravenously injected
in cynomolgus macaques .
animals were subjected to transient immune suppression
by cyclosporine a ( csa ) and rituximab treatment . immunological analysis ,
hfix antigen , anti - aav9
capsid igg , anti - hfix igg and bethesda assays are described in supplementary
methods .
gene expression levels were evaluated by qrt - pcr and normalized
to mrna levels of the endogenous monkey gapdh gene .
transduction
efficiency was determined by qpcr and transgene copy numbers were determined as
described previously .
nucleotide sequence of the hs - crm elements ( hs - crm1 to hs - crm14 ) .
hepatic hfix mrna levels and aav copy numbers following
intravenous injection of aav9-fix vectors in c57/bl6 mice ( n = 3 ) .
safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy
in non - human primates : analysis of coagulation parameters .
safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy
in non - human primates : analysis of hematological parameters .
. safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy
in non - human primates : blood chemistry .
evolutionary conserved hs - crms enriched in
tfbs associated with high liver - specific expression .
methods .
nucleotide sequence of the hs - crm elements ( hs - crm1 to hs - crm14 ) .
hepatic hfix mrna levels and aav copy numbers following intravenous injection
of aav9-fix vectors in c57/bl6 mice ( n = 3 ) . click here for additional data file .
safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy in non - human
primates : analysis of coagulation parameters .
safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy in non - human
primates : analysis of hematological parameters .
safety analysis of scaav9-hs - crm8-ttr - mvm - hfixco gene therapy in non - human
primates : blood chemistry .
evolutionary conserved hs - crms enriched in tfbs associated
with high liver - specific expression . | the robustness and safety of liver - directed gene therapy can be substantially
improved by enhancing expression of the therapeutic transgene in the liver . to
achieve this
, we developed a new approach of rational in silico vector
design .
this approach relies on a genome - wide bio - informatics strategy to
identify cis - acting regulatory modules ( crms ) containing
evolutionary conserved clusters of transcription factor binding site motifs that
determine high tissue - specific gene expression .
incorporation of these
crms into adeno - associated viral ( aav ) and non - viral vectors
enhanced gene expression in mice liver 10 to 100-fold , depending on the promoter
used . furthermore , these crms resulted in robust and sustained
liver - specific expression of coagulation factor ix ( fix ) , validating their
immediate therapeutic and translational relevance .
subsequent translational
studies indicated that therapeutic fix expression levels could be attained
reaching 2035% of normal levels after aav - based liver - directed gene
therapy in cynomolgus macaques .
this study underscores the potential of rational
vector design using computational approaches to improve their robustness and
therefore allows for the use of lower and thus safer vector doses for gene
therapy , while maximizing therapeutic efficacy . |
overweight and obesity in children are increasing in prevalence in both developing and developed countries ( 1 ) . abdominal obesity and insulin resistance
mets is a combination of disorders that increase the risk of cardiovascular disease and diabetes ( 2 ) .
in contrast to adults ; conflicting results were found regarding the relation between obesity and cardiac functions in children .
early recognition of premature atherosclerosis is important as subclinical atherosclerosis precedes the clinical manifestations of cardiovascular disease by many years . in patients with mets , carotid intima - media thickness ( imt )
left ventricular hypertrophy , left ventricular diastolic dysfunction , and myocardial dysfunction were reported in mets patients ( 3 ) .
previous studies found depressed ventricular functions in mets patients compared to normal subjects ( 4 ) .
the relationship of eatt with left ventricular mass index and myocardial performance index in obese adolescents with mets had not been documented ( 5 ) .
eatt , deriving from the same embryological origin as mesenteric and omental fat , is deposited around the heart and coronary vessels .
eatt has various endocrine and inflammatory functions and produce mediators which can cause endothelial dysfunction .
eatt is related to insulin resistance and dyslipidemia and leading to a higher risk of mets and cardiovascular disease ( 6 ) .
we aimed to evaluate eatt and carotid imt of obese children and to study the relationship of eatt with carotid imt and other echocardiographic findings as well as clinical parameters of the metabolic syndrome in obese children .
this study was conducted on 60 obese children and adolescent ( simple obesity ) their ages ranged from ( 8 - 16 years ) enrolled from pediatric outpatient clinic , al hada and taif military hospitals , saudia arabia between february 2013 to february 2014 . according to presence or absence of mets ,
they were divided into two groups , 22 ( 36.6% ) were found to show the criteria for mets diagnosis ( mets group ) and 38 ( 63.4% ) constituted non- mets group .
1 ) smoking ; 2 ) any chronic medication ; 3 ) family history of premature vascular disease ; 4 ) any endocrinal disorder ; 5 ) patients with underlying etiology ( secondary obesity ) .
1 ) history taking ; 2 ) clinical examination and anthropometry : all anthropometric measurements were taken ; body height and weight were measured in light clothes by a portable stadiometer .
body mass index was calculated as weight divided by the square of the height ( kg / m ) . in children ,
body mass index > 95th percentile is considered obese , so we used body mass index > 30 as obese , and body mass index < 25 as non - obese control level ( 7 ) .
waist circumference was measured at the high point of the iliac crest to the nearest 0.1 cm at the end of normal expiration with a non - stretch measure tape ( 8) .
mets was defined according to the criteria of international diabetes federation which required three or more of the following parameters : abdominal obesity , hypertension , hyperglycemia , high triglyceride and low high density lipoprotein levels .
measurement of fasting glucose by glucose hexokinase method ( 8) . measurement of serum lipids included serum total cholesterol , low density lipoprotein cholesterol , high density lipoprotein cholesterol and triglyceride levels .
they were measured by taking venous blood samples in the morning after overnight fasting ( 10 - 12 hour ) .
measures were done using standard enzymatic methods by boehringer mannheim gmbh and a fully automatic analyzer ( 9 ) .
high - sensitivity c - reactive protein was assessed by immune nephelometric assay method using company reagents ( 10 ) .
fasting serum insulin was measured using the chemiluminescent technique ( dpc kit on immulite 1000 ) .
using 3- and 7-mhz transducers echocardiographic measures were performed with vivid3 expert ( norway).the procedure was done without the need for sedative medication . according to the recommendations of the american society of echocardiography ,
chamber dimensions and wall thicknesses were obtained by 2-dimensionally guided m mode ( 12 ) .
systolic functions of the left ventricle ( ejection fraction and fractional shortening ) were evaluated by measuring left ventricular end - systolic and end - diastolic dimensions , interventricular septum systolic and diastolic thicknesses , and left ventricular posterior wall systolic and diastolic thicknesses . left ventricular mass and relative posterior wall thickness was calculated according to the standard formulae ( 13 ) .
diastolic functions of the left ventricle were evaluated by measuring peak early diastolic filling velocity ( e ) , peak late diastolic filling velocity ( a ) , e / a ratio , and deceleration time ( 14 ) .
epicardial adipose tissue was measured as the echo - free space over the pericardial layers using 2-dimensional echocardiography . whereas the epicardial adipose tissue thickness was measured on the free wall of the right ventricle , perpendicular to the wall from parasternal long and short axis views at end - diastole .
the measurement was done for 3 cardiac cycles ( 15 ) . using a 7-mhz probe ,
carotid imt was measured from the common carotid artery at a point 5 mm proximal to its bifurcation ( 16 ) . using a 3-mhz transducer ,
early ( e ) and atrial ( a ) transmitral maximal flow velocities were measured by pulsed - wave doppler . the ratio e / a was calculated .
early ( e ) and late ( a ) diastolic peak velocities were measured and we calculated the ratio of early and late diastolic annular velocities . cardiac time intervals - isovolumic contraction time , isovolumic relaxation time , and systolic ejection time were measured .
tissue doppler derived by myocardial performance index was calculated according to the formulae : ( isovolumic contraction time + isovolemic relaxation time ) /systolic ejection time .
1 ) smoking ; 2 ) any chronic medication ; 3 ) family history of premature vascular disease ; 4 ) any endocrinal disorder ; 5 ) patients with underlying etiology ( secondary obesity ) .
1 ) history taking ; 2 ) clinical examination and anthropometry : all anthropometric measurements were taken ; body height and weight were measured in light clothes by a portable stadiometer .
body mass index was calculated as weight divided by the square of the height ( kg / m ) . in children ,
body mass index > 95th percentile is considered obese , so we used body mass index > 30 as obese , and body mass index < 25 as non - obese control level ( 7 ) .
waist circumference was measured at the high point of the iliac crest to the nearest 0.1 cm at the end of normal expiration with a non - stretch measure tape ( 8) .
mets was defined according to the criteria of international diabetes federation which required three or more of the following parameters : abdominal obesity , hypertension , hyperglycemia , high triglyceride and low high density lipoprotein levels .
measurement of fasting glucose by glucose hexokinase method ( 8) . measurement of serum lipids included serum total cholesterol , low density lipoprotein cholesterol , high density lipoprotein cholesterol and triglyceride levels .
they were measured by taking venous blood samples in the morning after overnight fasting ( 10 - 12 hour ) .
measures were done using standard enzymatic methods by boehringer mannheim gmbh and a fully automatic analyzer ( 9 ) .
high - sensitivity c - reactive protein was assessed by immune nephelometric assay method using company reagents ( 10 ) .
fasting serum insulin was measured using the chemiluminescent technique ( dpc kit on immulite 1000 ) .
using 3- and 7-mhz transducers echocardiographic measures were performed with vivid3 expert ( norway).the procedure was done without the need for sedative medication . according to the recommendations of the american society of echocardiography , chamber dimensions and wall thicknesses were obtained by 2-dimensionally guided m mode ( 12 ) .
systolic functions of the left ventricle ( ejection fraction and fractional shortening ) were evaluated by measuring left ventricular end - systolic and end - diastolic dimensions , interventricular septum systolic and diastolic thicknesses , and left ventricular posterior wall systolic and diastolic thicknesses . left ventricular mass and relative posterior wall thickness was calculated according to the standard formulae ( 13 ) .
diastolic functions of the left ventricle were evaluated by measuring peak early diastolic filling velocity ( e ) , peak late diastolic filling velocity ( a ) , e / a ratio , and deceleration time ( 14 ) .
epicardial adipose tissue was measured as the echo - free space over the pericardial layers using 2-dimensional echocardiography . whereas the epicardial adipose tissue thickness was measured on the free wall of the right ventricle , perpendicular to the wall from parasternal long and short axis views at end - diastole .
the measurement was done for 3 cardiac cycles ( 15 ) . using a 7-mhz probe ,
carotid imt was measured from the common carotid artery at a point 5 mm proximal to its bifurcation ( 16 ) . using a 3-mhz transducer ,
early ( e ) and atrial ( a ) transmitral maximal flow velocities were measured by pulsed - wave doppler .
early ( e ) and late ( a ) diastolic peak velocities were measured and we calculated the ratio of early and late diastolic annular velocities . cardiac time intervals - isovolumic contraction time , isovolumic relaxation time , and systolic ejection time were measured .
tissue doppler derived by myocardial performance index was calculated according to the formulae : ( isovolumic contraction time + isovolemic relaxation time ) /systolic ejection time .
p3 , test significance between obese group with mets and obese group without mets . to the presence or absence of mets ,
obese adolescents were divided into two groups , 22 ( 36.6% ) were found to have the diagnosis of mets .
systolic and diastolic blood pressure values were significantly higher in obese group with mets than the non - mets and the control groups .
total cholesterol , triglyceride , low density lipoprotein cholesterol , fasting glucose , insulin levels , and homeostasis model assessment index of insulin resistance were significantly higher in mets obese group than the non- mets obese and the control groups , whereas high density lipoprotein cholesterol levels were significantly lower in the mets obese group than the non- mets and control groups .
blood pressure values , body mass index , body mass index - standard deviation score and waist circumference were significantly higher in the non - mets obese group than the control group .
also , total cholesterol , triglyceride , low density lipoprotein cholesterol , fasting glucose and insulin levels were significantly higher in the non - mets obese group than the control group .
high density lipoprotein cholesterol levels were significantly lower in the non - mets obese group than the control group .
homeostasis model assessment index of insulin resistance values were significantly lower in the control group than both non - mets and mets obese groups .
table 2 shows measurements of echocardiography in obese ( mets - non - mets ) and control groups .
the mets obese group had significantly higher eatt , carotid imt , left ventricular mass index and myocardial performance index values as compared to the both non - mets and control groups .
a statistically significant difference was found between the control and mets groups regarding the following parameters : acceleration time , deceleration time , diastolic early wave peak velocity ( e ) , diastolic late wave peak velocity ( a ) , ( e/a).also , isovolumic relaxation time , isovolumic contraction values and average myocardial performance index showed statistically significant difference between the control and mets groups .
the average isovolumic contraction values , isovolumic relaxation time , average myocardial performance index , and average deceleration time were significantly higher in mets obese group than in non - mets obese group ( p < 0.05).a significant correlation was found between eatt and carotid imt among obese patients with mets .
table 3 shows the correlation coefficient between ( eatt ) and clinical , laboratory and echocardiographic parameters in the obese mets patients and non - mets .
eatt was positively correlated with body mass index - standard deviation score , waist circumference , fasting glucose , fasting insulin , insulin resistance , triglyceride levels , left ventricular thickness , left ventricular mass index and myocardial performance index .
table 4 shows the multivariate regression model analysis , demonstrated that the only independent predictor of carotid imt among obese patients with mets is eatt ( coefficient = 0.62 , p = 0.0006 ) .
whit in red dashed shape is identified as the echo free space between the outer wall of the myocardium and the visceral layer of the pericardium in the parasternal long axis view .
figure 2 shows measurements of the intima - media thickness in the common carotid artery .
the gray scale ultrasound image shows the cursors placed perpendicular to the long axis of the common carotid artery to include only the intima and media in the thickness measurements .
with the rise in childhood obesity , the related cardiovascular disease in children is becoming more prevalent .
childhood obesity predisposes to an increased risk of morbidity and mortality related to cardiovascular diseases in later life .
it has been demonstrated that children may exhibit early signs of cardiovascular dysfunction as a result of their excess adiposity ( 18 ) .
eatt was significantly correlated with anthropometric measures as body mass index and waist circumference in obese adolescents with mets .
we found a high correlation between eatt and body mass index - standard deviation score .
the body mass index was used as a tool to identify individuals at risk of future cardiovascular disease and diabetes .
body mass reflects fat mass and lean body mass , but it does not discriminate the distribution of fat ( 22 ) . as a risk factor for cardiovascular and metabolic diseases ,
it was suggested that abdominal adiposity is more important than general adiposity ( 23 ) .
abdominal fat contributes to the risk of these diseases but the mechanisms are not fully understood . among metabolic elements of abdominal fat ,
is the visceral adipose tissue which probably plays a vital role in this process ( 24 ) . in our study ,
this is in agreement with other authors ( 3 , 19 , 20 ) who found that waist circumference is a good predictor of visceral fat mass and the strongest determinant of eatt.our data suggests that eatt can be used as indirect estimate of visceral fat . in our study
, there was a significant correlation between eatt and the following laboratory measures ; fasting insulin , insulin resistance , triglyceride , and high sensitivity c - reactive protein .
2013 ( 25 ) . in the present study , there is positive correlation between eatt and homeostasis model assessment index of insulin resistance .
in fact , this is an expected finding in the patient group with mets as epicardial adipose tissue is a component of abdominal fat and its increased amount in mets patients contributes to increase insulin resistance .
there are also some studies that have established a significant correlation between eatt and insulin resistance supporting the present study ( 26 , 27 ) .
eatt plays a role in the development of inflammation , which triggers the pathogenetic mechanisms of diabetes mellitus , cardiovascular disease , and mets . in the previous study by mazurek et al .
the expression of inflammatory mediators in the epicardial adipose tissue was greater than that released from subcutaneous adipose tissue in the patients with documented coronary artery disease ( 28 ) .
in the present study , the level of high sensitivity c - reactive protein , the indicator of a low - grade chronic inflammation , was higher compared to the control group . additionally , a positive correlation was established between eatt , which was higher in the mets patients , and high sensitivity c - reactive protein which is considered to be another important finding . in other words , visceral adipose tissue , such as eatt ,
may directly contribute to the development of mets due to its local and systemic pro - inflammatory effect . in our study ; eatt and carotid imt measurements were increased in obese adolescents with mets .
also , they have greater left ventricular mass index and myocardial performance index measurements in comparison to control and non - mets obese groups . in mets
obese patients , there were significant correlations between eatt and hypertension , echocardiographic parameters such as left ventricular thickness , left ventricular mass index , myocardial performance index and carotid imt .
, magnetic resonance imaging has been accepted as the gold standard for measuring eatt . however , its coast and radiation risk limit its use as a screening tool .
their study suggests that echocardiographic eatt is a simple and an easy measure in clinical practice ( 15 ) . in our study
, we showed that echocardiographic measurements of eatt significantly correlated with left ventricular mass index and systolic and diastolic function in obese adolescent with mets .
a recent meta - analysis demonstrated significantly higher echocardiographic eat thickness in patients with ms ( 29 ) .eat thickness is also related to obstructive coronary artery disease , cardiac ischemia , adverse cardiac events and subclinical coronary artery disease ( 30 , 31).other studies have also shown associations of eatt with diastolic function abnormalities and also left ventricular structural and functional changes ( 32 , 33 ) .
it can be used to evaluate both systolic and diastolic functions as it correlates well with invasive measurements ( 34 ) . in our study population ,
the correlates of eatt were body mass index , waist circumference and hip circumference , fasting glucose and insulin levels , homeostasis model assessment index of insulin resistance , high density lipoprotein , triglyceride levels , high sensitivity c - reactive protein , left ventricular thickness , left ventricular mass index , myocardial performance index and carotid imt.in multivariate logistic regression analysis , eatt was the only independent variable that was significantly associated with carotid imt .
eatt has been suggested as a new cardiometabolic risk factor as it reflects visceral fat tissue and obesity .
carotid imt is a noninvasive , feasible , reliable and inexpensive marker of subclinical atherosclerosis .
carotid imt was related to cardiovascular risk factors and could predict the possibility of future cardio - cerebrovascular disease ( 35 ) . in a recent study , carotid imt was significantly increased in the obese group compared with the lean group .
in addition , a statistically significant correlation was found between eatt and carotid imt measurements .
these findings suggested that eatt is a more reliable marker of development of subclinical atherosclerosis than the other parameters in obese patients ( 20 ) .
eatt leads to endothelial dysfunction , a key event in the development of atherosclerosis predating clinically obvious vascular disease ( 36 ) .
this may contribute to the initiation , progression and acceleration of coronary artery disease in patients with mets .
for this reason , eatt may be used as additional and easy diagnostic tools for the presence of endothelial dysfunction and the necessity for follow - up for possible future overt coronary artery disease . in a recent study , iacobellis et al . demonstrated that eatt was found to be a good predictor of steatosis of non - cardiac organs such as the liver ( 37 ) .
eatt is reported to be a marker for the presence and severity of coronary artery disease in recent studies ( 38 ) .
our results support these studies and suggest the possible association of eatt with subclinical atherosclerosis . in conclusion , we have observed that eatt , carotid imt , left ventricular mass index and myocardial performance index values were significantly higher in obese adolescents especially with mets .
eatt had a close link with carotid imt and early cardiac dysfunction in obese adolescents with mets .
assessment of eatt in routine echocardiography might be a feasible and reliable method for the evaluation of obesity and its related cardiovascular risks during childhood . | background : epicardial fat has a role in cardiovascular diseases.objectives:to assess epicardial fat and its relation with carotid intima - media thickness ( imt ) in obese adolescents with metabolic syndrome ( mets).patients and methods : the study included 60 obese adolescents and 25 control subjects . according to the presence or absence of mets ,
obese subjects were divided into two subgroups .
we measured weight , height , calculated body mass index , waist circumference , hip circumference , systolic blood pressure , diastolic blood pressure and biochemical parameters ( fasting glucose , total cholesterol , triglycerides , high density lipoprotein cholesterol , and low density lipoprotein cholesterol , high sensitivity c - reactive protein , fasting insulin , a homeostasis model assessment index of insulin resistance . plus an echocardiographic examination with measurement of epicardial adipose tissue thickness ( eatt).results : left ventricular mass index measurements were significantly higher in mets group than both non - ms and control groups .
the mets and non - mets obese patients had significantly higher carotid imt in comparison to the control group .
carotid imt measurements were significantly higher in mets group had than both non - mets and control groups . also , eatt was significantly increased in patients with mets compared to control group . among mets obese
group , eatt was positively correlated with body mass index - standard deviation score , waist circumference , fasting glucose , fasting insulin , insulin resistance , triglyceride levels , left ventricular thickness , left ventricular mass index and myocardial performance index .
eatt was found to be the only predictor of carotid imt.conclusions:eatt is closely related to carotid imt and early cardiac dysfunction in obese adolescents with mets . |
phosphate - based cathartic agents can be bought over the counter ( otc ) as remedial for constipation .
it is widely prescribed for colon preparation before any form of intra - abdominal surgery or colonoscopy .
we report a 49-year - old female who had a history of end - stage renal failure secondary to iga glomerulonephritis . she has been on regular hemodialysis since 2001 .
she was found to have right renal cell carcinoma during a routine 5 yearly ultrasound surveillance .
she was referred to a urologist who arranged for an elective laparoscopic nephrectomy of her right kidney .
the calcium and phosphate levels taken 1 month before the surgery were normal at 2.75 mmol / l and 0.97 mmol / l , respectively , and she had a complaint of severe constipation from tumor - related hypercalcemia .
one day before the planned surgery , she volunteered information that she had purchased two sachets of otc generic oral sodium phosphate ( osp ) for her severe constipation .
calcium and phosphate levels were 1.08 mmol / l and 5.88 mmol / l , respectively .
the strong temporal relationship between the otc consumption and the acute metabolic abnormalities led nephrologists to believe that this was an unusual case of acute hypocalcemia and hyperphosphatemia secondary to osp .
the repeat calcium and phosphate levels 4 h after dialysis were 2.34 mmol / l and 1.84 mmol / l .
laxatives are solutions of certain ingredients introduced orally or rectally to induce laxation . the different categories of laxatives include hyperosmotic chemicals ( glycerin , sorbitol ) , lubricant ( mineral oil ) , salines ( sodium phosphate / sodium biphosphate ) , and stool softeners ( docusate potassium ) .
it is frequently given in favor of standard polyethylene glycol - based lavage solutions because of the smaller required volume , which results in better patient compliance and improved colonic cleansing .
in this case , according to the objective causality assessment by the naranjo probability scale , the causal association between osp and the adverse event was probable ( naranjo score = 6 ) .
the adverse drug reaction was evaluated for causality assessment using the world health organization - uppsala monitoring center ( who - umc ) criteria .
the assigned causality category with the who - umc criteria for this adverse drug reaction was likely . in january 2014 , the united states food and drug administration issued a warning that using more than one dose of otc sodium phosphate product in 24 h can cause rare but serious kidney injury , arrhythmias , or even death .
various types of metabolic derangement , i.e. , hyperphosphatemia , hypocalcemia , hypernatremia , hypokalemia , and metabolic acidosis have been associated with osp .
hyperphosphatemia is more likely to occur in patients with renal insufficiency , who have decreased excretion of phosphate .
the other at - risk groups are those who are elderly , those who had intestinal obstruction , decreased intestinal motility , or on concomitant medications that can reduce renal perfusion ( diuretics , angiotensin converting enzyme inhibitor , or angiotensin receptor blocker ) . patients with history of congestive heart failure should also use osp with cautioned as the resulting hypernatremia could lead to acute fluid overload . even in patients with normal renal function , significant changes in the serum calcium and phosphate levels occurred after osp . in some occasions , this can lead to acute kidney injury . the sole causative factor could be explained by the case of biopsy - proven phosphate - induced nephrocalcinosis reported by desmeules et al .
, where the nature and localization of the lesions strongly suggest that phosphate ingestion led to obstructive calcium - phosphate crystalluria , followed by intratubular nephrocalcinosis .
phosphate - based cathartic agents should only be used in patients with normal renal function .
it may be prudent to evaluate serum calcium and phosphate levels before and after administration of sodium phosphate .
otc osp abuse should be suspected when a patient with renal impairment and constipation presents with sudden hypocalcemia and hyperphosphatemia .
| oral sodium phosphate ( osp ) , an effective bowel purgative , is available over the counter ( otc ) and requires a substantially lower volume than polyethylene glycol - based preparative agents . rarely , osp consumption has been associated with acute hypocalcemia and hyperphosphatemia .
we describe a case of chronic kidney disease patient developing symptomatic hypocalcemia following otc osp . |
the goals of surgical treatment for thoracolumbar burst fractures are to restore vertebral column stability and to decompress neural elements4 ) . due to several problems associated with extensive long - level instrumentation ,
posterior short - segment instrumentation has been recently preferred for the treatment of thoracolumbar burst fractures in cases mild communition or load sharing score5 ) . because , a fracture with mild communition or load sharing score indicates adequate sharing of load through the injured vertebral body itself along with the implant to permit short segment instrumentation and fusion5 ) .
short - segment instrumentation ( that is , pedicle screw instrumentation at the fracture level and the levels above and below ) is being selected among warrant surgical interventions .
pedicle screw insertion makes it possible to maintain vertebral column stability until the desired alignment is achieved , and simultaneously restores canal dimensions and vertebral body height6 ) .
furthermore , it has been assumed to facilitate early painless mobilization by providing substantial stabilization of the fractured vertebra .
some authors have hypothesized that bone grafting may be unnecessary in combination with short - segment fixation for the treatment of thoracolumbar burst fractures8 ) .
moreover , recent reports have demonstrated the efficacy of vertebral augmentation procedures in terms of preventing implant failure in osteoporotic patients1 ) .
in addition , to minimize the negative consequences of traditional open surgery , such as , muscular denervation and atrophy , screw fixation via percuaneous technique has been proposed and applied .
we conducted this study to compare and evaluate the efficacies of bone cement augmentation and bone fusion procedure for the treatment of thoracolumbar burst fractures accompanying osteopenia .
this retrospective study included 70 patients ( 47 males and 23 females ) with thoracolumbar burst fractures with osteopenia treated at our institute from january 2005 to january 2008 .
study inclusion criteria limited enrollment to single level thoracolumbar burst fractures with ; 1 ) a neurologically intact status , despite severe canal encroachment , 2 ) patients with osteopenia ( -2.5 < mean t - score by bone mineral densitometry < -1.0 ) , 3 ) a loss of anterior body height exceeding 40% or more than 30 of sagittal angulation , and 4 ) intact bilateral pedicles , enabling screw insertion at the fractured vertebra .
the exclusion criteria were major neurological deficits that required open decompression due to severe canal compromise and an accompanying pedicle fracture preventing screw insertion at the fracture level .
the 70 patients were divided into three groups by operation technique : 1 ) group i ( n=26 ) : short segment fixation with posterolateral bone fusion , 2 ) group ii ( n=23 ) : bone cement augmented short segment fixation with posterolateral bone fusion , 3 ) group iii ( n=21 ) : bone cement augmented short segment percutaneous screw fixation without bone fusion ( fig .
clinical outcomes were assessed using a visual analogue scale ( vas ) and modified mac nab 's criteria .
the effects of the procedures were analyzed preoperatively , 10 days postoperatively , and at final follow - up .
radiological findings , including height restoration , kyphotic angle , and other systemic complications , were analyzed .
extent of vertebral body collapse was determined using vertebral heights at the point of maximal collapse on lateral radiographs or on midsagittal computed tomography scans .
kyphotic angle was measured by drawing a straight line from the superior endplate of the vertebra one level above the fractured body and a straight line from the inferior endplate of the vertebra one level below the fractured body .
the three groups were compared with respect to age , gender , mean t - score , kyphotic angle , preoperative degree of pain , and vertebral height .
preoperative group differences were evaluated using the student 's t - test for unpaired data , and intra - group changes associated with the procedure were evaluated using the student 's t - test for paired data .
group demographics , that is , age , gender , pain , vertebral height , mean t - score , mean follow - up period , and mean kyphotic angle were not significantly different .
significant pain relief was achieved in all three groups at last follow - up ( p<0.01 ) .
for group i , mean vas scores were ; 7.6 before surgery , 3.0 at 10 days after surgery , and 3.0 at last follow - up . for group ii ,
mean vas scores were ; 7.5 before surgery , 2.7 at 10 days after surgery , and 2.5 at last follow - up . for group iii , mean vas scores were ; 7.5 before surgery , 2.5 at 10 days after surgery , and 2.6 at last follow - up ( fig .
1 ) . most patients achieved an ' excellent ' or ' good ' result at final follow - up according to modified macnab 's criteria .
however , patients that underwent short segment screw fixation with bone fusion ( group i ) showed higher complication rates for screw loosening and implant failure after the procedure than patients in group ii or iii ( p=0.002 ) .
the inter - group pairwise comparisons of complications revealed no significant difference between group ii and iii ( p=0.279 ) ( table 2 ) .
screw loosening or implant failure was detected in 7 group i patients ( 26.9% ) , 2 group ii patients ( 8.6% ) , and 2 group iii patients ( 9.5% ) during the follow - up period ( fig .
i , one male patient developed symptomatic infectious spondylitis that needed revision surgery at six months after screw fixation .
no major complications related to bone cement , such as , pulmonary embolism or epidural leakage , were encountered in group ii or iii , although asymptomatic cement leakage occurred into the disc space or posterior spinal canal in some patients .
mean kyphotic angle and vertebral height increased significantly at last follow - up in all three groups ( p<0.01 ) , but group i showed more correction loss of kyphotic deformities and vertebral height loss than with group ii or iii ( table 3 ) .
minimizing the number of vertebral levels in screw fixation is another important goal of internal fixation .
traditional short segment fixation , which is , pedicle screw fixation one level cephalad and one level caudad a fracture , is used to save the motion segments by limiting the number of the fusion segments . despite the theoretical benefits of motion preservation , clinical studies on traditional short segment instrumentation
failure to restore anterior column support can lead to secondary kyphosis , depending on the residual load transfer capacity of the fractured vertebral body .
however , the trend toward preservation of motion segments using short segment instrumentation has continued to gain popularity , especially when anterior column support is not severely compromised5 ) . in the present study ,
the radiographic and clinical outcomes of neurologically intact patients with a thoracolumbar burst fracture that underwent short segment fixation at the level of the fracture with or without posterolateral fusion and a vertebral augmentation procedure were analyzed . although kyphotic angle , vertebral height restoration , and clinical outcome were not significantly different in the three groups ,
our results show that bone cement augmentation seems to be essential in terms of reducing screw loosening and implant failure rates .
the pedicle screw - based instrumentation system has been used widely for the treatment of thoracolumbar fractures , and posterior fixation with pedicle instrumentation not only allows satisfactory fracture reduction but also provides immediate mechanical stability and alignment while preventing additional damage to non - injured segments . with regard to posterior fixation , spinal fusion
however , short segment fixation with bone has also been associated with considerable rates of implant failure and correction loss2,4 ) .
some authors have recently reported the results for posterior screw fixation without bone fusion for the treatment of thoracolumbar burst fractures .
sanderson et al.6 ) , in a retrospective study , examined 24 patients who had undergone short - segment fixation without fusion , and concluded that routine posterior or posterolateral fusion is unnecessary . in 2006 , wang et al.8 ) , also reported satisfactory results for short - segment fixation without fusion in surgically - treated thoracolumbar burst fractures , and found that low back outcome scores were no different for patients treatment with or without bone fusion .
however , in the osteoporotic or osteopenic spine , the screw anchoring effect is reduced and the probability of hardware failure is high , and thus , the possibility of non - union is increased7 ) .
therefore , screw loosening or vertebral collapse may occur more frequently after screw fixation without bone fusion in osteoporotic or osteopenic spines .
however , insertion of pedicle screws at the fracture level results in a segmental construct that improves biomechanical stability by protecting the fractured body and by supporting the anterior column indirectly3 ) .
in addition , the insertion of bone cement to augment pedicle screws at the fracture level probably results in a segmental construct with improved biomechanical stability by providing protection to the fractured vertebral body and indirectly supporting the anterior column3 ) . furthermore , bone cement augmentation at fractured level itself and adjacent vertebrae can significantly reduce pedicle screw bending and increase initial stiffness in the flexion - extension plane .
wittenberg et al.9 ) reported that pmma can increase pull - out strength from 96% to 262% and transverse bending stiffness by up to 153%9 ) . in the present study
mean kyphotic angle and vertebral height increased significantly at last follow - up in all three groups .
however , improved vertebral height and kyphotic angle were maintained during the follow - up and lower complication rates were observed in bone cement augmented screw fixation groups ( group ii and iii ) . as a result , bone cement augmented screw fixation procedure including fractured level was helpful for prevent screw loosening and implant failure .
moreover , due to the advance in minimally invasive instrumentation , bone cement augmented , percutaneous screw fixation was a safe and effective management to reduce screw loosening implant failure for the thoracolumbar burst fractures without neurologic deficits .
all surgical procedures in this study were performed by a single surgeon to eliminate the effects of different surgeons on outcome variables . however , larger - scale , multicenter studies involving more surgeons and fracture types are required to validate our results .
bone cement augmentation procedure is an effective and reliable operative technique for reducing screw loosening and implant failure after short segment fixation for thoracolumbar burst fractures accompanying osteopenia . | objectivethe purpose of this study was to compare the results of three types of short segment screw fixation for thoracolumbar burst fracture accompanying osteopenia.methodsthe records of 70 patients who underwent short segment screw fixation for a thoracolumbar burst fracture accompanying osteopenia ( -2.5 < mean t score by bone mineral densitometry < -1.0 ) from january 2005 to january 2008 were reviewed .
patients were divided into three groups based on whether or not bone fusion and bone cement augmentation procedure 1 ) group i ( n=26 ) : short segment fixation with posterolateral bone fusion ; 2 ) group ii ( n=23 ) : bone cement augmented short segment fixation with posterolateral bone fusion ; 3 ) group iii ( n=21 ) : bone cement augmented , short segment percutaneous screw fixation without bone fusion .
clinical outcomes were assessed using a visual analogue scale and modified macnab 's criteria .
radiological findings , including kyphotic angle and vertebral height , and procedure - related complications , such as screw loosening or pull - out , were analyzed.resultsno significant difference in radiographic or clinical outcomes was noted between patients managed using the three different techniques at last follow up .
however , group i showed more correction loss of kyphotic deformities and vertebral height loss at final follow - up , and group i had higher screw loosening and implant failure rates than group ii or iii.conclusionbone cement augmented procedure can be an efficient and safe surgical techniques in terms of achieving better outcomes with minimal complications for thoracolumbar burst fracture accompanying osteopenia . |
the traditional approach to drug discovery involves de novo identification and validation of new molecular entities ( nme ) , which is a time - consuming and costly process . despite huge investment in drug discovery and development and explosive advancement in biological / informational technologies during past decades ,
for example , while the total r&d expenditure for the drug discovery worldwide increased 10 times from 1975 ( us $ 4 billion ) to 2009 ( $ 40 billion ) , the number of nmes approved has remained largely flat ( 26 new drugs approved in 1976 and 27 new drugs approved in 2013 ) 1 , 2 .
it has been estimated that the average drug development time from discovery to market launch in us and eu countries was 9.7 years during 1990s , but has increased to 13.9 years from 2000 onwards 3 .
those hurdles in discovering and developing new drugs call for alternative approaches including drug repositioning .
drug repositioning refers to the identification of new indications from existing drugs and the application of the newly identified drugs to the treatment of diseases other than the drug 's intended disease .
a well - known example of drug repositioning is the use of sildenafil ( viagra ) in erectile dysfunctions .
sildenafil is an inhibitor of cyclic guanosine monophosphate ( cgmp)-specific phosphodiesterase type 5 ( pde5 ) and was originally developed for the treatment of coronary artery disease by pfizer in 1980s .
the side effect of sildenafil , marked induction of penile erections , was serendipitously found during the phase i clinical trials for the patients with hypertension and angina pectoris 4 .
after sildenafil failed in phase ii clinical trials for the treatment of angina , it was redirected to the treatment of erectile dysfunctions .
sildenafil received a us - food and drug administration ( fda ) approval and entered the us market in 1998 , quickly becoming a blockbuster .
thalidomide was originally developed as a sedative by the german pharmaceutical company grnenthal in 1957 .
not long after the drug was introduced , it was found to cause serious birth defects .
more than 10,000 children in 46 countries were born with malformation of the limbs and other body extremities due to the use of thalidomide , and around half of them died within a few months after birth 5 , leading to its withdrawal from the market . in the ensuing decades , several research groups found that thalidomide possesses anticancer activity .
it was found to inhibit angiogenesis in animal models by robert d'amato and judah folkman 6 and was subsequently shown to have promising therapeutic effect on refractory multiple myeloma and metastatic prostate cancer 7 , 8 . in 2006 , thalidomide received us - fda approval for the treatment of multiple myeloma in combination with dexamethasone .
several success stories of drug repositioning brought global attention to the existing drug space for potential off - target effects that may be beneficial to certain diseases such as cancer . since existing drugs have already been used in humans , they have well - established dose regimen with favorable pharmacokinetics ( pk ) and pharmacodynamics ( pd ) properties as well as tolerable side effects , making old drugs useful sources of new anticancer drug discovery . in
early 2000s , we launched a new initiative to assemble a library of existing drugs , dubbed the johns hopkins drug library ( jhdl ) 9 .
jhdl has about 2,200 drugs that have been approved by us - fda or by its foreign counterparts and about 800 non - approved drug candidates that have entered various phases of human clinical trials .
we note that nih chemical genomics center ( ncgc ) recently built a collection of existing drugs called ncgc pharmaceutical collection ( npc ) which contains 2,400 small molecular entities that have been approved for clinical use in us ( fda ) , eu ( ema ) , japan ( nhi ) , and canada ( hc ) 10 , 11 .
these clinical drug collections have proven to be useful sources to find new indications of existing drugs .
the term ' activity - based drug repositioning ' we shall use in this review refers to the application of actual drugs for screening .
in contrast , ' in silico drug repositioning ' utilizes public databases and bioinformatics tools to systematically identify interaction networks between drugs and protein targets 12 .
this latter approach has become successful since a large amount of information on the structure of proteins and pharmacophores has been accumulated over the past few decades along with the advancement of bioinformatics and computational science .
most pharmaceutical companies have already adopted the in silico models for drug discovery from diverse chemical spaces . in silico drug repositioning
is a potentially powerful technology and has some advantages over the activity - based drug repositioning , including increased speed and reduced cost .
however , it also has some limitations since it requires high - resolution structural information of targets .
it also requires disease / phenotype information or gene expression profiles of drugs when a screen does not involve protein targets .
in contrast , activity - based drug repositioning can employ both protein target - based and cell / organism - based screens without requiring structural information of target proteins or database .
thus , activity - based and in silico drug repositioning represent two alternative and complementary approaches to new drug discovery ( table 1 ) . here
, we briefly summarize a few recent discoveries of new anti - angiogenic and anticancer activities of existing drugs through activity - based screening of the jhdl along with the subsequent mechanistic and translational follow - up studies .
itraconazole is a triazole antifungal drug developed in 1980s . like other azole family of antifungal drugs , it is effective in a variety of systemic fungal infections 13 .
it is known to inhibit cytochrome p450-dependent lanosterol 14--demethylation ( 14 dm ) in the ergosterol biosynthesis pathway in fungi 14 .
ergosterol is the main sterol in most yeasts and fungi , being responsible for their membrane integrity and function .
, itraconazole and related azole compounds cause the depletion of ergosterol and induce accumulation of 14- methylsterols that can impair membrane functions , thereby suppressing the fungal growth 14 , 16 .
although itraconazole is a well - tolerated drug , it has some side effects including hepatotoxicity ( rare but sometimes serious ) , cardiovascular toxicity and diarrhea ( when prepared with cyclodextrin ) 17 . anticancer activity of itraconazole was first reported by chong et al . in 2007 due to its newly discovered anti - angiogenic activity 18 . in this study
, the jhdl was screened for inhibitors of human umbilical vein endothelial cell ( huvec ) proliferation , a proxy for angiogenesis , and itraconazole was identified as one of the most potent hits . in follow - up studies , itraconazole , either alone or in combination with other anticancer drugs , showed strong anticancer activities in preclinical models including non - small cell lung cancer ( nsclc ) , medulloblastoma , and basal cell carcinoma 19 , 20 . prompted by such encouraging preclinical results , itraconazole has entered several phase ii clinical studies for the treatment of various types of cancer .
most recently , positive clinical results have been reported from advanced lung cancer and prostate cancer trials ( both at johns hopkins sidney kimmel comprehensive cancer center ) and from basal cell carcinoma trial ( at stanford university ) 21 - 23 .
itraconazole in combination with pemetrexed showed significant survival benefit in patients with progressive nonsquamous non - small - cell lung cancer compared to the control arm of pemetrexed alone 21 .
high dose ( 600 mg / day ) of itraconazole also showed modest anticancer activity in patients with metastatic castration - resistant prostate cancer ( crpc ) 22 . in the basal cell carcinoma trial , patients were received oral itraconazole 200 mg twice per day for 1 month or 100 mg twice per day for an average of 2.3 months . in this exploratory trial ,
overall , itraconazole was well - tolerated by the patients in all three aforementioned phase ii trials with a few common toxicities including fatigue , nausea and anorexia .
recent study about therapeutic drug monitoring ( tdm ) of itraconazole suggests that serum concentration of 5 g / ml ( 7 m ) is associated with 26% probability of adverse effect 24 .
classification and regression tree ( cart ) analysis suggests that serum itraconazole level of 17.1 g / ml ( 24.4 m ) is upper limit for tdm 24 .
considering that itraconazole 's ic50 values for angiogenesis range from sub - micromolar to single digit micromolar concentrations ( 0.5 ~ 3 m ) , it has a moderate therapeutic window .
however , occurrence of some rare but serious side effects such as hepatotoxicity and congestive heart failure should be monitored in the clinical settings , though it is interesting that the higher dose of itraconazole ( 600 mg daily ) did not cause those major side effects in prostate cancer patients as would have been anticipated 17 , 22 .
although itraconazole showed promising anticancer activity in several types of cancer , its precise anticancer mechanism has remained elusive . to date , two anticancer mechanisms of itraconazole have been proposed ; inhibition of angiogenesis and inhibition of hedgehog signaling pathway in certain cancer cells ( figure 1 ) .
26 showed that itraconazole inhibited cholesterol trafficking in human endothelial cells , leading to inhibition of mammalian target of rapamycin ( mtor ) and vascular endothelial growth factor receptor type 2 ( vegfr2 ) signaling pathways that are critical for endothelial cell proliferation and angiogenesis . in a separate study , kim et al . demonstrated that itraconazole inhibited hedgehog signaling pathway , thereby suppressing the growths of medulloblastoma and basal cell carcinoma 19
ongoing studies are being focused on identifying the molecular target of itraconazole in mammalian cells , which will further our understanding of the precise mode of action of itraconazole for its anticancer activity , facilitating its development as a new anticancer and anti - angiogenic drug .
nelfinavir is a competitive inhibitor of human immunodeficiency virus ( hiv ) aspartyl protease and is being used in combination with other antiretroviral drugs to treat patients with hiv infection 27 .
it received the us - fda approval in 1997 for an oral dose regimen of 750 mg three times daily .
it was later modified to a regimen of 1250 mg twice daily as recommended by us - fda .
the average peak plasma level of nelfinavir is around 8 m and the bioavailability is known to be increased when taken with food 29 .
nelfinavir is a well - tolerated drug with some common side effects such as insulin resistance , hyperglycemia and lipodystrophy . from early 2000s , researchers have found potential anticancer activity of nelfinavir .
it was reported to inhibit the growths of kaposi 's sarcoma 30 , multiple myeloma 31 , nsclc 32 , 33 , prostate cancer 34 , and breast cancer 35 , 36 .
nelfinavir exhibited a broad - spectrum anticancer activity in vivo , being efficacious in several preclinical cancer models .
a common side effect of nelfinavir was insulin resistance , which was later found to be through inhibition of phosphatidylinositol-3-kinase ( pi3k)/akt signaling pathway .
recently conducted a phase i clinical trial of nelfinavir and chemoradiation for locally advanced pancreatic cancer 37 . in this trial ,
nelfinavir showed potent radiosensitizing and antitumor activities without adding toxicity in patients with pancreatic cancer .
although nelfinavir is known to inhibit akt signaling pathway , it does not directly inhibit the kinase activity of akt .
gupta et al . showed that nelfinavir down - regulates akt phosphorylation by inhibiting 20s proteasome activity 38 .
hamel et al . also showed that nelfinavir inhibited chymotrypsin- and trypsin - like activities of the rat proteasome preparation in vitro
39 .
however , whether proteasome is the relevant target of nelfinavir for its anticancer and anti - akt activity has been debated since several conflicting results have been reported .
nelfinavir was shown to inhibit cyclin - dependent kinase 2 ( cdk2 ) activity by enhancing proteasome - dependent degradation of cdc25a phosphatase 40 .
in addition , known proteasome inhibitors including mg132 and bortezomib did not recapitulate effects of nelfinavir in breast cancer cells , but , instead , they rescued the akt inhibition by nelfinavir 36 . recently , srirangam et al .
36 demonstrated that nelfinavir is a novel inhibitor of heat shock protein-90 ( hsp90 ) .
. showed that ritonavir , a structurally related hiv protease inhibitor to nelfinavir , bound to hsp90 and inhibited interaction between hsp90 and akt .
shim et al . conducted a pharmacological profiling of seven genotypically different breast cancer cell lines using jhdl and found that nelfinavir selectively inhibited the proliferation of human epidermal growth factor receptor 2 ( her2)-positive breast cancer cells over her2-negative ones .
in her2-positive breast cancer cells , nelfinavir caused degradation of her2 and akt by inhibiting their association with hsp90 .
in addition to her2 and akt , the study further showed that nelfinavir decreased the levels of other known hsp90 client proteins including cdk4 and cdk6 36 .
other proposed anticancer mechanisms of nelfinavir include induction of endoplasmic reticulum ( er ) stress and autophagy in cancer cells and inhibition of angiogenesis through down - regulation of hypoxia - inducible factor-1 ( hif-1 ) 32 , 42 , 43 .
since nelfinavir can potentially interact with multiple proteins in cells , its anticancer activity might be a consequence of simultaneous inhibition of multiple pathways essential for cancer cell proliferation and survival ( figure 2 ) .
although the anticancer mechanism of nelfinavir remains to be completely elucidated , promising anticancer activities have been reported from the clinical studies 44 - 46 .
it has a long history of use in the treatment of various heart conditions including heart failure and arrhythmia .
digoxin is known as a potent inhibitor of na / k - atpase pump in cell membrane 48 .
na / k - atpase regulates sodium ion gradient across the cell membrane to efflux intracellular ca ions .
inhibition of na / k - atpase by digoxin causes an increase in intracellular ca concentration in myocardiocytes and pacemaker cells , thereby lengthening cardiac action potential 49 . from early 1980s
, a few cohort studies with a small group of breast cancer patients have shown that the use of digoxin decreased the breast cancer recurrence and aggressiveness 50 , 51 .
it was believed that digoxin , as a phytoestrogen , could interfere with estrogen receptor ( er ) signaling in cancer cells , thereby suppressing the growth of breast cancer 52 , 53 .
showed that the population who were taking digitoxin , another cardiac glycoside , had a higher incidence of cancer compared to the control population 54 .
56 reported that the use of digoxin significantly increased the breast cancer incidence among women in denmark . among the digoxin users
, there was the higher risk for developing er - positive breast cancers than er - negative breast cancers 56 .
these data suggested that digoxin , in certain conditions , might act as an estrogen - like molecule rather than an anti - estrogen in women , thus increasing er - positive breast cancer risk . in a recently study ,
conducted two - stage multidisciplinary studies to identify possible anti - prostate cancer drugs from jhdl 57 .
the authors screened jhdl and identified digoxin as one of the most potent inhibitors of prostate cancer cell proliferation . a subsequent large - scale cohort study with long - term follow - up demonstrated that digoxin significantly reduced the incidence of prostate cancer by 25% among men 57 .
moreover , men who had used digoxin for longer than 10 years showed 46% lower incidence of prostate cancer , suggesting a potential anti - prostate cancer activity of digoxin .
this encouraging observation led to the recent phase 2 clinical trial for recurrent prostate cancer .
the fact that digoxin increased the risk of only estrogen sensitive cancers including breast and uterus cancers , but not ovary or cervix cancer , suggests that the tumor promoting mechanism is mediated through its estrogenic effect 58 .
hedelin et al . reported that intake of dietary phytoestrogens significantly reduced prostate cancer risk among the population in sweden 60 .
moreover , digoxin and other cardiac glycosides decreased secretion of prostate specific antigen ( psa ) in androgen receptor ( ar)-dependent prostate cancer cells 61 .
these observations strongly suggest that estrogenic effect of digoxin is beneficial for the treatment of androgen - dependent cancers , such as prostate cancer .
in addition to the estrogenic effect , other anticancer mechanisms of digoxin have been also proposed , such as inhibition of na / k - atpase and hif-1 synthesis 62 , 63 . proposed anticancer mechanisms of digoxin
digoxin is known to have a narrow therapeutic index ( 2 to 3 ) , suggesting that doubling or tripling its recommended dose may cause toxicity 64 .
the therapeutic serum level of digoxin for heart rate control is about 2 ng / ml ( 2.6 nm ) . however , ic50 of digoxin for prostate cancer cell proliferation was about 5 - 10 times the therapeutic serum level , suggesting a discrepancy between in vitro and in vivo anti - prostate cancer activity of digoxin 57 .
although the mechanism by which digoxin exerts anticancer activity in vivo with its therapeutic serum level remains unclear , it is intriguing to postulate that digoxin may accumulate in prostate tissue or that it may indirectly inhibit prostate cancer growth through other mechanisms such as inhibition of angiogenesis during the long - term , low - dose treatment .
nonetheless , it is clear that digoxin has a beneficial effect on patients with certain types of cancer and is currently undergoing several clinical trials for the treatment of cancer as a monotherapy or in combination with other chemotherapy drugs ( http://clinicaltrials.gov/ ) .
nitroxoline is an old antibiotic which has been widely used in european , asian and african countries from 1960s .
it is particularly effective for the treatment of urinary tract infections ( uti ) due to the drug 's unique pk property . when administered orally , nitroxoline is rapidly absorbed into the plasma and is subsequently excreted into urine 65 .
it has a long retention time in urine , thus making it ideal for uti treatment .
nitroxoline is known to be able to chelate divalent metal ions such as mg and mn , which is appreciated as a possible mechanism for its antibacterial activity 66 .
the authors conducted two distinct screens , a target - based ( methionine aminopeptidase-2 or metap2 as a target ) and cell - based ( huvec ) screens to identify novel anti - angiogenic agents from a diverse chemical compound library and jhdl , respectively .
nitroxoline was found to be a common hit from both screens 67 . as it was identified from the metap2 inhibitor screen , it is not surprising that nitroxoline potently inhibited metap2 activity in vitro ( ic50 = 54 nm ) and in endothelial cells .
it is well established that inhibition of metap2 activity in endothelial cells causes an increase in p53 level and an activation of retinoblastoma protein ( prb ) by decreasing its phosphorylation , leading to the inhibition of endothelial cell proliferation 68 .
similar to a known metap2 inhibitor tnp-470 , nitroxoline increases the level of p53 and induces hypo - phosphorylation of prb in huvec .
in addition , nitroxoline also causes an increase in acetylation of p53 ( k382 ) , -tubulin and histone h3 , hallmarks of inhibition of human sirtuins 1 and 2 .
subsequent in vitro and in vivo studies showed that nitroxoline inhibited angiogenesis and the growth of cancer xenograft in mouse models . given that nitroxoline has a long retention time in urine
, it was postulated that the drug might be particularly effective in urological cancers such as bladder cancer .
nitroxoline was tested in an orthotopic bladder cancer model in mice and was administered orally for two weeks to assess its anticancer activity .
cancers from control group grew continuously , whereas the cancer growth was significantly delayed in nitroxoline treated group , suggesting a potential anticancer activity of nitroxoline against bladder cancer in vivo . from the translational perspective , the concentration of nitroxoline required for inhibition of endothelial cell proliferation ( ic50 = 1.9 m ) was well below the maximal clinically achievable concentration ( cmax > 10 m ) in both human plasma and urine .
taking into account that antibacterial activity of nitroxoline was shown at greater than 10 m and that daily nitroxoline dosage of 400 - 750 mg ( for adult ) was sufficient to show antibacterial activity in human , the current nitroxoline dosage regimen for uti treatment is likely to be sufficient for blocking angiogenesis in vivo .
cathepsin b plays a role in degradation of extracellular matrix ( ecm ) and is implicated in tumor cell migration , invasion and metastasis .
however , the kivalues of nitroxoline for endopeptidase activity of cathepsin b were 154.4 m ( for dissociation of ei complex ) and 39.5 m ( for dissociation of esi complex ) , calling into question the relevance of this effect of nitroxoline to its anti - angiogenic activity .
however , it can not be ruled out that the anti - cathepsin b effect of nitroxoline contributes to its anticancer activity in vivo by suppressing tumor cell migration and invasion .
the proposed mechanisms of anticancer activity of nitroxoline are summarized in figure 4 . in a separate study , jiang et al .
recently reported that nitroxoline showed strong anticancer activity against lymphoma , leukemia , pancreatic cancer and ovarian cancer cells 70 .
nitroxoline has been used in many european countries as uti drug for over 50 years and no apparent human toxicity has been reported , making the drug an excellent candidate for anticancer drug repositioning . with the unique pk property and current dosage regimen , human clinical studies of nitroxoline for the treatment of cancer ,
itraconazole is a triazole antifungal drug developed in 1980s . like other azole family of antifungal drugs , it is effective in a variety of systemic fungal infections 13 .
it is known to inhibit cytochrome p450-dependent lanosterol 14--demethylation ( 14 dm ) in the ergosterol biosynthesis pathway in fungi 14 .
ergosterol is the main sterol in most yeasts and fungi , being responsible for their membrane integrity and function .
, itraconazole and related azole compounds cause the depletion of ergosterol and induce accumulation of 14- methylsterols that can impair membrane functions , thereby suppressing the fungal growth 14 , 16 .
although itraconazole is a well - tolerated drug , it has some side effects including hepatotoxicity ( rare but sometimes serious ) , cardiovascular toxicity and diarrhea ( when prepared with cyclodextrin ) 17 . anticancer activity of itraconazole was first reported by chong et al . in 2007 due to its newly discovered anti - angiogenic activity 18 . in this study
, the jhdl was screened for inhibitors of human umbilical vein endothelial cell ( huvec ) proliferation , a proxy for angiogenesis , and itraconazole was identified as one of the most potent hits . in follow - up studies , itraconazole , either alone or in combination with other anticancer drugs , showed strong anticancer activities in preclinical models including non - small cell lung cancer ( nsclc ) , medulloblastoma , and basal cell carcinoma 19 , 20 . prompted by such encouraging preclinical results , itraconazole has entered several phase ii clinical studies for the treatment of various types of cancer .
most recently , positive clinical results have been reported from advanced lung cancer and prostate cancer trials ( both at johns hopkins sidney kimmel comprehensive cancer center ) and from basal cell carcinoma trial ( at stanford university ) 21 - 23 .
itraconazole in combination with pemetrexed showed significant survival benefit in patients with progressive nonsquamous non - small - cell lung cancer compared to the control arm of pemetrexed alone 21 .
high dose ( 600 mg / day ) of itraconazole also showed modest anticancer activity in patients with metastatic castration - resistant prostate cancer ( crpc ) 22 . in the basal cell carcinoma trial , patients were received oral itraconazole 200 mg twice per day for 1 month or 100 mg twice per day for an average of 2.3 months . in this exploratory trial ,
overall , itraconazole was well - tolerated by the patients in all three aforementioned phase ii trials with a few common toxicities including fatigue , nausea and anorexia .
recent study about therapeutic drug monitoring ( tdm ) of itraconazole suggests that serum concentration of 5 g / ml ( 7 m ) is associated with 26% probability of adverse effect 24 .
classification and regression tree ( cart ) analysis suggests that serum itraconazole level of 17.1 g / ml ( 24.4 m ) is upper limit for tdm 24 .
considering that itraconazole 's ic50 values for angiogenesis range from sub - micromolar to single digit micromolar concentrations ( 0.5 ~ 3 m ) , it has a moderate therapeutic window .
however , occurrence of some rare but serious side effects such as hepatotoxicity and congestive heart failure should be monitored in the clinical settings , though it is interesting that the higher dose of itraconazole ( 600 mg daily ) did not cause those major side effects in prostate cancer patients as would have been anticipated 17 , 22 .
although itraconazole showed promising anticancer activity in several types of cancer , its precise anticancer mechanism has remained elusive . to date , two anticancer mechanisms of itraconazole have been proposed ; inhibition of angiogenesis and inhibition of hedgehog signaling pathway in certain cancer cells ( figure 1 ) .
26 showed that itraconazole inhibited cholesterol trafficking in human endothelial cells , leading to inhibition of mammalian target of rapamycin ( mtor ) and vascular endothelial growth factor receptor type 2 ( vegfr2 ) signaling pathways that are critical for endothelial cell proliferation and angiogenesis . in a separate study , kim et al . demonstrated that itraconazole inhibited hedgehog signaling pathway , thereby suppressing the growths of medulloblastoma and basal cell carcinoma 19
ongoing studies are being focused on identifying the molecular target of itraconazole in mammalian cells , which will further our understanding of the precise mode of action of itraconazole for its anticancer activity , facilitating its development as a new anticancer and anti - angiogenic drug .
nelfinavir is a competitive inhibitor of human immunodeficiency virus ( hiv ) aspartyl protease and is being used in combination with other antiretroviral drugs to treat patients with hiv infection 27 .
it received the us - fda approval in 1997 for an oral dose regimen of 750 mg three times daily .
it was later modified to a regimen of 1250 mg twice daily as recommended by us - fda .
both regimens were proven to be equally effective 28 . the average peak plasma level of nelfinavir is around 8 m and the bioavailability is known to be increased when taken with food 29 .
nelfinavir is a well - tolerated drug with some common side effects such as insulin resistance , hyperglycemia and lipodystrophy . from early 2000s , researchers have found potential anticancer activity of nelfinavir . it was reported to inhibit the growths of kaposi 's sarcoma 30 , multiple myeloma 31 , nsclc 32 , 33 , prostate cancer 34 , and breast cancer 35 , 36 .
nelfinavir exhibited a broad - spectrum anticancer activity in vivo , being efficacious in several preclinical cancer models .
a common side effect of nelfinavir was insulin resistance , which was later found to be through inhibition of phosphatidylinositol-3-kinase ( pi3k)/akt signaling pathway .
brunner et al . recently conducted a phase i clinical trial of nelfinavir and chemoradiation for locally advanced pancreatic cancer 37 . in this trial , nelfinavir showed potent radiosensitizing and antitumor activities without adding toxicity in patients with pancreatic cancer .
although nelfinavir is known to inhibit akt signaling pathway , it does not directly inhibit the kinase activity of akt .
gupta et al . showed that nelfinavir down - regulates akt phosphorylation by inhibiting 20s proteasome activity 38 .
hamel et al . also showed that nelfinavir inhibited chymotrypsin- and trypsin - like activities of the rat proteasome preparation in vitro
39 .
however , whether proteasome is the relevant target of nelfinavir for its anticancer and anti - akt activity has been debated since several conflicting results have been reported .
nelfinavir was shown to inhibit cyclin - dependent kinase 2 ( cdk2 ) activity by enhancing proteasome - dependent degradation of cdc25a phosphatase 40 .
in addition , known proteasome inhibitors including mg132 and bortezomib did not recapitulate effects of nelfinavir in breast cancer cells , but , instead , they rescued the akt inhibition by nelfinavir 36 . recently ,
36 demonstrated that nelfinavir is a novel inhibitor of heat shock protein-90 ( hsp90 ) .
showed that ritonavir , a structurally related hiv protease inhibitor to nelfinavir , bound to hsp90 and inhibited interaction between hsp90 and akt .
shim et al . conducted a pharmacological profiling of seven genotypically different breast cancer cell lines using jhdl and found that nelfinavir selectively inhibited the proliferation of human epidermal growth factor receptor 2 ( her2)-positive breast cancer cells over her2-negative ones .
in her2-positive breast cancer cells , nelfinavir caused degradation of her2 and akt by inhibiting their association with hsp90 .
in addition to her2 and akt , the study further showed that nelfinavir decreased the levels of other known hsp90 client proteins including cdk4 and cdk6 36 .
other proposed anticancer mechanisms of nelfinavir include induction of endoplasmic reticulum ( er ) stress and autophagy in cancer cells and inhibition of angiogenesis through down - regulation of hypoxia - inducible factor-1 ( hif-1 ) 32 , 42 , 43 .
since nelfinavir can potentially interact with multiple proteins in cells , its anticancer activity might be a consequence of simultaneous inhibition of multiple pathways essential for cancer cell proliferation and survival ( figure 2 ) .
nelfinavir is now under more than 20 phase i / ii clinical trials for cancer ( http://clinicaltrials.gov/ ) .
although the anticancer mechanism of nelfinavir remains to be completely elucidated , promising anticancer activities have been reported from the clinical studies 44 - 46 .
it has a long history of use in the treatment of various heart conditions including heart failure and arrhythmia .
digoxin is known as a potent inhibitor of na / k - atpase pump in cell membrane 48 .
na / k - atpase regulates sodium ion gradient across the cell membrane to efflux intracellular ca ions .
inhibition of na / k - atpase by digoxin causes an increase in intracellular ca concentration in myocardiocytes and pacemaker cells , thereby lengthening cardiac action potential 49 . from early 1980s
, a few cohort studies with a small group of breast cancer patients have shown that the use of digoxin decreased the breast cancer recurrence and aggressiveness 50 , 51 .
it was believed that digoxin , as a phytoestrogen , could interfere with estrogen receptor ( er ) signaling in cancer cells , thereby suppressing the growth of breast cancer 52 , 53 .
showed that the population who were taking digitoxin , another cardiac glycoside , had a higher incidence of cancer compared to the control population 54 .
56 reported that the use of digoxin significantly increased the breast cancer incidence among women in denmark . among the digoxin users
, there was the higher risk for developing er - positive breast cancers than er - negative breast cancers 56 .
these data suggested that digoxin , in certain conditions , might act as an estrogen - like molecule rather than an anti - estrogen in women , thus increasing er - positive breast cancer risk . in a recently study ,
conducted two - stage multidisciplinary studies to identify possible anti - prostate cancer drugs from jhdl 57 .
the authors screened jhdl and identified digoxin as one of the most potent inhibitors of prostate cancer cell proliferation . a subsequent large - scale cohort study with long - term follow - up demonstrated that digoxin significantly reduced the incidence of prostate cancer by 25% among men 57 .
moreover , men who had used digoxin for longer than 10 years showed 46% lower incidence of prostate cancer , suggesting a potential anti - prostate cancer activity of digoxin .
this encouraging observation led to the recent phase 2 clinical trial for recurrent prostate cancer .
the fact that digoxin increased the risk of only estrogen sensitive cancers including breast and uterus cancers , but not ovary or cervix cancer , suggests that the tumor promoting mechanism is mediated through its estrogenic effect 58 .
hedelin et al . reported that intake of dietary phytoestrogens significantly reduced prostate cancer risk among the population in sweden 60 .
moreover , digoxin and other cardiac glycosides decreased secretion of prostate specific antigen ( psa ) in androgen receptor ( ar)-dependent prostate cancer cells 61 .
these observations strongly suggest that estrogenic effect of digoxin is beneficial for the treatment of androgen - dependent cancers , such as prostate cancer .
in addition to the estrogenic effect , other anticancer mechanisms of digoxin have been also proposed , such as inhibition of na / k - atpase and hif-1 synthesis 62 , 63 . proposed anticancer mechanisms of digoxin
digoxin is known to have a narrow therapeutic index ( 2 to 3 ) , suggesting that doubling or tripling its recommended dose may cause toxicity 64
. the therapeutic serum level of digoxin for heart rate control is about 2 ng / ml ( 2.6 nm ) .
however , ic50 of digoxin for prostate cancer cell proliferation was about 5 - 10 times the therapeutic serum level , suggesting a discrepancy between in vitro and in vivo anti - prostate cancer activity of digoxin 57 .
although the mechanism by which digoxin exerts anticancer activity in vivo with its therapeutic serum level remains unclear , it is intriguing to postulate that digoxin may accumulate in prostate tissue or that it may indirectly inhibit prostate cancer growth through other mechanisms such as inhibition of angiogenesis during the long - term , low - dose treatment .
nonetheless , it is clear that digoxin has a beneficial effect on patients with certain types of cancer and is currently undergoing several clinical trials for the treatment of cancer as a monotherapy or in combination with other chemotherapy drugs ( http://clinicaltrials.gov/ ) .
nitroxoline is an old antibiotic which has been widely used in european , asian and african countries from 1960s .
it is particularly effective for the treatment of urinary tract infections ( uti ) due to the drug 's unique pk property . when administered orally , nitroxoline is rapidly absorbed into the plasma and is subsequently excreted into urine 65 .
it has a long retention time in urine , thus making it ideal for uti treatment .
nitroxoline is known to be able to chelate divalent metal ions such as mg and mn , which is appreciated as a possible mechanism for its antibacterial activity 66 .
the authors conducted two distinct screens , a target - based ( methionine aminopeptidase-2 or metap2 as a target ) and cell - based ( huvec ) screens to identify novel anti - angiogenic agents from a diverse chemical compound library and jhdl , respectively .
nitroxoline was found to be a common hit from both screens 67 . as it was identified from the metap2 inhibitor screen , it is not surprising that nitroxoline potently inhibited metap2 activity in vitro ( ic50 = 54 nm ) and in endothelial cells .
it is well established that inhibition of metap2 activity in endothelial cells causes an increase in p53 level and an activation of retinoblastoma protein ( prb ) by decreasing its phosphorylation , leading to the inhibition of endothelial cell proliferation 68 .
similar to a known metap2 inhibitor tnp-470 , nitroxoline increases the level of p53 and induces hypo - phosphorylation of prb in huvec .
in addition , nitroxoline also causes an increase in acetylation of p53 ( k382 ) , -tubulin and histone h3 , hallmarks of inhibition of human sirtuins 1 and 2 .
subsequent in vitro and in vivo studies showed that nitroxoline inhibited angiogenesis and the growth of cancer xenograft in mouse models . given that nitroxoline has a long retention time in urine
, it was postulated that the drug might be particularly effective in urological cancers such as bladder cancer .
nitroxoline was tested in an orthotopic bladder cancer model in mice and was administered orally for two weeks to assess its anticancer activity .
cancers from control group grew continuously , whereas the cancer growth was significantly delayed in nitroxoline treated group , suggesting a potential anticancer activity of nitroxoline against bladder cancer in vivo . from the translational perspective , the concentration of nitroxoline required for inhibition of endothelial cell proliferation ( ic50 = 1.9 m ) was well below the maximal clinically achievable concentration ( cmax > 10 m ) in both human plasma and urine .
taking into account that antibacterial activity of nitroxoline was shown at greater than 10 m and that daily nitroxoline dosage of 400 - 750 mg ( for adult ) was sufficient to show antibacterial activity in human , the current nitroxoline dosage regimen for uti treatment is likely to be sufficient for blocking angiogenesis in vivo .
cathepsin b plays a role in degradation of extracellular matrix ( ecm ) and is implicated in tumor cell migration , invasion and metastasis .
however , the kivalues of nitroxoline for endopeptidase activity of cathepsin b were 154.4 m ( for dissociation of ei complex ) and 39.5 m ( for dissociation of esi complex ) , calling into question the relevance of this effect of nitroxoline to its anti - angiogenic activity .
however , it can not be ruled out that the anti - cathepsin b effect of nitroxoline contributes to its anticancer activity in vivo by suppressing tumor cell migration and invasion . the proposed mechanisms of anticancer activity of nitroxoline are summarized in figure 4 . in a separate study , jiang et al .
recently reported that nitroxoline showed strong anticancer activity against lymphoma , leukemia , pancreatic cancer and ovarian cancer cells 70 .
nitroxoline has been used in many european countries as uti drug for over 50 years and no apparent human toxicity has been reported , making the drug an excellent candidate for anticancer drug repositioning . with the unique pk property and current dosage regimen , human clinical studies of nitroxoline for the treatment of cancer ,
in this review , we provided an overview of drug repositioning for anticancer applications with a particular emphasis on activity - based drug repositioning of non - cancer drugs . several successful case studies including those exemplified in this review are summarized in table 2 .
although drug repositioning should significantly reduce the time and cost associated with drug development processes , benefits are limited to a certain process between preclinical to phase ii study . many challenges still exist after phase ii trials .
phase iii studies involves much larger number of patients compared to phase i and ii studies . due to the size and relatively long duration ,
phase iii studies are the most expensive and time - consuming trials and these hurdles in phase iii studies have not changed over the years .
another challenge that should be considered for drug repositioning has to do with intellectual property ( ip ) protection of the repositioned drugs , especially for those drugs that are off patents .
off - patent drugs can be protected in part by method - of - use ( mou ) patents which contain one or more claims directed to a method of use .
mou patents are much weaker than the composition - of - matter ( com ) patents in terms of the exclusionary right .
nitroxoline , for example , used to be off patent , but is currently under mou patent protection for anticancer applications since it was found to have anticancer properties 71 .
currently , an estimated number of 4,000 of active pharmaceutical ingredients ( api ) have been approved for human use in the world 10 .
approved drugs keep accumulating over the years ; on average 20 to 30 nmes each year have been approved by us - fda 2 further expanding the space for drug repositioning .
since more diverse and selective cancer drug targets are being discovered and developed , the approved drug collections will be particularly useful to quickly identify clinically advanced anticancer drugs against those targets .
a major problem of conventional cancer chemotherapy drugs ( mainly dna damaging agents ) is notorious side effects that significantly reduce the quality of life of patients .
as most of non - cancer drugs have little or tolerable side effects in human , repositioning of non - cancer drugs for anticancer therapy as exemplified in this review will be an excellent strategy for future anticancer drug development . | drug repositioning ( also referred to as drug repurposing ) , the process of finding new uses of existing drugs , has been gaining popularity in recent years .
the availability of several established clinical drug libraries and rapid advances in disease biology , genomics and bioinformatics has accelerated the pace of both activity - based and in silico drug repositioning .
drug repositioning has attracted particular attention from the communities engaged in anticancer drug discovery due to the combination of great demand for new anticancer drugs and the availability of a wide variety of cell- and target - based screening assays . with the successful clinical introduction of a number of non - cancer drugs for cancer treatment ,
drug repositioning now became a powerful alternative strategy to discover and develop novel anticancer drug candidates from the existing drug space . in this review , recent successful examples of drug repositioning for anticancer drug discovery from non - cancer drugs will be discussed . |
six percent of the world population is affected by diabetes mellitus ( dm ) which is a chronic metabolic disorder .
the who defines diabetes mellitus as a metabolic disorder of multiple aetiology characterized by chronic hyperglycemia with disturbances of carbohydrate , fat and protein metabolism resulting from defects in the insulin secretion , insulin action , or both .
a survey on indian population shows that 4% of the adults suffered from diabetes mellitus in the year 2000 and it is expected to rise to 6% by the year 2025 .
in developing country like india , the majority of diabetics are in the age group of 45 - 64 years in contrast to developed countries it is highly prevalent in more than 65 years of age .
the management of type 1 diabetes mellitus depends mainly on insulin , whereas the oral antidiabetic drugs ( oads ) are the first line treatment for type 2 diabetes mellitus .
complications due to hyperglycemia in diabetes mellitus can be prevented by using rational use of oral antidiabetic drugs ( oads ) and insulin .
rational use of the drugs is a complex issue with a goal that is difficult to achieve , defined as follows : that patients receive medications appropriate to their clinical needs , in doses that meet their own individual requirements for an adequate period of time , and at the lowest cost to them and their community .
rational use of the drugs in populations can be effectively studied with drug utilization reviews . the world health organization ( who ) defines drug utilization as the marketing , distribution , prescription and use of the drugs in a society considering its consequences , either medical , social , and economic .
drug utilization studies is an invaluable investigational resource to study pharmacoepidemiology , pharmacovigilance , pharmacoeconomics and pharmacogenetics .
the world health organization ( who ) has formulated a set of core prescribing indicators for improvement in the rational drug use in the outpatient practice .
so the cost of antidiabetic drug is the major deciding factor for the patients compliance .
selection of oral antidiabetic drugs as first - line drug or combined therapy should be based on both the pharmacological properties of the compounds like efficacy , safety profile and also on the clinical characteristics of the patient like stage of disease , body weight , bmi etc .
, there exists a wide range of variation in the prices of drugs marketed in india and other countries of the world .
percentage cost variation is an effective tool to find out the difference between the various brands prescribed by prescriber in the same setting . in 2005 ,
indian council of medical research has given guidelines for treatment of diabetes in which the selection of drugs are mainly based on body mass index ( bmi ) .
judicious use of anti - diabetic drugs by adhering to these guidelines will decrease the complications and cost of the drug therapy .
there are many studies carried out on drug utilization in diabetic patients but a limited number of studies had focus on analyses of cost and adherence to treatment guidelines .
so , we planned to carry out this study drug utilization studies in diabetic patient with focus on cost analyses and adherence to standard treatment guideline . to study prescription pattern , calculate cost of antidiabetic agents and to evaluate the adherence to treatment guidelines in diabetic patients attending the medicine outpatient department in a tertiary care teaching hospital .
to study prescription pattern , calculate cost of antidiabetic agents and to evaluate the adherence to treatment guidelines in diabetic patients attending the medicine outpatient department in a tertiary care teaching hospital .
a prospective , cross - sectional study was carried out over the duration of 5 months from october 2012 to february 2013 .
all the diabetic patients attending the medicine outdoor department were enrolled in the study after explaining the aim of the study . written informed consent
prior approval of institutional ethics committee , hospital superintendent and from the head of the medicine department was obtained .
patients receiving any of the anti - diabetic drugs were included in the study irrespective of their gender and those patients who were pregnant and having insufficient data or records were excluded from the study .
for those prescriptions whose generic name of the drugs and price were not mentioned in the prescription , they were obtained from cims ( current index of medical specialty ) and indian drug review ( idr ) [ 2012 issues ] .
we collected the patient 's pharmacy bills and analyzed the retail cost of a particular drug being manufactured by different companies , in the same strength , number and dosage form was compared .
the difference in the maximum and minimum price of the same drug manufactured by different pharmaceutical companies was calculated .
the drugs being manufactured by only one company or being manufactured by different companies however , in different strengths were excluded .
for calculation of adherence to standard diabetes guidelines we used indian council of medical research guidelines , 2005 .
the data was calculated using mystat software 12.0 and statistical package for social sciences software 21.0 ( spss ) .
we used unpaired t - test and fischer 's exact test to evaluate the difference between two groups .
the data was calculated using mystat software 12.0 and statistical package for social sciences software 21.0 ( spss ) .
we used unpaired t - test and fischer 's exact test to evaluate the difference between two groups .
a total of 250 prescriptions were collected in the study during the period of 5 months .
highest numbers of the patients were found in the age group of 51 - 60 ( 36% ) years . out of the 250
the age difference between two gender groups ( p 0.05 ) was statistically significant as shown in table 1 .
demographic variables ( n=250 ) number of drugs prescribed was 1,391 ( range 2 - 11 ) .
number of drugs prescribed ranged from 2 - 14 drugs , with a mean of 5.56 2.52 drugs .
a total of 539 antidiabetic drugs were used as shown in table 2 and figure 1 ( range 1 - 6 ) .
most commonly used drug combination was of glimepiride and metformin in 119 ( 76.28% ) patients .
most commonly used drugs other than antidiabetic were aspirin 146 ( 18.9% ) and atorvastatin 119 ( 15.41% ) .
drug use pattern of anti - diabetic drugs ( n=250 ) number of anti - diabetic drug prescribed per patient in our study , therapy cost for a diabetic patient ranged from 15 to 2501 inr per month and most of the patients fall in the cost range of 100 - 400 inr i.e. in 68.4% of the patients .
percentage cost variation is shown in table 3 which ranges from 1.47 to 181.81 for glimepiride + metformin + pioglitazone and glimepiride alone , respectively . who drug prescribing indicators are shown in table 4 and adherence to icmr guidelines on diabetes is shown in table 5 .
percentage cost variation of anti - diabetic agents who core prescribing indicators adherence of treatment to indian council of medical research guidelines , 2005
this study was carried out with aim to analyze the drug utilization pattern in diabetic patients in medicine outpatient department in a tertiary care teaching hospital .
drug utilization research study will help for improvement in drug usage , more focused generic brand prescribing pattern .
selecting proper cost effective brand will help in quality of the drug usage , cost reduction , proper dose selection and better health outcome .
although lifestyle modifications play an important role in diabetes management , drugs become unavoidable in many patients .
this study was focusing on the prescription pattern in diabetic patients attending the outpatient departments in a hospital .
demography details shows male patients were 126 ( 50.4% ) and females patients were 124 ( 49.60% ) . in our study ,
however in earlier study male predominance was seen in the study population which is not in agreement with the results of our study
majority of 90 ( 36% ) of the patients were found in 51 - 60 years of age group and among 43 ( 17.2% ) newly diagnosed patients with type - ii dm , 30% were of the age group of 41 - 50 years indicating that the risk of type - ii dm increases after the age of 40 years , this result was similar to study of roy v et al ( 1998 ) .
middle age preponderance was seen in our study which was similar to an earlier study of das p et al . , ( 2011 )
average age of male was 59 years and female was 56 years . it was reported in near to study of wu et al .
average bmi observed in our study was 27.16 2.85 in male patients and 26.40 3.65 in female patients during course of antidiabetic therapy , which implies that the patients were overweight ( bmi 25 kg / m2 ) and were on the borderline of becoming obese ( bmi 30 kg / m2 ) , which in itself is a well - recognized significant risk factor for diabetes mellitus . in our study , average fbs and ppbs notified was 113.19 27.80 and 209.5 46.92 mg / dl respectively in female and 115.85 31.46 and 0.89 35.78 mg / dl respectively in male .
it was higher than that reported in the study done by dave dj et al .
similar result was obtained by the study conducted by rataboli p et al . , ( 2007 ) stated that among all diabetic complications , cardiovascular complications ( hypertension ) pose a major threat .
, we also found out that the genetic basis i.e. , majority of the patients had either of their parents suffering from diabetes mellitus this finding was further substantiated by study of kannan et al . , (
most of our patients had a history of diabetes around 5 - 15 years which an earlier study also reported . in our study , most commonly used drug group used was biguanides and sulfonylureas and not insulin as it was opd based as compared to other studies .
the most common drug prescribed was metformin as compared to previous studies done a decade back .
this suggests the gradual takeover of metformin as a first - line agent for type-2 dm in a decade . among sulfonylureas ,
selection of glimepiride and glipizide has been recommended by texas diabetes council because these agents have lower incidence of hypoglycemia .
johnson ja et al . ( 2006 ) stated that a study from spain reported that 25.3% of the patients were prescribed insulin .
this difference is due to difference in presentation in different study populations . in this study
most commonly used drug combination was of glimepiride and metformin which was a common finding seen with earlier study of v. sivasankari et al . ,
and das p et al . , ( 2011 ) which also suggest combination of biguanides and sulfonylureas was most frequently used combination and most effective one .
this combination is most desirable and having a rational basis of use i.e. both of this drugs act through different mechanism one is insulin sensitizer and other insulin secretogogue .
most commonly used drug other than anti - diabetic drugs was aspirin , atorvastatin and clopidogrel which suggest association of cardiovascular disease especially hypertension and higher blood cholesterol level in diabetes mellitus patients . whereas certain other drugs like pregabalin and mecobalamin were prescribed for patients having signs and symptoms of diabetic neuropathy .
mean drugs prescribed per patient was 5.56 2.52 whereas study by das p et al
out of all drugs 619 ( 44.50% ) antidiabetic drugs were prescribed which was a similar finding of an previous study conducted by upadhyay d et al .
( 2007 ) where anti - diabetics accounted for 314 ( 45.84% ) of the total drugs .
average number of antidiabetic drugs per prescription was 1.45 which was similar to an early indian studies .
our study and another study of upadhyay d et al . , ( 2007 ) reported that 95.62% of the drugs were prescribed in oral dosage form and only insulin was prescribed by parenteral route .
data was analyzed for who drug utilization indicators , we have seen a trend of using brand name for prescribing and a very less amount of drugs was prescribed by generic names .
more and more amount of drugs should be prescribed by generic names as it increases uniformity and decreases cost of drug therapy . in
earlier study essential drugs were prescribed to a large extent but in our study nearly half numbers of drugs were prescribed from essential drug list which suggest a trend of using newer drugs in prescribing and also suggest the influence of pharmaceutical companies in prescribing .
a very less amount of drugs was given in injections as compared to earlier study .
this finding can be explained as we collected only outpatient prescriptions and insulin was the most frequently prescribed drug by injection route which is a cornerstone of type 1 dm and also this also suggest better knowledge of the doctors about the risks and high cost of these injections .
only few antibiotics ( 0.79% ) were prescribed out of all drugs it was an expected finding as patient in outpatient department arrives only for refilling of prescription and present mostly without infection .
the percentage of generics and drug use from essential drug list are higher when compared to those from a study reported in delhi by kumar r et al ( 2013 ) .
cost of therapy per month was higher as compared to study by kannan et al .
cost of drug therapy was a cause for non - adherence . in this study cost of the drugs per prescription
the cost of prescription can be reduced by choosing most economic drugs without changing its quality .
cost of prescription is important in chronic diseases like diabetes . in our study therapy cost for a diabetic patient ranged from 15 to 2501 inr and
most of the patients fall in the cost range of 100 - 400 inr per month .
mean cost of therapy for a diabetic patient was 354.60 305.72 inr , in our study it was found higher as expected as no anti - diabetic drug was given free of cost in our institute as compared to other government hospitals .
we found out the percentage cost of variation an estimation of variability in cost for different brands prescribed to the patients .
glimepiride , metformin and pioglitazone combination had the least percentage cost variation i.e. 1.47 whereas glimepiride had highest percentage cost variation i.e. 181.81 , this finding suggest availability of more brands in glimepiride as compared to other drugs and total cost of treatment can be reduced drastically by using the cheapest brand of glimepiride and prescriber should avoid writing the costliest brand of glimepiride in order to curtail the total cost of drug therapy .
( 2013 ) stated that glimepiride ( 1 mg ) shows maximum price variation of 655.38% .
icmr guidelines have suggestion that in patient less than 18.5 body mass index metformin should not be used , in patients in between 18.6 to 24.9 bmi guidelines suggest that metformin should be used as second line oad only in the resistance cases after using other oad and metformin should be combined when used .
whereas in patients above 25 bmi first - line drug used should be metformin . in our study majority ( 15.6% ) of the non - adherence was due to prescribing of other drugs in place of metformin in the patients having bmi more than 25 . prescribing drugs other than metformin in inappropriate as literature showed that using in obese patient metformin should be preferred .
sulfonylurea and biguanide combination drugs were used . in these glimepiride and metformin combination drugs were prescribed and used commonly .
oral dosage form was the most commonly used to increase the patient compliance in type 2 dm .
the cost of prescription can be reduced by choosing the most economic drugs ( generic ) without changing its quality .
prescribers followed the icmr guidelines to a large extent . the pattern of prescription for diabetic patients should be more rational as per our study and compliant with current evidence and clinical guidelines . | introduction : diabetes mellitus is on alarming rise in india .
drug utilization studies help to identify the adherence to standard treatment guidelines and to evaluate the rational drug usage.objective:to study prescription pattern , calculate the cost of antidiabetic drugs and to evaluate the adherence to treatment guidelines in diabetic patients attending the medicine outpatient department in a tertiary care teaching hospital.materials and methods : a prospective observational study was carried out for a period of 5 months .
the diabetic patients who visited the medicine outdoor department were included .
demographic data and complete prescription details were recorded in the structured case record form .
cost of the drug therapy was calculated from the patient 's bills .
indian council for medical research guidelines-2005 for diabetes management was used to evaluate the adherence.results:a total of 250 patients were enrolled in the study with mean age 57.91 9.37 . out of 250
patients 126 ( 50.4% ) were male and rest were female .
a total of 1,391 drugs were prescribed , with mean of 5.56 2.52 drugs and out of which 539 drugs were antidiabetics with mean of 2.18 0.96 . in monotherapy , metformin was frequently 218 ( 40.45% ) prescribed .
glimepiride and metformin was the most frequently prescribed in 119 ( 76.28% ) out of 156 antidiabetic drug combinations .
most commonly used drugs other than antidiabetics were aspirin 146 ( 18.9% ) and atorvastatin 119 ( 15.41% ) .
mean cost of therapy for a month for a diabetic patient was 354.60 305.72 inr .
majority 209 ( 83.6% ) of prescriptions was in accordance to guidelines.conclusion:metformin was the most frequently prescribed drug in the diabetes patient .
metformin and glimeperide being the most frequent combination used .
majority of the prescriptions followed standard guidelines . |
a male neonate weighing 3.9 kg who exhibited whole body cyanosis and desaturation upon birth was diagnosed with dextro - transposition of the great arteries , atrial septal defect , and patent ductus arteriosus associated with a single coronary artery pattern .
the leiden classification is often used in clinical contexts to describe coronary patterns . in this classification , the right hand of an observer standing within the noncoronary sinus points to sinus 1 and the left hand points to sinus 2 . in this patient ,
a single coronary artery originated from the left sinus ( sinus 2 ) , had a proximal left circumflex arterial branch , and passed anteriorly to the right side of the aorta , further branching into the right coronary and left anterior descending arteries .
the left circumflex artery passed posteriorly to the pulmonary artery , and the left anterior descending branch passed anteriorly to the aorta ( fig .
after cardioplegic arrest with antegrade cold blood cardioplegia under moderate hypothermic cardiopulmonary bypass , the ascending aorta was transected and the coronary ostia were visualized . a single coronary artery originated from the left sinus , giving rise to a proximal left circumflex arterial branch that passed posterior to the pulmonary artery , then branched into the right coronary artery , and finally branched into the left anterior descending artery , passing anteriorly to the aorta .
after the first dose of cardioplegia , the root catheter was removed and the ascending aorta was transected distally .
the pulmonary artery was transected and brought anteriorly to the aorta using the lecompte maneuver .
the distal ascending aorta was then anastomosed to the proximal pulmonary artery . marking sutures were placed on the left sinus .
a cardioplegic catheter was then placed at the coronary sinus of the aorta ( the neopulmonary artery ) , and a second dose of cardioplegia was infused while obliterating the aorta with forceps .
the single coronary artery was removed from the aortic root along with a button from the coronary os .
the coronary button was formed in a long u - shape instead of the usual round shape ( fig .
an arteriotomy was performed in the pulmonary artery ( neoaorta ) , which was aligned with the opposite side of the left sinus and extended with potts scissors .
coronary button reimplantation was performed by tube reconstruction of the coronary os with an aortic autograft patch ( fig .
then , the defect resulting from the resection of the proximal aorta ( neopulmonary artery ) to create the button was reconstructed using a u - shaped patch of glutaraldehyde - fixed autologous pericardium .
all other steps of the procedure were routine , including the lecompte maneuver , transatrial closure of the atrial septal defect , and division of the patent ductus arteriosus . after an aortic cross - clamping time of 120 minutes and a cardiopulmonary bypass time of 167 minutes
, the child was weaned from cardiopulmonary bypass with low - dose inotropes , and the chest was primarily closed .
the child was extubated on postoperative day five , transferred to the ward on postoperative day 10 , and discharged home on postoperative day 15 in good clinical condition .
coronary computed tomography angiography showed a normal tube graft of the reimplanted coronary arteries ( fig .
the arterial switch operation has become the surgery of choice for correcting transposition of the great arteries . due to variations in the pattern of coronary arteries associated with the transposition of the great arteries and transfer of the coronary arteries during the arterial switch operation ,
much attention has been paid to the relationship between original patterns of coronary arteries and surgical outcomes .
therefore , the coronary artery transfer technique is the most important step in attaining successful outcomes in arterial switch operations correcting transposition of the great arteries .
many techniques have been described , but each has its own advantages and disadvantages [ 58 ] .
it is debatable whether autologous pericardium is a suitable material to use as a flap or tunnel in the coronary artery transfer , because the pericardium might cause long - term calcification , contraction , or thrombosis .
recently , a technique using the patients own arterial wall , from either the aorta or the pulmonary artery , has become standard .
no postoperative torsion or obstruction has been identified , and no thrombosis was found at a long - term follow - up .
the introduction of this technique reduced the frequency of complications and prevented coronary artery torsion and deformation .
in addition , adapting arterial vessel walls from either the aorta or the pulmonary artery into lateral walls or roofs has the benefit of allowing growth and recovery at the sites where the coronary artery was cut .
the circumflex coronary artery arose from the single coronary artery , extending from the rightward posterior - facing sinus and passing behind the pulmonary artery .
next , the right coronary artery branched , and finally , the left anterior descending artery passed in front of the aorta .
some authors have even suggested placing the coronary button at or above the neoaortic anastomosis . in this patient ,
resecting the coronary button in the usual round shape would have led to the possibility of the circumflex artery kinking and creating tension on the right coronary and left anterior descending arteries .
tube graft reconstruction was performed to avoid kinking or tension on the coronary arteries as well as to prevent postoperative myocardial ischemia . when performing tube graft reconstruction , using autologous pericardium is an option , although an aortic autograft was used in the present case .
autologous arterial tissue has the benefit of eliminating the risk of overstretching while preserving growth potential .
tube graft reconstruction is a useful surgical technique for coronary translocation in patients with transposition of the great arteries associated with abnormal coronary artery variation .
likewise , the use of an autologous aortic patch in tube reconstruction led to satisfactory results . | an eight - day - old neonate was diagnosed with dextro - transposition of the great arteries , atrial septal defect , patent ductus arteriosus , and a single sinus origin of the coronary arteries .
the single coronary artery originated from the left sinus ( sinus 2 ) , had a proximal left circumflex arterial branch , and passed anteriorly to the right side of the aorta , further branching into the right coronary and left anterior descending arteries .
we successfully performed an arterial switch operation and coronary transfer by tube graft reconstruction with autologous aortic tissue to treat the dextro - transposition of the great arteries and atrial septal defect with a single - sinus origin of the coronary arteries . |
although the exact embryological mechanism for this condition remains unclear , it is thought to relate to a disruption of development of the lateral folds of rathke during hindgut development.1 several anatomical variants have been described , and the classification system proposed by effman , lebowitz , and colodny has been widely adopted2 ( fig .
type 1 : blind ending channels or incomplete duplication type 1a : a blind ending channel opening on the dorsal or ventral surface of the penis in the midline , without communicating with either the bladder or urethra .
type 2 : patent and complete duplication type 2a : two urethral meati ( which may open anywhere along the midline ) .
2a - ii : the accessory urethra divides from the main urethra and maintains a separate course 2a- ii y - type : the ventral urethra opens in the perineum type 2b : the two urethrae unite and form a single channel before opening at the skin type 3 : urethral duplication associated with caudal duplication ( i.e. , duplication of the bladder ) some of these lesions may be totally asymptomatic ; obviating the need for surgery .
however , the more complex variants may require multiple operations to obtain functionality and these interventions entrain future risks of incontinence and stricture.3
4
5 in this case we describe the management of a urethral duplication with two hypospadic meati in a 21-month - old boy .
a 27-week premature boy was referred to the pediatric urology clinic at the age of 9 months for assessment of a proximal hypospadias .
he had a complicated neonatal course ; spending 11 weeks in the neonatal intensive care unit , initially requiring a ventilator .
he had neonatal complications of jaundice requiring phototherapy , and necrotizing enterocolitis , which was managed medically .
however , closer inspection revealed what appeared to be a second urethral meatus opening within the glans ( fig .
( a ) preoperative picture ( the arrows show the position of the two urethral meati ) . ( b )
( d ) follow - up 21 months postoperatively . a preoperative contrast study ( fig .
3 ) revealed two distinct urethral channels originating as a single channel from the bladder neck and then running separately by definition an effman type 2a - ii .
surgery was performed at the age of 21 months : the challenge was the presence of a hypospadic rudimentary dorsal urethra , chordee , and a hooded foreskin ( fig .
2b ) , the dorsal urethra was opened in the midline from the meatus to the level of the proximal urethral ending .
a micturating cystourethrogram demonstrating a single distinct channel arising from the bladder , splitting into two ( arrowed ) .
the bridge between proximal urethra and duplicated urethra was divided and a urethra - urethroplasty was performed a complete distal urethral tubularization , layered closure , and glanuloplasty resulted in a single , glanular urethral opening ( fig . 2c ) .
a urinary catheter was left in place for 1 week and then removed without complication . at follow - up
21 months postoperatively , there was a good cosmetic result with no postsurgical complications ( fig .
2d ) . the mother reported that her son was passing urine with a thick and straight stream with no evidence of fistula .
various theories to explain the origin of a urethral duplication have been suggested , with different types of classification proposed,6
7
8 the most widely adopted being that proposed by effman.2 of the cases of urethral duplication reported in the literature , type i lesions are thought to be the most common , although since these are generally asymptomatic , their true incidence is unknown.2 there have been cases reported that are associated with a single hypospadic urethra , commonly associated to an atretic accessory channel distally , such as in type 1a lesions.9
10
the type 2 a - ii group is characterized by the presence of a second urethra which divides from the main urethra , and maintains a separate course .
this group includes a subgroup of patients in whom the ventral urethra opens in the perineum ( y - type duplication ) ( fig .
the ventral urethra opened at the base of the shaft , thus representing a variant that could be considered an intermediate between the classic type 2 a - ii and the y - type duplication .
the ventral urethra was the more functional , while the dorsal urethra was less developed .
several established techniques have been described for the correction of a duplicated urethra.5
11
12 a general consensus is that each patient should be considered individually , with no standard fit - all approach really being suitable for all cases of a particular lesion.13 some authors would tend toward using the orthotopic urethra , even if it is hypoplastic ; ortolano and nasrallah first proposed progressive urethral dilation to achieve adequate caliber.14 we believe , as suggested by salle et al , that after dilatation of the accessory urethra the risk of inadequate urine flow is high.10 therefore in the case described , we opened the dorsal urethra and performed a tubularization over a 8 fr catheter , as in the classic repair for hypospadias .
the use of a voiding cystourethrogram is typically adequate to trace the course and caliber of the two channels , however , retrograde contrast may be required in cases ( as the one presented ) with a hypoplastic urethral opening.15
postsurgical complications tend to manifest as stricture at the anastomosis or related to the hypoplastic channel .
there have been reported cases of fistula formation much like that documented in penoscrotal hypospadias repair.5
15
we believe this case of duplicated urethra is unique for the particular anatomy of the two urethral channels , with the main urethra being the one opening at the base of the shaft .
a distal blind pit is common in hypospadias and should always prompt thorough examination for accessory urethra , in particular , in proximal hypospadias cases .
this report demonstrates a new variant of urethral duplication of which pediatric surgeons should be aware . in cases of proximal hypospadias ,
the surgeon should consider the possibility of a distal accessory urethra , which may require consideration when planning surgery .
this case highlights that this unusual variant can be successfully managed in a single - stage procedure with good functional and cosmetic results . | duplication of the urethra is a rare congenital anomaly , with approximately 300 cases reported in the literature .
we report a unique case of this condition in a male infant .
this case differs from the classical effman type ii - a2 duplication because of the presence of two hypospadic urethral meati , as opposed to a ventral or dorsal accessory meatus with a normally positioned distal urethra .
the patient underwent a single - stage repair consisting of a proximal urethra - urethral anastomosis and distal urethral tubularization at 21 months of age with excellent results in terms of both function and cosmesis . |
the ageing process is accompanied by an increased prevalence of the signs and symptoms of physical fragility , including sarcopenia , a decrease in exercise tolerance , osteopenia , an increase in visceral adiposity and a worsening of quality of life .
adults with growth hormone deficiency ( ghd ) exhibit similar clinical signs , but it is possible to treat them with gh to ameliorate these signs and symptoms .
the gh secretion declines gradually with age , whereby studies demonstrate a progressive reduction of 14% secretion per decade of life beginning in the second decade [ 2 , 3 ] .
these findings suggest a possible association between ghd and the ageing process [ 4 , 5 ] .
the association between the physiological alterations of the ageing process and the reduction of gh secretion is called somatopause .
gh replacement is well known to improve body composition , leading to a decrease in total body fat and an increase in lean body mass .
however , gh replacement has only shown an effect on muscle strength in gh - deficient adults subjected to long - term gh therapy .
few studies have evaluated the effect of gh replacement on muscle strength in elderly people engaged in a program of exercise training [ 2 , 710 ] .
specific training that aims to increase muscle strength can potentiate the anabolic effect of gh , which leads to a greater impact on body composition in comparison to gh replacement alone [ 11 , 12 ] .
these subjects showed increased exercise capacity with improvements in oxygen uptake and ventilatory threshold after gh therapy .
this improvement was most likely due to some combination of increased muscle strength , improved body composition , and improved thermoregulation .
gh has been speculated to improve physical capacity in subjects without ghd through stimulation of collagen synthesis in the tendon and skeletal muscle , which leads to better exercise training and increased muscle strength .
administration of supraphysiological doses of gh to athletes increases fatty acid availability , reduces oxidative protein loss , particularly during exercise , and increases lean body mass .
our purpose was to evaluate the effect of gh therapy on muscle strength in healthy , nonsedentary men over 50 years old .
healthy and nonsedentary men ageing 5070 years were recruited by newspaper advertisements with information that patients were needed for a study of hormonal evaluation .
exclusion criteria were pituitary disease , gh use in the last 12 months , severe acute disease , hepatic and/or renal chronic disease , uncontrolled systemic arterial hypertension , diabetes mellitus , psychiatric disorders , history of cancer , nontreated hypogonadism , and the presence of any other disease that could interfere with the somatotrophic axis . all patients practiced some kind of exercise ( minimum of three times per week , aerobic and resistance exercise ) to be eligible to be included in the study .
twenty - nine subjects were screened for a project about somatotrophic profile in healthy men over 50 years old ( which includes this paper ) and have resulted in other published studies [ 15 , 16 ] .
after that , fourteen from the 29 initial patients agreed to participate in this part of the project that included the evaluation of muscle strength in gh x placebo replacement ; those subjects who gave up had personal problems , such as work compromises and address change .
all patients signed the informed written consent approved by the ethics committee of the clementino fraga filho hospital / ufrj . at baseline , subjects were submitted to evaluation of gh secretion , testosterone level , body composition , and muscle strength .
absence of pituitary disease and normal testosterone levels were necessary for the inclusion in the study .
gh secretion was evaluated through insulin tolerance test ( itt ) or glucagon stimulation test ( gst ) and igf - i levels .
the itt was performed by intravenous injection of regular insulin at a dose of 0.10.15 iu / kg .
blood samples were drawn every 30 minutes from baseline until 120 minutes for the determination of glucose and gh levels .
all patients had hypoglycemia ( glucose nadir lower than 40 mg / dl ) during the test .
severe ghd was considered when gh peak was lower than 3 g / dl , and it was an exclusion criterion .
g / l and the intra - assay coefficient of variation ( cv ) was 5.3% at 1.7 g / l .
serum igf - i concentrations were determined by an immunoradiometric assay ( kit dsl-5600 ) , with acid - ethanol extraction .
the minimal detection limit was 0.80 ng / ml and the intra - assay was 3.4% at 9.38 ng / ml and 3.7% at 255.8 ng / ml . all subjects had gh peak at gst higher than 3
g / l and had igf - i levels normal according to their age ; that is , none of them had severe ghd ( table 1 ) .
subjects were then randomised according to their arrival to the medical evaluation : one patient to each group ( gh and placebo groups ) , sequentially .
the gh group received an initial dose of 0.5 ui / day ( 0.2 mg / day ) , with readjustments to 1.0 ui / day ( 0.4 mg / day ) and 1.5 ui / day ( 0.6 mg / day ) after 1 and 2 months of treatment , respectively .
these gh dosages were based on the dose used for adults of the same age with ghd at the time of this research .
subjects were reevaluated after 6 months of gh therapy or placebo according to the parameters above .
pfizer laboratory also provided the placebo . during treatment , every month the patients should take their used gh flask to the medical consult in order to realize a counting flask for control of the correct use of gh .
body mass index was calculated using quetelet 's formula ( weight / height ) and the percentage of body fat was calculated using the adipometer on seven cutaneous folds ( tricipital , subscapular , suprailiac , pectoral , axillary , abdominal , and thigh ) according to the pollock and jackson protocol .
abdominal circumference was measured using a nonelastic tape at the greatest diameter between the last ribs and the iliac crests .
muscle strength was evaluated for the maximum strength based on the concept of maximum repetition ( 1 mr ) ( i.e. , the maximum load that can be performed using the correct technique for an exercise ) . the exercises evaluated were bench press and leg press using a righetto machine ( so paulo , brazil ) , which are dynamic exercises .
the bench press focuses on the strengthening of the pectoralis major muscle as well as other muscles including anterior deltoids , serratus anterior , coracobrachialis , scapulae fixers , trapezzi , and the triceps .
the bench press was performed according to the following technique : ( a ) initial position : dorsal decubitus , elbows in extension with hands sustaining the barbell , knees and hips semiflexed , and feet on the equipment base ; ( b ) exercise development : elbows flexion , to form a 90 degree angle with the arm and forearm , followed by complete extension of the elbows and horizontal flexion of the shoulders , returning to the initial position .
the leg press works the following muscles groups : quadriceps , hamstring , gluteus maximus , and calves ( partially ) .
the leg press was performed according to the following technique : ( a ) initial position : hips and knees flexed at a 90 degree angle , feet on the platform , and inferior members slightly separated and lined up within the limits of the iliac crests ; ( b ) exercise development : complete extension of the knees then returning to the initial position . before the use of the equipment
, subjects could familiarise themselves with the exercises and the equipment without the load . to determine the load associated with 10 mr ,
each subject initially performed 10 repetitions of each exercise with a submaximum load , which was considered a load that was possible to mobilise .
then , the loads were progressively increased and , with a maximum of 3 trials , the load of 10 mr was reached .
if the 10 mr load was not reached after 3 attempts , the subject was asked to come to the laboratory for another opportunity . to reduce the error margin ,
the following strategies were adopted : ( a ) standardised instructions were given before the test to make the subjects aware of the data collection routine ; ( b ) subjects were instructed on the execution technique of the exercises ; ( c ) the examiner was attentive to the correct position of the patients ' joints at the time of measurement to avoid small variations in joint positioning that could put action on other muscles and lead to erroneous interpretations of the obtained scores ; and ( d ) verbal stimuli were given during the exercise to maintain a high level of motivation . during the 10 mr test , a 25 minute interval was provided between the attempts of each exercise . after the load was obtained for a determined exercise , intervals
comparisons between the groups and timepoints were performed using two - way anova for repetitive measures , followed by fischer post - hoc evaluation if necessary .
the differences between the baseline and the last measure ( delta ) were compared by student 's t - test for independent variables .
thirteen subjects completed the study ( 6 subjects in the placebo group and 7 subjects in the gh group ) .
one subject in the placebo group dropped out of the study because of the development of arthralgia .
subjects of both groups were similar in age , weight , height , and bmi at baseline ; they were overweight according to bmi ( between 2530 kg / m ) , except one subject who was obese ( bmi 38.5 kg / m)table 1 .
all subjects of both groups were nonsedentary ; that is , they performed planned physical activity for more than 150 minutes per week .
after 6 months of therapy , the evaluation of the gh and placebo groups did not show evidence of significant differences in weight , bmi , waist , and the sum of the cutaneous folds ( triceps , biceps , subscapular , and iliac folds ) .
furthermore , there were no differences on the delta ( 6 months baseline ) for these parameters between the groups ( table 2 ) .
muscle strength in the upper body part , as evaluated by supine horizontal rising , did not increase after 6 months in both groups .
muscle strength in the lower body part , as measured by the leg press , showed a statistically significant increase in the gh group when compared to placebo ( table 2 ) .
the effects of ageing cause undesirable changes in body composition , a reduction of bone mineral density and muscle strength ( with greater falling risk ) , and worsening exercise capacity , which is associated with a decrease in gh and igf - i levels .
however , gh replacement to minimise the adverse effects of ageing must be based on cost - benefit and risk - benefit analysis .
furthermore , randomised studies demonstrated that the effectiveness of gh replacement is modest , either on its own or in combination with sex steroids or exercises [ 2 , 4 , 7 ] .
our study evaluated muscle strength in men over 50 years of age after 6 months of gh therapy , and we observed a statistically significant increase in leg press responsive muscles ( quadriceps being the mainly muscle evaluated ) when compared to placebo .
there was no evidence of alterations in body composition in either group ( gh therapy or placebo ) .
the gh group had a bmi and body fat percentage ( represented by the sum of the cutaneous folds ) slightly greater than the placebo group but without statistical significance .
therefore , our results differed from those found in the literature , in which an improvement in body composition and no difference on muscle strength during gh replacement are described [ 7 , 9 ] .
the effects of gh therapy on body composition ( increased lean body mass and decreased fat mass ) are very well reported [ 1 , 1924 ] , but the effects on muscle strength are unclear , which was one of the objectives of our study . it has already been reported that recombinant human gh administered for 6 months to healthy older men decreases the percentage of body fat and increases lean body mass and igf - i levels to values observed in young adult males .
a recent study using a low - dose sustained - release recombinant human gh administered during 26 weeks to subjects with the so - called somatopause showed improvements in body composition and quality of life .
many studies in ghd adults demonstrated the normalisation of muscle strength after 10 years of gh replacement [ 6 , 26 , 27 ] but did not show an increase in muscle strength with the use of gh for short periods of time .
however , a study of 18 elderly men submitted to progressive weight training for 14 weeks and then randomised to receive either gh or placebo during a further 10 weeks of strength training showed that gh had no effect on muscle strength at any time , but lean body mass increased and fat mass decreased in the gh group .
frail older people subjected to gh treatment with or without a structured resistance exercise program had a significantly increase in muscle strength in both gh / exercise as well as the exercise alone groups , but there was a significant increase in the proportion of type 2 fibers at the end of the study in the combined gh / exercise treated subjects .
there are few studies in the elderly that combine gh replacement and specific training for muscle strength [ 7 , 8 ] .
this type of training could potentiate the anabolic effects of gh , which would cause a greater impact on body composition than gh replacement alone as well as greater impact on muscle strength .
gh has been speculated to improve physical capacity in subjects without ghd through its stimulation of collagen synthesis in the tendon and skeletal muscle .
furthermore , gh appears to be more important for the reinforcement of the tissue matrix than for muscle cell hypertrophy .
thus , the gh use could attenuate or prevent muscle fibre injuries , causing a lower frequency of training disruption and leading to greater training in athletes or nonathletes with the use of gh , which leads to a muscle fibre hypertrophy and consequently an increase in strength .
this could explain the increase in leg press responsive muscles in the gh group of the present study .
although no participants of our study were athletes or performed supervised physical training before and during the gh x placebo use , none of the subjects were considered sedentary and all practiced some type of exercise ( minimum of three times per week150 minutes per week , aerobic and resistance exercises ) . we can postulate that this group , even without supervised training , had fewer injuries and consequently performed more exercises to increase muscle strength .
moreover , the majority of subjects realized walk and running as exercise , which prioritizes the lower body part muscles . another point that corroborates
the gain of muscle strength in this group is the documented evidence that adults with ghd have lower fatigue sensation when subjected to gh replacement , which increases the adherence to exercise and leads to cardiopulmonary benefits .
although there are conflicting results regarding adults without ghd , it could be another fact that increased the disposition to exercise and consequently to an increase in lower body part strength in subjects in the gh group .
the median age of the subjects in our study was lower than the studies cited above .
although our objective did not include evaluation of the gonadic axis , subjects were required to have normal serum levels of testosterone to be included in the study .
thus , we can attribute the observed amelioration of strength in leg press responsive muscles to the gh therapy , independent of gonadic function .
another point is that the majority of the studies were performed for periods less than 6 months , and this could be an explanation for the different results obtained in our study .
another consideration is the gh dosage used in our study ; we used a fixed dosage with increases in the first and second months , independent of the patient 's weight .
the final gh dosage ( 0.6 mg / day ) was the median dosage used for adults ( men with an average age of 50 years ) with ghd at the time that our study began .
however , at the present data , gh dosages are lower in ghd adult replacement ( initial dose of 0.10.3 mg / day ) . we can not discard a pharmacologic effect of this higher gh dosage on muscle strength .
although anthropometry is an important method of nutritional evaluation in subjects over 50 years of age , one limitation of our study is that we did not use a more precise method of body composition evaluation .
fat tissue redistribution is more prominent in women , and reduction in body water content is more common in men .
in addition , the lack of references for anthropometric measures in the brazilian population can reduce the precision of such evaluations . the use of dual energy x - ray absorptiometry ( dexa ) would improve evaluation of modifications in body composition , mainly lean mass .
even still , the results could still be vulnerable to error because dexa is not able to estimate body water accumulation caused by gh therapy .
the bmi was demonstrated to be well correlated with anthropometric indicators of nonvisceral fat ( tricipital and subscapular folds ) and abdominal fat ( abdominal circumference ) , in addition to being in direct relation with total body fat mass [ 33 , 34 ] .
the fact that we did not find a change in weight in the study group despite an improvement of strength in leg press responsive muscles might suggest that there was an increase in muscle mass and a decrease in fat mass .
our study differs from the literature in the following parameters : ( 1 ) age ( our population was younger ) ; ( 2 ) exercise training ( subjects in our study were not considered sedentary and did some type of exercise but not a specific training ) ; ( 3 ) gh dose ( similar to the dose used for ghd adult men at the time of the study ) ; and ( 4 ) longer duration of gh use ( 6 months ) .
our study demonstrated an increase in leg press responsive muscles in men over 50 years old after gh therapy , which is important for reducing falls and fractures in aged populations .
this finding must be considered and tested in frail older populations , whose physical incapacity is caused primarily by proximal muscle weakness .
considering that ageing is related to loss of strength in lower body part and a worsening quality of life , our results can be taken into account for future studies with higher numbers of subjects .
however , we must emphasise that gh therapy has been absolutely contraindicated for aesthetic or antiageing reasons until now . | growth hormone ( gh ) use has been speculated to improve physical capacity in subjects without gh deficiency ( ghd ) through stimulation of collagen synthesis in the tendon and skeletal muscle , which leads to better exercise training and increased muscle strength . in this context , the use of gh in healthy elderly should be an option for increasing muscle strength . our aim was to evaluate the effect of gh therapy on muscle strength in healthy men over 50 years old .
fourteen healthy men aged 5070 years were evaluated at baseline for body composition and muscle strength ( evaluated by leg press and bench press exercises , which focus primarily on quadriceps lower body part and pectoralis major upper body part
muscles , resp . ) .
subjects were randomised into 2 groups : gh therapy ( 7 subjects ) and placebo ( 7 subjects ) and reevaluated after 6 months of therapy .
thirteen subjects completed the study ( 6 subjects in the placebo group and 7 subjects in the gh group ) .
subjects of both groups were not different at baseline .
after 6 months of therapy , muscle strength in the bench press responsive muscles did not increase in both groups and showed a statistically significant increase in the leg press responsive muscles in the gh group .
our study demonstrated an increase in muscle strength in the lower body part after gh therapy in healthy men .
this finding must be considered and tested in frail older populations , whose physical incapacity is primarily caused by proximal muscle weakness .
the trial was registered with nct01853566 . |
squamous cell carcinoma ( scc ) is a common malignancy , and cutaneous scc accounts for up to 20% of non - melanoma skin cancers in the united states , making it the second most common skin cancer .
other primary sites of scc are locations where squamous epithelium is normally found , including the lung , oropharynx , cervix , esophagus , and anal canal .
metastatic scc without a known primary site constitutes about 5% of all cancers of unknown primary site .
gastrointestinal involvement as a manifestation of metastatic scc is relatively uncommon , yet has been reported in the literature , including several cases identifying colonic involvement from known primary locations including the esophagus , lung [ 5 , 6 ] , and cervix .
however , this is the first report that identifies a gastrointestinal metastasis manifesting as recurrent disease from a cutaneous primary source without evidence of active disease elsewhere on multiple imaging studies .
the patient was a 65-year - old male with a history of cutaneous scc involving the right upper chest diagnosed in 2009 and initially treated with resection followed by mohs surgery .
two years later he had a cutaneous recurrence confirmed to be carcinoma in situ . in october 2014
ct of the chest , abdomen , and pelvis showed bulky right axillary and retropectoral lymphadenopathy .
lymph node dissection in november 2014 confirmed 7 of 22 positive nodes for metastatic disease .
postoperative pet scan demonstrated mildly fdg - avid right pectoral lymph nodes believed to be reactive and a small area of increased uptake in the transverse colon of uncertain significance .
colonoscopy was recommended based on the pet scan but deferred by the patient as he began chemoradiation using radiosensitizing doses of carboplatin and paclitaxel , including the right subpectoral lymph node in the treatment field .
this therapy was completed in february 2015 , and subsequent imagining including pet / ct scans in august 2015 and february 2016 demonstrated low - grade metabolic uptake in the region of the right shoulder and axilla , consistent with posttreatment changes without any evidence of active disease , including no evidence of increased colonic uptake . on february 22 , 2016
the patient underwent a routine screening colonoscopy , which did not demonstrate any lesion in the transverse colon , but a small area of nodular polypoid tissue within a diverticulum of the sigmoid colon was identified ( fig 1 ) .
these findings were confirmed on histology and immunohistochemical stains ( pancytokeratin aw1/ae3 and p63 positive , ck20 negative ) .
the subsequent management decisions were discussed at several specialized cancer centers , and it was ultimately recommended that the patient undergo local resection of the colonic metastasis , as this was the only identified site of recurrent neoplasm .
preoperative marking of the site was accomplished on repeat endoscopic evaluation with repeat biopsy confirmation of the site . in june 2016 ,
laparoscopy with a robotically assisted low anterior resection was performed and a previously unidentified small bowel metastatic lesion was noted in the jejunum .
the sigmoid resection confirmed metastatic scc at the base of a deep diverticulum with 3 of 4 positive lymph nodes and clear margins of resection .
postsurgically in july 2016 the patient was enrolled in a trial with pembrolizumab and is currently undergoing systemic immunotherapy .
repeat ct / pet imaging in october 2016 demonstrated unremarkable colocolonic and small bowel anastomotic sites without evidence of any new metastatic disease .
scc is a common malignancy with several potential primary locations , including the oropharynx , nasopharynx , lung , esophagus , cervix , and anus , with metastasis typically involving regional lymph nodes .
cutaneous scc constitutes up to 20% of non - melanoma skin cancers , with principal epidemiologic risk factors that include uv exposure , age , and phenotypic variations ( including fair skin , blue eyes , and northern european origin ) .
other less common but significant risk factors include prior therapy with immunosuppressive agents and immunosuppressed populations such as transplant recipients .
in general , the spread of metastatic cutaneous scc occurs via lymphatics , with occasional distant metastases to organs .
primary colonic scc has been described in case reports [ 12 , 13 ] and several small series and has been estimated to constitute up to 0.2% of primary colon cancers .
however metastatic colonic involvement , though reported to rarely occur with lung [ 5 , 6 ] , esophageal , and cervical primary cancers , has not been reported from a cutaneous scc .
a single case of a synchronous metastatic scc to the colon was reported by ito et al . in 2016 .
however , their patient presented with concurrent cervical lymph node involvement , no identifiable primary site , and a colonic submucosal mass .
the diagnosis was established based only on surgical resection and not mucosal biopsies , in contrast to the currently reported patient in whom a very subtle mucosal abnormality was identified arising within a diverticular orifice .
in addition , this is the first reported case to date of a previously treated cutaneous scc to manifest solely as a colonic metastasis .
the secondary metastatic findings in this case are also noteworthy . discovering the colonic lesion prompted laparoscopic surgical resection , which revealed a second gastrointestinal lesion in the small bowel . though uncommon , the presence of a gastrointestinal metastatic lesion in this setting should prompt consideration to exclude other synchronous lesions and the need for possible additional systemic therapy .
the authors declare that there are no conflicts of interest or conflicts of financial support . | metastatic squamous cell carcinoma ( scc ) involving the gastrointestinal tract as the sole site of metastatic disease is exceedingly rare .
we report a patient with known cutaneous scc that metastasized to regional lymph nodes who , after therapy , appeared to be disease free until a small metastatic lesion was identified on colonoscopy within a diverticular orifice .
he was subsequently noted to have more diffuse gastrointestinal involvement , including a small bowel lesion not previously identified on imaging .
the presence of a gastrointestinal metastatic lesion in this setting should prompt consideration to exclude other synchronous lesions and the need for possible additional systemic therapy . |
giant cell tumor of the bone ( gctb ) is a benign bony tumor accounting for 5 to 8% of all bone tumors ; it is generally observed at the epiphysis of long bones .
it is rare before maturity , and it has a female predilection.1 the axial skeleton above the sacrum , especially the upper cervical spine , is an uncommon location for gctb.1
2
3
4
5
although it is benign , its locally aggressive nature makes its management demanding , especially in the upper cervical spine due to the critical surrounding neural and vascular structures . complete en bloc excision is usually the ideal treatment option , but this method is also challenging due to its critical location.3
6 in cases with a residual tumor , radiation therapy or chemotherapy may be recommended.7
the few case reports and small series in the literature highlight the rarity of this tumor .
we present this rare case involving the upper cervical spine in a 13-year - old girl that was managed surgically with a combined posterior and anterior cervical left retropharyngeal approach ; the tumor was determined to be successfully controlled after 2 years of follow - up .
a 13-year - old girl , who was previously diagnosed with thoracolumbar idiopathic scoliosis , was admitted to the regional hospital . during her preoperative evaluation , she was found to have a lytic lesion in her second cervical vertebra ( c2 ) . after a full workup that included an x - ray , a computed tomography ( ct ) scan , a magnetic resonance imaging ( mri ) scan , and a bone scan , an open biopsy of the lesion
a bone graft was applied , and the patient was immobilized in a halo vest . a week later , the pathology report revealed a gctb , and she was referred to our institution for further management .
the ct scan revealed the details of the bony expansile lytic lesion of c2 involving the body , pedicle , and odontoid process with extensive thinning of the cortex .
the lesion was associated with a soft tissue mass extending to the inside of the neuronal canal and causing effacement of the spinal cord .
computed tomography ( ct ) scan of the c2 axis showing the destructive lesion involving the odontoid , c2 body , and lateral masses with an element of atlantoaxial rotational instability .
( a ) ct scan sagittal cut ; ( b ) ct scan coronal ; ( c ) ct scan axial cut at the odontoid level ; ( d ) ct scan axial cut at the c2 body level . the mri with contrast revealed the extension of the lesion inside the canal with moderate compromise of the cervical spinal cord with effacement and extension of the lesion to the pedicles and lateral masses encasing both vertebral arteries
. the lesion showed a low - intensity signal on t1 , intermediate heterogeneous signal on t2 , and intense homogenous contrast enhancement , as shown in fig .
2a , b , c , d , and e.
magnetic resonance imaging of the upper cervical spine showing low signal intensity on t1 , intermediate signal on t2 , and intensely enhanced postcontrast .
( a , b ) t2-weighted images , sagittal and axial cuts ; ( c , d ) t1-weighted images , sagittal and axial cuts ; ( e ) t1-weighted image , postcontrast , sagittal cut .
an interventional radiologist performed a vertebral artery occlusion test , and the results revealed that both arteries were patent with good collaterals .
a musculoskeletal metastatic workup was performed ; the bone scan revealed increased uptake in the c2 body and no other uptake throughout the skeleton .
the biopsy of the referring hospital was reviewed and the diagnosis was confirmed by our pathology department . however , because of the uncommon location of the lesion , the tumor board recommended another biopsy of the lesion .
the interventional radiologist deferred the biopsy because of the critical location of the tumor , and he performed preoperative vertebral artery stenting to enable easy identification of the vessels during surgery and to prevent occlusion by the tumor .
an open biopsy using a posterior cervical approach with occipitocervical fusion was preferred because the spine was posteriorly explored in the initial procedure , which was done at the referring hospital .
in addition , because malignancy was expected , the surgical team did not want to contaminate the anterior structures with potentially malignant cells and preferred to perform a long occipitocervical fixation down to c5 to achieve solid fusion , as there was no plan for another surgery at this stage .
microscopically , the lesion was full of multinucleated giant cells in the mononuclear stroma , consistent with a gctb .
after confirmation of the diagnosis of gctb , the management plan involved complete excision of the tumor and reconstruction and stabilization of the spine , so a left retropharyngeal approach ( submandibular ) was preferred over a transoral approach because a wide exposure was required for the tumor excision and reconstruction using bone graft with instrumentation .
surgery revealed a near completely destroyed c2 body and odontoid , and the tumor mass was an eggshell cortex overlapping the c3 .
the turmor was firm , brownish yellow , and it was friable and bled easily .
nearly complete excision of the tumor was accomplished , with excision of the c2 body , odontoid , and part of the lateral mass .
a 3-mm - deep trough was created at the upper end of the graft to fit the anterior arch of c1 to maximize stability , and another trough was created at the body of c3 to fit the lower end of the graft .
additional stability was applied by adding a plate from the c1 arch to the body of c3 ( fig .
( a ) postoperative x - ray ; ( b ) postoperative sagittal magnetic resonance imaging ; ( c ) postoperative sagittal computed tomography scan .
an elective tracheostomy was performed and maintained for 2 months , when it was weaned gradually until it was removed .
the final pathology supported the diagnosis of gctb at our pathology laboratory and was confirmed by an international pathology laboratory , as shown in fig .
4a and b. our institution 's tumor board advised regular radiologic follow - up with no additional adjuvant therapy .
( b ) high - power field of the histopathology showing the giant cell tumor .
the patient was regularly followed every 6 months with no evidence of recurrence or cervical spine instability for 2 years ( fig .
5a and b ) .
follow - up computed tomography ( ct ) of craniocervical junction after 2 years shows no tumor recurrence .
( a ) ct scan sagittal cut ; ( b ) ct scan axial cut at the c2 body level .
the spine is not a common site for a gctb,8 with a 2.5% incidence in the sacrum and 2.9% in the vertebrae above the sacrum.9 in the cervical spine , the incidence is extremely low and has been reported to be less than 1% in the literature.1
5 gctb usually occurs after bone maturity , is slightly more prevalent in females , and is rare in the pediatric population.1
the gross appearance of a gctb is described as having a vascular , friable tissue with a surface that is reddish brown , yellowish brown , or yellowish gray.10 based on histologic study , the red spots appear to be hemorrhagic , whereas the brown regions represent cellular stroma .
microscopically , there are moderately vascularized networks of round , oval , or spindle - shaped stromal cells and multinucleated giant cells that can be modified by hemorrhage and necrosis.11 stromal cells , each containing a single large nucleus located in an indistinct cytoplasm , are loosely embedded in a sparse intercellular substance , and occasional mitosis may be observed .
multinucleated giant cells show pyknosis of their nuclei , and the number of nuclei in the giant cells varies greatly , with potentially as many as 50.4
11
12
differentiation between gctbs and aneurysmal bone cysts ( abcs ) may be challenging .
a young age suggests abcs , whereas an older age suggests gctb.12 lesions above the sacrum were once believed to be abcs until proven otherwise.4 histologically , both lesions contain giant cells , which make them difficult to differentiate .
cavernous vascular spaces are the hallmark of abcs but are absent in gctb.4
11 microscopically , the giant cells in a gctb lie among the stromal cells , which do not have intercellular spaces , whereas the giant cells in an abc tend to be small with small nucleoli unlike the giant stromal cells in gctb.4
11
12
it is not easy to differentiate gctb radiologically , as it may be confused with abc , brown tumor , osteoid osteoma , plasmacytoma , or even metastases , chordoma , and lymphoma of the spine.4
13
the radiographic features of a gctb of the spine always start in the vertebral body with possible vertebral body collapse or extension into the nearby structures like intervertebral disk , adjacent vertebral body , spinal canal , or paraspinal soft tissues.14
the x - ray appearance of a gctb in the spine , usually involving the body of the vertebra , is cystlike , with or without compression fractures of the involved vertebrae and with possible involvement of the pedicle or other portion of the arch of the vertebra and the body.4 ct is superior to conventional radiography and will outline the tumor extent , especially in the extraosseous portions , and reveal the bony integrity and stability.15
16
on mri , gctb may show heterogeneous or homogeneous signal intensity on t1-weighted images with possible areas of high - signal intensity caused by recent hemorrhage .
the solid areas of the tumor have heterogeneous low to intermediate signal intensity on t2-weighted images . in more than 50% of cases ,
areas of low signal intensity may be exaggerated on t2-weighted , spin - echo images due to the presence of hemosiderin.11
13
abcs may appear cystic , and trabeculae in the cyst may create a soap - bubble appearance in the lesion .
the air - fluid levels may also account for the cystic component , and the lesions may be rim - enhanced with contrast on mri.13
17
because of its benign nature , the standard treatment is complete en bloc excision of the tumor , but the complex anatomy with proximity to vital neural and vascular structures in the cervical spine usually limits the en bloc removal of the lesion .
a combined approach may be recommended to maximize the chance of tumor excision as well as the spine reconstruction and stabilization.18
in the presented case , the surgical team preferred to perform long occipitocervical fusion down to c5 to maximize the spinal fusion because the spine was posteriorly explored ( cervical laminectomy ) in the initial procedure that was done in the referring hospital .
in addition , the pathology was uncertain ( malignancy was still suspected ) , and there was no plan for another surgery at this stage .
the anterior submandibular retropharyngeal approach for tumor resection and spinal fusion was planned after confirmation of the histopathology as gctb .
in retrospect , if the pathology was confirmed as a benign lesion , the occipitocervical arthrodesis in the pediatric age group should be limited to the minimum number of levels necessary to achieve satisfactory stabilization .
moreover , decision making usually depends on the diagnosis , tumor type , extension of the lesion , presentation , and stability.3
7
a submandibular retropharyngeal approach provides excellent exposure to the anterior cervical spine from the craniovertebral junction up to c4 and facilitates reconstruction of the spine .
in contrast , the transoral approach is considered only for biopsy and/or limited excisions of the lesion.7 the submandibular retropharyngeal approach has no connection with the oral cavity and allows placement of a graft to reconstruct or stabilize the spine with hardware at the same time , which is an advantage over the transoral approach .
the disadvantages include the extensive dissection , prolonged exposure time , and a deep working field .
in addition , a traction injury to the ninth and tenth cranial nerves and compression of the pharynx may cause transient deglutition impairment.7
local recurrence is high when total resection is not achieved , especially when the tumor extends around the neural elements or adjacent vascular structures .
the overall recurrence rate in the spine is between 25 and 45%.19 in the upper cervical spine , the recurrence rate is slightly higher because complete excision is not always feasible .
some suggest complete excision must be attempted because of the benign nature of gctb and because radiation therapy , which is considered for unresectable tumors and those with multilevel involvement , may increase the tendency for aggressive transformation of the residual tumor.8 however , some authors have had good results with primary radiation therapy followed by surgical excision,7 with no recurrence or malignant transformation at the latest follow - up.20
the lesion was surgically resected , and the spine was reconstructed using a combined posterior occipitocervical arthrodesis and anterior cervical retropharyngeal c2 corpectomy and fusion using fibular bone graft and fixation from c1 to c3 , with no recurrence or eventful outcomes at the final follow - up 2 years after surgery .
the presented case was unique in terms of the tumor location , the patient 's age , and surgical management . | study design case report .
objective the purpose of this work is to report the case of a giant cell tumor involving the second cervical vertebra in a pediatric patient .
surgical management included a combined posterior and anterior cervical approach .
there has been no recurrence in 2 years of follow - up .
case report a 13-year - old girl presented with scoliosis with incidentally lytic lesion involving the second cervical vertebra .
the radiologic investigations and biopsy result indicated a giant cell tumor of the bone .
a combined posterior and anterior cervical approach was performed to resect the lesion , reconstruct the spine , and restore stability .
two years of follow - up revealed no recurrence of the lesion with stable reconstruction of the spine .
results the lesion was surgically managed for excision and spinal fusion by combining a posterior occipitocervical arthrodesis with an anterior retropharyngeal cervical approach .
the final histopathology result confirmed a giant cell tumor of the bone .
conclusions giant cell tumor involving the second cervical vertebra is uncommon ; this tumor can be managed surgically by using a combined posterior and anterior cervical retropharyngeal approach .
the presented case was unique in terms of the tumor location , patient age , and surgical management . |
pedicle screw fixation is a necessary part of various spinal surgeries . despite many advantages of using pedicle screws , their usage in the spine is replete with danger and has the potential for permanent neurologic deficit , especially when placing screws near the spinal cord at the concave apex of a scoliotic spine5151620 ) .
there are several methods of pedicle screw insertion that have been developed to enhance the safety of its usage .
some surgeons first identify anatomical landmarks by intraoperative c - arm image intensifier or k - wire placement into the pedicles1221 ) .
others utilize stereotactic image guided systems that are based on preoperative computerized tomographic ( ct ) scan or fluoroscopy1919 ) .
the incidence of pedicle screw misplacement ranges from 2% to 44.2% , with screw - related neurologic complications in the 0 - 0.9% range , using the aformentioned methods25678111314151820212223 ) .
previous studies on the morphometry of thoracic pedicles indicate that there is a high inter - individual variability in the pedicle dimensions17 ) .
the pedicles exhibited significant variability in their shape and orientation , not only between different vertebrae within the thoracic spine , but also in the same vertebra and even in the same pedicle among individuals .
therefore , the starting point for each thoracic screw is slightly variable and is based on the anatomy of the posterior elements that are visualized intraoperatively .
it is time consuming to take c - arm images after each pedicle screw placement and to rearrange the drapes each time an image is taken .
the stereotactic image guided systems requires preoperative ct scan with exposure to high levels of radiation , higher cost , the need for expensive equipment , and prolonged operation time .
exposure to radiation is considered to be a serious health hazard1919 ) . to improve pedicle screw placement accuracy with minimal radiation and maintain low cost
1 ) . we can identify the desired starting and aiming points using this guide pin .
the purpose of this study was to describe a novel pedicle screw placement technique using a special guide - pin and present the results of our first 183 pedicle screw placement analyzed by ct scan .
in the first phase , 2 thoracic spine cadaver specimens were assigned to test the accuracy of the guide - pin for transpedicular screw fixation .
a specially designed guide - pin with a marker was inserted into the pedicle to identify the correct starting ( 2 - 3 mm lateral to the center of the pedicle ) and aiming points ( center of the pedicle isthmus ) in posteroanterior ( pa ) and lateral x - rays ( fig .
2 ) . after confirming the safety and accuracy of transpedicular screws using ct scan and naked eye examination in cadavers , eighteen patients underwent posterior stabilization utilizing transpedicular screws by two surgeons from two institutions using the same methods described above ( phase 2 ) .
all pedicle screws were inserted using the free hand technique after confirmation of the guide - pin on pa and lateral portable / c - arm images1011 ) . to objectively evaluate the position of the screws inserted , postoperative computed axial tomography ( cat ) scans were performed .
the relative position of the screw inside the pedicle was graded as follows : 1 ) contained : axis of the pedicle screw between the medial and lateral pedicle walls completely ; 2 ) medial violation : axis of the pedicle screw is out of medial wall of the pedicle ; and 3 ) lateral violation : axis of the pedicle screw is out of the lateral pedicle wall .
the positions of the scanned pedicle screws were independently determined by two senior spine surgeons .
the surgical technique of free hand transpedicular screw placement using guide - pin can be broken down into specific steps that are repeated at each level
. the first component of successful thoracic screw placement is meticulous exposure of the posterior elements .
the spine was exposed to the tips of the transverse processes bilaterally , staying strictly subperiosteal to reduce bleeding .
we placed the guide - pins into the pedicles according to the recommended starting point following 215 mm hole by cortical burr10 ) .
we checked pa and lateral portable x - rays to adjust the starting and aiming points after all the guide - pins were placed ( fig .
4 ) . we started gearshift from the dorsal lamina 2 mm lateral to the center of the pedicle .
gearshift was advanced from the desired starting point toward the center of the pedicle isthmus , which was the center of pedicle on a pa x - ray and isthmus on the lateral radiograph . after inserting the gearshift tip approximately 20 mm ( past the pedicle isthmus ) ,
we confirmed the integrity of 5 bony walls with a flexible , ball - tipped probe .
the pedicle finder was then advanced further with an ultimate depth averaging 45 - 55 mm for the lumbar spine and 25 - 45 mm in the thoracic region .
a flexible ball - tipped pedicle sounding or palpating device was utilized to palpate five distinct bony borders : a floor and four walls ( medial , lateral , superior , and inferior ) . at this point , if a soft tissue breach was palpated , this provided an opportunity to redirect the screw into an appropriate position into the pedicle .
the pedicle tract was undertapped with a 1 mm less diameter tap than the intended screw . following this ,
the pedicle tract was palpated again to make sure that the five osseous borders were intact .
this second inspection often allowed palpation of distinct bony ridges confirming intraosseous position and the tract length was remeasured with a hemostat .
the coronal plane radiograph is evaluated for the harmonious position of all screws especially when any rotational scoliosis deformity exists . in cases of shorter or longer screws compared to adjacent screws ,
the screw length as viewed on the lateral x - ray is mandatory to check for harmonious position . on the lateral x - ray
, the screws should be parallel to the superior endplates , and not extending past the anterior border of the vertebral body .
these two plane radiographs should confirm the harmonious position of the screws as noted by the surgeon in vivo ( fig .
in addition , various osteotomy procedures were used depending on the etiology of the deformity .
the surgical technique of free hand transpedicular screw placement using guide - pin can be broken down into specific steps that are repeated at each level .
the first component of successful thoracic screw placement is meticulous exposure of the posterior elements .
the spine was exposed to the tips of the transverse processes bilaterally , staying strictly subperiosteal to reduce bleeding .
we placed the guide - pins into the pedicles according to the recommended starting point following 215 mm hole by cortical burr10 ) .
we checked pa and lateral portable x - rays to adjust the starting and aiming points after all the guide - pins were placed ( fig .
4 ) . we started gearshift from the dorsal lamina 2 mm lateral to the center of the pedicle .
gearshift was advanced from the desired starting point toward the center of the pedicle isthmus , which was the center of pedicle on a pa x - ray and isthmus on the lateral radiograph . after inserting the gearshift tip approximately 20 mm ( past the pedicle isthmus ) ,
we confirmed the integrity of 5 bony walls with a flexible , ball - tipped probe .
the pedicle finder was then advanced further with an ultimate depth averaging 45 - 55 mm for the lumbar spine and 25 - 45 mm in the thoracic region . once the pedicle seeker was removed , the tract was visualized .
a flexible ball - tipped pedicle sounding or palpating device was utilized to palpate five distinct bony borders : a floor and four walls ( medial , lateral , superior , and inferior ) . at this point , if a soft tissue breach was palpated , this provided an opportunity to redirect the screw into an appropriate position into the pedicle .
the pedicle tract was undertapped with a 1 mm less diameter tap than the intended screw . following this ,
the pedicle tract was palpated again to make sure that the five osseous borders were intact .
this second inspection often allowed palpation of distinct bony ridges confirming intraosseous position and the tract length was remeasured with a hemostat .
the coronal plane radiograph is evaluated for the harmonious position of all screws especially when any rotational scoliosis deformity exists . in cases of shorter or longer screws compared to adjacent screws ,
the screw length as viewed on the lateral x - ray is mandatory to check for harmonious position . on the lateral x - ray
, the screws should be parallel to the superior endplates , and not extending past the anterior border of the vertebral body .
these two plane radiographs should confirm the harmonious position of the screws as noted by the surgeon in vivo ( fig .
in addition , various osteotomy procedures were used depending on the etiology of the deformity .
the first component of successful thoracic screw placement is meticulous exposure of the posterior elements .
the spine was exposed to the tips of the transverse processes bilaterally , staying strictly subperiosteal to reduce bleeding .
we placed the guide - pins into the pedicles according to the recommended starting point following 215 mm hole by cortical burr10 ) .
we checked pa and lateral portable x - rays to adjust the starting and aiming points after all the guide - pins were placed ( fig .
we started gearshift from the dorsal lamina 2 mm lateral to the center of the pedicle .
gearshift was advanced from the desired starting point toward the center of the pedicle isthmus , which was the center of pedicle on a pa x - ray and isthmus on the lateral radiograph . after inserting the gearshift tip approximately 20 mm ( past the pedicle isthmus ) ,
we confirmed the integrity of 5 bony walls with a flexible , ball - tipped probe .
the pedicle finder was then advanced further with an ultimate depth averaging 45 - 55 mm for the lumbar spine and 25 - 45 mm in the thoracic region .
a flexible ball - tipped pedicle sounding or palpating device was utilized to palpate five distinct bony borders : a floor and four walls ( medial , lateral , superior , and inferior ) . at this point , if a soft tissue breach was palpated , this provided an opportunity to redirect the screw into an appropriate position into the pedicle .
the pedicle tract was undertapped with a 1 mm less diameter tap than the intended screw . following this ,
the pedicle tract was palpated again to make sure that the five osseous borders were intact .
this second inspection often allowed palpation of distinct bony ridges confirming intraosseous position and the tract length was remeasured with a hemostat .
the coronal plane radiograph is evaluated for the harmonious position of all screws especially when any rotational scoliosis deformity exists . in cases of shorter or longer screws compared to adjacent screws ,
the screw length as viewed on the lateral x - ray is mandatory to check for harmonious position . on the lateral x - ray
, the screws should be parallel to the superior endplates , and not extending past the anterior border of the vertebral body .
these two plane radiographs should confirm the harmonious position of the screws as noted by the surgeon in vivo ( fig .
after the insertion of the pedicle screws , deformity correction was carried if necessary . in addition , various osteotomy procedures were used depending on the etiology of the deformity .
there were 18 patients with a mean age of 58.2 years ( range , 34 - 68 years ) at the time of the surgical treatment .
the diameter of the screws used in the thoracic spine ranged from 4.0 mm to 7.5 mm .
the number of screws inserted were as follows ( total n=183 ) : t1 - 6 n=28 ; t7 - 12 n=70 ; l1 - 5 n=85 .
the diagnoses were spondylolisthesis ( 7 patients ) , infection ( 3 ) , fracture ( 3 ) , spinal stenosis ( 3 ) , degenerative scoliosis ( 1 ) , and tumor ( 1 ) .
all 183 thoracolumbar transpedicular screws inserted into the spine were evaluated by cat scan to assess for screw position .
ninety - nine percent ( 181/183 ) of screws were contained within the pedicle . among 183 pedicle screws inserted , thoracic pedicle screws were accurately placed in 98% by the 2 surgeons ( 96 of 98 ) . only two screws ( 2% ) showed moderate cortical perforation which meant the central line of the pedicle screw was out of the outer cortex of the pedicle wall and included 1 screw ( 1% ) that violated the medial wall ( 1 lateral violation in a patient and 1 medial violation in a cadaver specimen ) . eighty - five screws inserted into the lumbar spine showed 100% accuracy without any medial or lateral pedicle wall violation . there were no screws ( out of the 135 thoracolumbar pedicle screws inserted into 18 patients ) that caused neurologic or vascular complications .
there were no instances of cerebrospinal fluid ( csf ) emanating from the initial pedicle tract during the preparation of the screw holes .
there were 18 patients with a mean age of 58.2 years ( range , 34 - 68 years ) at the time of the surgical treatment .
the diameter of the screws used in the thoracic spine ranged from 4.0 mm to 7.5 mm .
the number of screws inserted were as follows ( total n=183 ) : t1 - 6 n=28 ; t7 - 12 n=70 ; l1 - 5 n=85 .
the diagnoses were spondylolisthesis ( 7 patients ) , infection ( 3 ) , fracture ( 3 ) , spinal stenosis ( 3 ) , degenerative scoliosis ( 1 ) , and tumor ( 1 ) .
all 183 thoracolumbar transpedicular screws inserted into the spine were evaluated by cat scan to assess for screw position .
ninety - nine percent ( 181/183 ) of screws were contained within the pedicle . among 183 pedicle screws inserted , thoracic pedicle screws were accurately placed in 98% by the 2 surgeons ( 96 of 98 ) . only two screws ( 2% ) showed moderate cortical perforation which meant the central line of the pedicle screw was out of the outer cortex of the pedicle wall and included 1 screw ( 1% ) that violated the medial wall ( 1 lateral violation in a patient and 1 medial violation in a cadaver specimen ) .
eighty - five screws inserted into the lumbar spine showed 100% accuracy without any medial or lateral pedicle wall violation .
there were no screws ( out of the 135 thoracolumbar pedicle screws inserted into 18 patients ) that caused neurologic or vascular complications .
there were no instances of cerebrospinal fluid ( csf ) emanating from the initial pedicle tract during the preparation of the screw holes .
pedicle screw fixation has the advantage of obtaining purchase of all three spinal columns without encroaching into the spinal canal4 ) .
this theoretical advantage has been translated to superior clinical results in fracture fixation as well as in deformity correction111516172021 ) .
however , their usage in the spine has the potential for permanent neurologic deficit , especially when placing screws near the spinal cord at the concave apex of a scoliotic spine35151823 ) .
the safety margin for this technique has been improved through the use of image - guided techniques1919 ) .
nevertheless , these newer techniques require additional equipment as well as the use of fluoroscopy which increases the radiation exposure .
we developed specially designed guide - pin with a ball maker as it allows identification of the starting and aiming points as well as its direction without the problems mentioned earlier .
the incidence of pedicle screw misplacement ranges from 1.5% to 25% using the k - wire guided methods31621 ) .
although previous research showed only 1.5% misplacement , they admitted that the actual rate would be higher as their patients were primarily evaluated by the postoperative radiographs21 ) .
conventional k - wire guide technique could not allow direct visualization of the starting point on the intraoperative fluoroscopy and/or radiography .
in the present study , only two pedicle screws of 183 ( 1.0% ) demonstrated violations ( 1 lateral violation in a patient and 1 medial violation in a cadaver ) as confirmed by postoperative ct scan evaluation of all pedicle screws enrolled .
ball tip is corresponding to " starting point " of the dorsal cortex of the pedicle . all previous guide - pin except ours failed to show starting point .
some guide - pin had notch(es ) in the middle of guide - pin but they did not demonstrate starting point .
tip of the guide - pin is placed in the isthmic part of the pedicle , which is 15 mm or 20 mm away from ball tip .
the position of the tip should be located around isthmic part of the pedicle which is 15 mm to 20 mm away from starting point .
this special guide - pin can show exact starting point in the dorsal cortex of the pedicle with ball tip .
also tip of the guide pin can show the exact spot around the isthmic part of the pedicle .
thus , operator can adjust the starting point and aiming spot with zero violation of the medial wall of the pedicle .
kim et al.1011 ) reported a series in which 8000 screws had been placed by the free - hand technique without neurovascular complications , and only 8% had significant breaches .
the free - hand technique relies on anatomy and the tactile feedback for pedicle screw insertion .
we could identify the desired starting point and aiming spot using this specially designed guide - pin .
thus , we feel this novel guide - pin provides additional clinically relevant information and is helpful to the spinal surgeon to undertake pedicle screw fixation using the free - hand technique even more safely . the weak point of this study was that confirmation of screw placement could only be performed by postoperative cat scan and not by direct inspection in real patients .
previous studies reported accuracy rates of 68% to 87% in the ct evaluation of pedicle screw placement in cadaver scans using titanium versus cobalt chrome screws24 ) .
a simple , specially designed guide - pin with portable x - rays can provide correct starting and aiming points to place the accurate pedicle screw without preoperative ct scan and intraoperative fluoroscopic assistance .
the placement of thoracolumbar pedicle screws using this guide - pin is an accurate , reliable , and safe without increased radiation or surgical time . | objectiveto improve pedicle screw placement accuracy with minimal radiation and low cost , we developed specially designed k - wire with a marker . to evaluate the accuracy of thoracolumbar pedicle screws placed using the novel guide - pin and portable x-rays.methodsobservational cohort study with computerized tomography ( ct ) analysis of in vivo and in vitro pedicle screw placement .
postoperative ct scans of 183 titanium pedicle screws ( 85 lumbar and 98 thoracic from t1 to l5 ) placed into 2 cadavers and 18 patients were assessed .
a specially designed guide - pin with a marker was inserted into the pedicle to identify the correct starting point ( 2 mm lateral to the center of the pedicle ) and aiming point ( center of the pedicle isthmus ) in posteroanterior and lateral x - rays .
after radiographically confirming the exact starting and aiming points desired , a gearshift was inserted into the pedicle from the starting point into the vertebral body through the center of pedicle isthmus.resultsninety-nine percent ( 181/183 ) of screws were contained within the pedicle ( total 183 pedicle screws : 98 thoracic pedicle screws and 85 lumbar screws ) .
only two of 183 ( 1.0% ) thoracic pedicle screws demonstrated breach ( 1 lateral in a patient and 1 medial in a cadaver specimen ) .
none of the pedicle breaches were associated with neurologic or other clinical sequelae.conclusiona simple , specially designed guide - pin with portable x - rays can provide correct starting and aiming points and allows for accurate pedicle screw placement without preoperative ct scan and intraoperative fluoroscopic assistance . |
we identified 27 hips in 27 patients who underwent revision tha and showed metallosis from a catastrophic failure of a pe liner in their hip joints during revision tha among all the patients who underwent revision thas in seoul national university hospital between january 1996 and august 2004 .
three patients were lost to follow - up during the 5 years period after surgery and could not be reached by telephone or mail . excluding these 4 patients , 23 patients ( 12 men and 11 women ) had tha at the average age of 38.4 years ( range , 15 to 63 years ) , had index revision thas at the average age of 48.1 years ( range , 23 to 78 years ) and the average interval between tha and revision tha was 9.7 years ( range , 4.0 to 14.2 years ) .
patients were followed for at least 6.5 years after the index revision tha ( average duration of follow - up , 12.5 years ; range , 6.5 to 18.0 years ) .
nineteen of the 23 hips underwent clinical and radiographic evaluations and four hips underwent clinical evaluation only , which was conducted over the telephone and with a questionnaire sent by mail .
the initial cause of primary tha was osteonecrosis of the femoral head in 7 patients , degenerative arthritis in 5 patients , legg - calve - perthes disease sequelae in 3 patients , developmental dysplasia of the hip in 2 patients , infection sequelae in 2 patients , tuberculosis sequelae in 1 patient , developmental coxa valga in 1 patient , dysplasia in 1 patient , and rheumatoid arthritis in 1 patient .
metal - on - pe bearings were used for primary tha in 22 patients , and ceramic - on - pe bearing was used in 1 patient .
nineteen thas were done in our hospital and their implants were the aml hip system ( depuy , warsaw , in , usa ) in 8 hips and osteonics hip system ( stryker , kalamazoo , mi , usa ) in 11 hips .
implants of remaining 4 hips are not identifiable because the thas were performed at other hospitals and the corresponding medical records were insufficient . considering the time of the primary thas ( 1988 , 1994 , 1995 , and 1996 ) , the unidentifiable 4 liners probably were conventional pe .
the revision thas were performed due to osteolysis without loosening in 21 hips and osteolysis with loosening of the cup in 2 hips .
two hip surgeons who had more than 15 years of experience with hip operations each , performed all the revision thas . the bubble sign , which is known to be related with metallosis , was observed in 10 hips on plain radiographs taken before revision thas.12 ) the extent and location of the osteolysis were evaluated on preoperative radiographs , and bone deficiency around the cup and stem was categorized according to the system of the american association of orthopaedic surgeons.13,14 ) osteolysis was defined as periprosthetic cystic or scalloped lesions with a diameter exceeding 2 mm that had not been noted on the immediate postoperative radiograph.15,16 ) among the 23 hips , 15 hips showed type iii acetabular defects .
type ii acetabular defects were observed in 7 hips , and a type i acetabular defect was observed in one hip .
osteolysis around the femur was also observed in all 23 hips before revision thas with type i osteolysis in 6 hips and type ii osteolysis in 17 hips .
total revision and cup revision procedures were performed in 13 and 10 hips , respectively .
posterolateral and transtrochanteric approaches were used for revision thas in 10 hips and in 13 hips , respectively .
cementless acetabular implants were used in 22 hips and an acetabular reinforcement ring was used in one hip for revision thas .
chang et al.7 ) classified the severity of metallosis as grade i ( mild : black spotting in the soft tissues ) , grade ii ( moderate : geographically patterned black stain in soft tissues ) , and grade iii ( severe : black spotting throughout the soft tissues and bones ) .
we used the same grading criteria and all 23 hips in this study showed grade iii metallosis ( fig .
1 ) during revision thas . we irrigated the operative field copiously and removed the functionless black - stained tissue as best we could .
bone grafts were applied to both the acetabular and femoral defects in all hips . during revision surgery ,
metal - on - pe bearings were used in 12 hips , ceramic - on - ceramic bearings were used in 5 hips , ceramic - on - pe bearings were used in 4 hips , and metal - on - metal bearings were used in 2 hips .
three of 16 pe liners were crossed linked pe and the others were conventional pe .
clinical evaluations were performed using harris hip scores.17 ) when the patient could not visit the hospital , the evaluation was completed via telephone or by mail .
radiographs were evaluated by two independent observers , who had completed their residency and fellowship programs and were working as adult hip reconstruction specialists for 22 years and 5 years , respectively , and were well - experienced in assessing the presence of osteolysis and loosening .
a change in the angle of more than 4 , or vertical or horizontal migration of more than 3 mm were taken as evidences of an unstable cup.15 ) instability of the femoral stem was defined as subsidence or vertical migration of more than 2 mm or a change in the stem angle of more than 2 , on the anteroposterior radiographs.18 ) kaplan - meier survival analysis was performed to assess the implant survival after revision .
the 15-year survival rates of revision tha were measured using osteolysis or loosening as the endpoint , respectively .
demographic and clinical variables were compared by using chi - square or fisher exact tests for categorical variables such as gender , bubble sign , revision type , bearing for revision tha and bony defect severity between patients demonstrating osteolysis or loosening and the others .
the t - test was also used to compare continuous variables such as age at revision tha and body mass index ( bmi ) .
we identified 27 hips in 27 patients who underwent revision tha and showed metallosis from a catastrophic failure of a pe liner in their hip joints during revision tha among all the patients who underwent revision thas in seoul national university hospital between january 1996 and august 2004 .
three patients were lost to follow - up during the 5 years period after surgery and could not be reached by telephone or mail . excluding these 4 patients , 23 patients ( 12 men and 11 women ) had tha at the average age of 38.4 years ( range , 15 to 63 years ) , had index revision thas at the average age of 48.1 years ( range , 23 to 78 years ) and the average interval between tha and revision tha was 9.7 years ( range , 4.0 to 14.2 years ) .
patients were followed for at least 6.5 years after the index revision tha ( average duration of follow - up , 12.5 years ; range , 6.5 to 18.0 years ) .
nineteen of the 23 hips underwent clinical and radiographic evaluations and four hips underwent clinical evaluation only , which was conducted over the telephone and with a questionnaire sent by mail .
the initial cause of primary tha was osteonecrosis of the femoral head in 7 patients , degenerative arthritis in 5 patients , legg - calve - perthes disease sequelae in 3 patients , developmental dysplasia of the hip in 2 patients , infection sequelae in 2 patients , tuberculosis sequelae in 1 patient , developmental coxa valga in 1 patient , dysplasia in 1 patient , and rheumatoid arthritis in 1 patient .
metal - on - pe bearings were used for primary tha in 22 patients , and ceramic - on - pe bearing was used in 1 patient .
nineteen thas were done in our hospital and their implants were the aml hip system ( depuy , warsaw , in , usa ) in 8 hips and osteonics hip system ( stryker , kalamazoo , mi , usa ) in 11 hips .
implants of remaining 4 hips are not identifiable because the thas were performed at other hospitals and the corresponding medical records were insufficient . considering the time of the primary thas ( 1988 , 1994 , 1995 , and 1996 ) , the unidentifiable 4 liners probably were conventional pe .
the revision thas were performed due to osteolysis without loosening in 21 hips and osteolysis with loosening of the cup in 2 hips .
two hip surgeons who had more than 15 years of experience with hip operations each , performed all the revision thas .
the bubble sign , which is known to be related with metallosis , was observed in 10 hips on plain radiographs taken before revision thas.12 ) the extent and location of the osteolysis were evaluated on preoperative radiographs , and bone deficiency around the cup and stem was categorized according to the system of the american association of orthopaedic surgeons.13,14 ) osteolysis was defined as periprosthetic cystic or scalloped lesions with a diameter exceeding 2 mm that had not been noted on the immediate postoperative radiograph.15,16 ) among the 23 hips , 15 hips showed type iii acetabular defects .
type ii acetabular defects were observed in 7 hips , and a type i acetabular defect was observed in one hip .
osteolysis around the femur was also observed in all 23 hips before revision thas with type i osteolysis in 6 hips and type ii osteolysis in 17 hips .
total revision and cup revision procedures were performed in 13 and 10 hips , respectively .
posterolateral and transtrochanteric approaches were used for revision thas in 10 hips and in 13 hips , respectively .
cementless acetabular implants were used in 22 hips and an acetabular reinforcement ring was used in one hip for revision thas .
chang et al.7 ) classified the severity of metallosis as grade i ( mild : black spotting in the soft tissues ) , grade ii ( moderate : geographically patterned black stain in soft tissues ) , and grade iii ( severe : black spotting throughout the soft tissues and bones ) .
we used the same grading criteria and all 23 hips in this study showed grade iii metallosis ( fig .
1 ) during revision thas . we irrigated the operative field copiously and removed the functionless black - stained tissue as best we could .
bone grafts were applied to both the acetabular and femoral defects in all hips . during revision surgery ,
metal - on - pe bearings were used in 12 hips , ceramic - on - ceramic bearings were used in 5 hips , ceramic - on - pe bearings were used in 4 hips , and metal - on - metal bearings were used in 2 hips .
three of 16 pe liners were crossed linked pe and the others were conventional pe .
clinical evaluations were performed using harris hip scores.17 ) when the patient could not visit the hospital , the evaluation was completed via telephone or by mail .
radiographs were evaluated by two independent observers , who had completed their residency and fellowship programs and were working as adult hip reconstruction specialists for 22 years and 5 years , respectively , and were well - experienced in assessing the presence of osteolysis and loosening .
a change in the angle of more than 4 , or vertical or horizontal migration of more than 3 mm were taken as evidences of an unstable cup.15 ) instability of the femoral stem was defined as subsidence or vertical migration of more than 2 mm or a change in the stem angle of more than 2 , on the anteroposterior radiographs.18 )
the 15-year survival rates of revision tha were measured using osteolysis or loosening as the endpoint , respectively .
demographic and clinical variables were compared by using chi - square or fisher exact tests for categorical variables such as gender , bubble sign , revision type , bearing for revision tha and bony defect severity between patients demonstrating osteolysis or loosening and the others .
the t - test was also used to compare continuous variables such as age at revision tha and body mass index ( bmi ) .
they both died of an unrelated medical condition at 6.5 years and 16 years after their revision tha procedures , respectively .
one of these two patients could not visit the outpatient clinic due to a geographical reason and did not complain of any hip problems over the phone before death .
the other patient died 16 years after the revision tha and used a cane due to hemiplegia from a cerebrovascular attack .
the median harris hip score increased from 60 points ( range , 35 to 89 points ) before revision tha to 90 points ( range , 3 to 98 points ) at the final follow - up ( fig .
three patients reported a decrease in the harris hip score at the last follow - up .
osteolysis was detected on average 9.3 years ( range , 5.3 to 13.5 years ) after revision thas in 13 hips ( fig .
, the acetabular cup was loosened an average of 9.8 years ( range , 5.7 to 18.3 years ) after revision tha in 9 hips .
re - revision of the cup was performed in 5 hips and re - revision of both the cup and stem was performed in 2 of the 9 loosened hips ( fig .
, cup removal and cutting of the artificial femoral neck were performed due to severe bone loss and vessel injury following cup re - revision due to infection on two occasions . among the other two patients with loosening
, one patient could perform his daily activities with a cane and refused to undergo re - revision surgery .
the other patient was recently recommended revision tha , but did not undergo the procedure under the time constraints of this study .
one of the 4 patients in whom osteolysis without loosening was detected underwent a head and liner change with allogeneic bone grafting into the acetabular bone defect .
the remaining 3 patients who showed osteolysis around the well - fixed implant did not complain of any discomfort and were followed - up without any further operations .
, the 15-year survival rate was 28.2% ( 95% confidence interval [ ci ] , 8.3 to 52.6 ) and 56.0% ( 95% ci , 27.3 to 77.2 ) , respectively ( fig .
statistical analysis did not reveal that any variables among age at revision tha , gender , bmi , bony defect severity , bubble sign , bearings used for revision tha , and revision type had a significant association with osteolysis or loosening after revision tha .
metallosis from a catastrophic failure of a pe liner is a devastating condition . during our study period ,
the revision rate was 30.4% after revision tha , which was very high compared to the re - revision rate of other recent studies reporting the results of revision tha in the presence or absence of metallosis.19,20 ) moreover , the incidences of osteolysis and loosening after revision tha in this study were 56.5% and 39.1% , respectively , and they were also higher than those in other studies.20 ) this study has some limitations that need to be mentioned .
first , there was no control group that included patients who had only catastrophic liner wear without metallosis , for comparing and determining the exact effect of metallosis on the results of revision tha .
however , this study demonstrated that severe metallosis resulting from catastrophic liner wear by itself is a risk factor for the failure of revision tha .
second , we could not identify any factors which affect the results of revision tha following severe metallosis .
twenty - three cases do not constitute an adequate sample size to allow for analysis of other factors that may affect the results of revision tha .
quantification of metallosis or metal wear might give further information on the volumetric effect of the metal problem .
in fact , it is difficult to define the margin of metallosis because there is grossly invisible metallic invasion . during revision tha
, all of the hips showed severe metallosis classified as grade iii in this study .
bony defects before revision tha were also severe , and acetabular defects were noted in all 23 hips .
in contrast , chang et al.7 ) reported osteolysis around the acetabular cup in only 24 of 31 hips ( 77.4% ) demonstrating metallosis , and only 4 of these hips showed a type iii acetabular defect .
their results were not in agreement with those of the current study , in which 15 of 23 hips showed type iii acetabular bony defect.14 ) this may be related to the mechanism of metallosis .
it is known that liner dissociation , catastrophic liner wear , or direct impingement between components , such as shell and stem or shell and screw , can cause metallosis.7,12 ) among them , pe liner wear usually does not cause symptoms that are different from those of liner dissociation or restricted motion due to impingement .
metallosis from catastrophic liner wear can continue to progress , generating many pe and metal wear particles .
pe wear particles are known to cause an inflammatory reaction and subsequent osteolysis.21 ) moreover , metal wear particles and pe wear particles can exert a synergistic effect.10 ) metallosis due to catastrophic liner wear leads to osteolysis and loosening .
the problems caused by metal wear particles have been identified recently due to failed metal - on - metal tha and resurfacing tha.22,23,24,25 ) pseudotumor is one such problem .
pseudotumor was first described as a soft - tissue mass associated with the metal - on - metal resurfacing implant , and it is neither malignant nor infective in nature.25 ) substantial necrosis and a heavy macrophage infiltrate were noted around the pseudotumor .
the adaptive immune response also plays an important role in the development of pseudotumor.23 ) direct cytotoxicity of metal particles and immunologic reaction play an important role in problems related to metal ions.22,24 ) the increased incidence of osteolysis after revision tha following severe metallosis could be partially explained by the same phenomenon . in conclusion ,
the survival rate of revision tha in patients with metallosis following a catastrophic failure of a pe liner was found to be low .
a substantial amount of pe wear debris and the infiltration of metallic wear particles in the periprosthetic tissues might lead to progressive bone loss and implant loosening , even after revision tha . the patient , in whom this catastrophic condition is detected during revision surgery ,
should be informed that he / she has to regularly attend follow - up visits due to a high possibility of additional revision surgeries . | backgroundwear can not be completely prevented after total hip arthroplasty .
if severe polyethylene ( pe ) liner wear develops , the so - called catastrophic failure occurs and metallosis develops .
we postulated that longevity of the new implant may be affected after revision surgery for metallosis following a catastrophic failure of a pe liner due to the substantial amount of pe wear particles and infiltration of the metal particles in this catastrophic condition.methodstwenty-three hips of 23 patients were identified because they showed metallosis during revision total hip arthroplasties performed in seoul national university hospital between january 1996 and august 2004 .
they were followed for at least 6.5 years after the index revision total hip arthroplasty .
the clinical and radiological results of revision total hip arthroplasties in these patients were evaluated.resultsthe median harris hip score increased from 60 points before revision total hip arthroplasties to 90 points at the final follow - up .
osteolysis was detected at an average of 9.3 years after revision total hip arthroplasties in 13 hips and acetabular cup loosening at average 9.8 years after revision total hip arthroplasties in 9 hips . with radiographic evidence of osteolysis and loosening as the end points , the 15-year survival rates were 28.2% and 56.0% , respectively.conclusionsthe survival rate of revision total hip arthroplasty in patients with metallosis following a catastrophic failure of a pe liner was low . |
classic neuroimaging findings described in gm2 gangliosidosis is hyperdense signal in bilateral thalami in non - contrast computed tomography ( ncct ) scan and hypointensity in t2 weighted mri images .
hyperdens thalamus in ncct head in a child with neuroregrssion , facial dysmorphism and organomegaly , should lead us to think about gm1 gangliosidosis .
a 16-month - old girl presented with history of regression of mile stones for last 6 months .
psychomotor development was normal until 12 months of age ; subsequently , she gradually lost ability to sit , neck control , and social smile .
she had attained vocabulary of single syllable by 1 year , but subsequently lost it .
there was no significant family history . on examination , she had coarse facies , horizontal nystagmus , hepatosplenomegaly , and multiple mongolian spots [ figure 1 ] .
on fundus examination , she had bilateral macular cherry red spots [ figure 2 ] .
skeletal survey revealed dysostosis multiplex at dorsolumbar junction , as superiorly notched ( inferiorly beaked ) vertebral bodies in the lateral view at the thoracolumbar junction [ figure 3 ] .
clinical diagnosis of gm1gangliosidosis was supported with mri brain findings and confirmed with absence of -galactosidase enzyme activity in leukocytes [ figure 4 ] .
clinical pictures of a 16-month - oldgirl showing massive hepatosplenomegaly , excessive mongolian spots , and severe failure to thrive photograph of retina in patient with gm1gangliosidosis showing cherry red spot and optic disc edema x - ray of same patient showing dysostosis multiplex at lumbar spine and ncct scan of brain ( axial sections ) at level of thalamus showing bilateral hyperdense thalamus axialt1-weighted image and t2-weighted image at the level of thalamus show diffuse dysmyelination of white matter with bilaterally symmetric thalamic signal change , which appear hyperintense on t1-weighted and hypointense on t2-weighted images
gm1gangliosidosis is a rare autosomal recessive lysosomal storage disorder caused by deficiency of lysosomal enzyme -galactosidase , resulting in progressive neural and visceral accumulation of gm1gangliosides , its asialo derivative ga1 , and other minor glycolipids and glycopeptides .
three clinical phenotypes can be distinguished and classified by age of onset : infantile , late infantile / juvenile , and adult .
the age of onset and rate of progression of the disease depend on the residual activity of enzyme -galactosidase .
infantile gm1gangliosidosis , the most common and severe form , is characterized by facial and skeletal abnormalities and neurological deterioration before the age of 6 months .
this disorder can be diagnosed by several ways , including lysosomal enzyme assay of low -galactosidase activity in peripheral blood leukocytes or cultured skin fibroblasts , detection of abnormal urinary oligosaccharide excretion , and rectal biopsy .
prenatal diagnosis by measurement of enzyme activity in amniotic fluid and cultivated amniotic fluid cells has also been established .
the classic neuroimaging findings in patients with infantile gm1gangliosidosis include thalamic hyper density on non contrast computed tomography ( ncct ) scan and hypointense signal of the thalami with persistent high signal intensity of the white matter on t2-weighted images , indicating severely defective myelination[13 ] [ figure 4 ] .
significant white matter abnormalities are present only in the infantile form of gm1gangliosidosis , but they are also described in other infantile - onset neuronal storage disorders , such as gm2gangliosidosis and infantile neuronal ceroid lipofuscinosis . a paucity of myelin in gm1gangliosidosis has been demonstrated by several classical neuropathologic and neuroimaging studies of patients and animal models . | gm1gangliosidosis is a rare autosomal recessive lysosomal storage disorder caused by deficiency of enzyme -galactosidase , resulting in progressive neural and visceral accumulation of gm1gangliosides .
coarse facial features , bilateral cherry red spots , and dysostosis multiplex are important clue to diagnose this condition .
we describe a case of gm1gangliosidosis with dysostosis multiplex and characteristic magnetic resonance imaging findings . |
malignancy is a recognized complication of renal transplantation . in australia , it is estimated that 25% of all renal transplant recipients surviving for 20 years will develop a non - skin cancer .
renal cell carcinoma ( rcc ) accounted for 4.6% of de novo cancer in solid organ transplant recipients , but the majority occurred in native kidneys and less than 10% in allograft kidneys .
a number of case reports have been published on partial nephrectomy as a treatment option particularly when the rcc is less than 4 cm [ 36 ] .
we report a case of rcc in a kidney allograft compromised by previous segmental infarction and chronic allograft nephropathy ( can ) , which was treated successfully with nephron - sparing partial nephrectomy .
a 47-year - old woman with end - stage renal disease due to reflux nephropathy underwent deceased donor renal transplantation in 1994 .
sirolimus was changed to tacrolimus 2 years later due to heavy proteinuria ( 4.5 g / day ) .
eleven years after transplantation , a 3.3 cm mass was found in the lower pole of the renal allograft during transabdominal ultrasound for investigation of pelvic pain .
magnetic resonance imaging confirmed the presence of a complex cystic mass that was highly suspicious for a rcc .
there was no radiological evidence of local and distant metastases or involvement of the native kidneys . at that time
post - surgery , she developed acute tubular necrosis of the allograft and required three haemodialysis sessions .
she subsequently made an uneventful recovery with serum creatinine 272 mol / l on discharge .
three years after surgery , there is no evidence of rcc recurrence and she remains free of renal replacement therapy .
traditionally , the treatment for rcc arising from kidney allografts has been allograft nephrectomy , withdrawal of immunosuppressive therapy and resumption of dialysis . however , recent data suggest that partial nephrectomy is emerging as a viable alternative option .
butler et al . reported that radical nephrectomy and partial nephrectomy provided equally effective treatment for single , localized rcc of less than 4 cm in native kidneys .
one long - term follow - up study has found the cancer - specific survival to be 100% at 10 years in patients who underwent partial nephrectomy with tumours less than 4 cm .
rcc involving transplant recipients appear to have similar biological behaviour as those involving patients without immunosuppression . as a result
, the use of partial nephrectomy has been extrapolated into the management of rcc in kidney allografts .
there were 18 cases of tumour in renal allografts treated by partial nephrectomy reported in the worldwide literature .
of these cases , 13 ( 81% ) had rcc , 2 had oncocytoma , 1 had angiomyolipoma and the pathology was not reported in 2 cases .
of the rcc cases , 11 cases ( 85% ) were small lesions measuring less than 4 cm while one was 6 cm and the other 9 cm .
mean time of diagnosis after transplantation was 11.1 years ( range : 1 month to 21.5 years ) . in this series ,
mean follow - up after partial allograft nephrectomy was 32 months ( range : 0.5138 months ) with no patient reported to develop local recurrence , renal failure or distant dissemination .
although the data are limited , the evidence so far indicates that nephron - sparing surgery provides a durable cancer control of tumours less than 4 cm without significant compromise to graft function .
therefore , partial graft nephrectomy may be proposed as the treatment of choice for tumours less than 4 cm .
this would avoid returning patients to dialysis that is associated with inferior life expectancy and quality of life when compared with a functioning graft .
moreover , partial graft nephrectomy avoids overtreatment of unexpected non - malignant tumours . in the majority of previous cases ,
partial graft nephrectomy was performed on good functioning allograft with serum creatinine between 79 and 158 mol / l .
however , in the case described , the graft function is compromised because of segmental infarction and can .
partial graft nephrectomy in allograft kidneys with severe compromised function has only been reported in two previous cases , one with creatinine of 211 mol / l and 222 mol / l in the other .
graft function in these two cases was still preserved after 6 and 22 months , respectively . to our knowledge ,
the described case is the first to have undergone successful partial allograft nephrectomy in the setting of segmental infarction .
it is performed in a non - nave surgical field where severe inflammation and scarring are usually present , making kidney mobilization , pedicle control and parenchymal resection often difficult .
the continuous perfusion of the allograft with the cooled ross solution preserved the residual kidney function , as seen in the merkel 's case and the current case .
this continuous perfusion also made it easier to identify and individually ligate all vessels in the cut surface to minimize blood loss .
intra - operative ultrasound can assist in gaining satisfactory clearance and preserve renal parenchyma for optimal renal function .
it also allowed exact determination of the extent of these lesions if polycentricity is present .
rcc arising from a renal allograft is rare . data are accumulating to suggest that preservation of renal function can be achieved by partial nephrectomy without sacrificing control of the malignancy . | the increased risk of malignancies is a well - recognized complication of organ transplantation .
when renal cell carcinoma ( rcc ) occurs in kidney transplant recipients , less than 10% of it affects the allograft .
recent experience suggests that partial allograft nephrectomy for tumours less than 4 cm may be considered the treatment of choice .
we report a case of a 3.3 cm rcc discovered in a renal allograft .
limited allograft function was due to segmental infarction after transplant surgery and chronic allograft nephropathy .
she underwent successful partial allograft nephrectomy . at 36 months post - surgery , there is no evidence of rcc recurrence and she remains free of renal replacement therapy . |
it is recognized that the growing epidemic of obesity is linked to a recent decline in physical activity levels .
socioeconomic changes have led to profound changes in individual 's lifestyle including the adoption of unhealthy food consumption patterns , prevalent tobacco use , alcohol abuse , and physical inactivity .
regular physical activity is an important aspect for prevention of noncommunicable disease ( ncd ) , and inactivity is the fourth largest contributor to global mortality and morbidity .
physical activity is defined as any force exerted by skeletal muscles that results in energy expenditure above resting level .
it can be in the form of lifestyle activity ( such as climbing stairs or brisk walking ) or structured exercise or sport , or a combination of these . in daily life ,
physical activity is undertaken in several domains such as work , transport , and leisure time .
the centre for disease control recommends at least 30 min of moderate - intensity physical activity for at least 5 days per week for adults . according to the world health organization ( who ) health report ,
globally around 31% of adults aged 15 years and over were insufficiently physically active in 2008 .
noncommunicable disease risk factor surveillance reported that the job - related moderate- and vigorous- intensity physical activity in urban , slum , and rural population was 35.8% , 55.2% , and 61% , respectively , whereas leisure time , moderate- and vigorous - intensity physical activity was 15.6% , 12.1% , and 14% , respectively .
health professionals have an important role in guiding patients to adopt healthy lifestyles through behavior modification and mediated intervention .
the more the health - care professionals carry out good personal health habits , the more likely they are to counsel their patients on a range of behaviors such as physical activity , smoking , alcohol abusing , and having a balanced diet .
mckenna et al . concluded that health - care professionals who are physically active themselves are three times more likely to regularly promote physical activity in their patients .
frank et al . reported that when doctors demonstrate their own personal healthy habits ; patients find them to be more believable and better able to motivate changes in their diet and their physical activity levels .
dental health professionals are at a greater risk to develop musculoskeletal disorders due to stress onto certain muscles contributed by awkward postures during dental procedures , prolonged static and unsupported sitting , extended work hours , and the impact of working with thin instruments in overextended positions throughout the day . in a study by sharma and golchha ,
75% of dental practitioners were at a risk for developing work - related musculoskeletal disorders , and the prevalence and severity of these disorders decreased by performing regular specific exercises by 20% and 80% , respectively , thus participation in physical activity throughout life can help in maintaining musculoskeletal health . recognizing this importance of physical activity among health professionals , the present study aimed to assess physical activity among dental health professionals in hyderabad city , using global physical activity questionnaire ( gpaq ) .
a cross - sectional questionnaire study was conducted to determine the level of physical activity among dentists .
participants were personally interviewed by a single trained interviewer to gather information on demographics data and levels of physical activity using 16-item gpaq .
show cards with photographs of different physical activities were used to provide a more objective measure to the respondents .
gpaq developed by the who , measures the frequency ( in days ) and time ( in minutes / hours ) spent in doing vigorous and moderate - intensity physical activity in a typical week in three domains and sedentary behavior ( p16 ) .
the three domains include :
work - related physical activity ( p1-p6)travel to and from places ( p7-p9)discretionary leisure time ( recreational ) physical activity ( p10-p15 ) .
work - related physical activity ( p1-p6 ) travel to and from places ( p7-p9 ) discretionary leisure time ( recreational ) physical activity ( p10-p15 ) .
the unit metabolic equivalents ( mets ) were applied for measuring physical activity energy expenditure . met
one met is defined as 1 kcal / kg / h and is equivalent to the energy cost of sitting quietly . the frequency , intensity , and the met values in all three domains and sedentary behavior and the levels of physical activity based on physical activity cutoff value were calculated using gpaq analysis guide .
the cut off values for vigorous activity is 8 , for moderate intensity is 4 and for transport activity is 4 .
the questionnaire was pilot tested and proved to have a good internal validity with a cronbach 's value of 0.8 .
the study was approved by the ethical committee of the institutional review board ( pmvids / phd/0018/2013 ) .
statistical analysis was done using statistical package for social sciences software ( spss version 12.0 ) , statistical data 2003 spss , inc .
independent student 's t - test and one - way analysis of variance were used for comparison among the variables .
chi - square test was used to determine the association between the variables . the level of significance ( p value )
overall , 85.7% of the 365 dental health professionals agreed to participate in an interviewer - based questionnaire study .
the study included 163 ( 52.1% ) males and 150 ( 47.9% ) females with a mean age of 32.64 5.20 years .
the majority of the study population belonged to the age group of 3140 years ( 52.7% ) and about 73.2% of dental health professionals were married .
majority of the study population had a master 's degree ( mds : 236 [ 75.4% ] ; bds : 77 [ 24.6% ] ) with 33.9% in private practice , 21.1% in the teaching field , and 45% in both private practice and teaching field . out of the total sample ,
16.6% of participants carried out vigorous - intensity activity at work and statistically significant association was observed with gender ( p = 0.00 ) , age ( p = 0.002 ) , and marital status ( p = 0.00 ) . it was observed that 26.4% of males , 25.2% of participants in the age group of 2130 years and 37.5% of professionals who were single did vigorous - intensity physical activity . when moderate - intensity physical activity at work was considered ,
51.1% of participants carried out and significant difference was noted with gender ( p = 0.01 ) , age ( p = 0.04 ) , and employment ( p = 0.0002 ) , where 57.7% of males , 56.6% of participants in the age group of 2130 years and 59.6% of professionals employed in both private practice and teaching field performed moderate - intensity activity .
a high number of participants in the age group of 4150 years reported that they did not perform any vigorous - intensity ( 95.2% ) and moderate - intensity ( 71.4% ) activity at work [ table 1 ] .
responses of study population based on physical activity at work , commuting , and recreational activities when activity during commuting was considered , the majority of participants carried out ( 68% ) and further significant difference was observed with all demographic variables except employment . more number of females ( 76.6% ) and around 90% of professionals who were single and those possessing bachelor 's degree either walked or cycled to places [ table 1 ] .
a very few participants carried out recreational activity ( vigorous 8.63% , moderate 20.1% ) wherein gender and employment ( p = 0.001 ) were significant for both vigorous- and moderate - intensity recreational physical activities , whereas marital status ( p = 0.03 ) also played a significant influence in moderate - intensity recreational activity [ table 1 ] . the total mean number of days spent on vigorous physical activity was 4.51 1.83 and the mean minutes were 42.6 40.9 .
a statistically significant association in mean days spent was observed only with the level of education ( p = 0.03 ) , with professionals having bachelor 's degree spending on more days ( 5.31 1.44 ) .
however , mean time spent on vigorous physical activity was not significant with any demographic variable . on the other hand , mean days spent on moderate physical activity at work was 4.98 1.52 and the mean minutes were 46.7 61.6 .
the mean number of days spent was significant with all demographic variables except with gender ( p = 0.11 ) .
although professionals in private practice did the activity at work on more days ( 5.78 1.20 ) , the mean time spent was more with those in teaching field ( 77.3 103.4 ) [ table 2 ] .
duration spent on activity at work , commuting , and recreational activities by study population in case of traveling to and from places , mean days spent was 5.6 1.42 and the mean minutes were 46.7 61.6 .
the mean number of days spent showed a significant association with all demographic variables , i.e. , gender ( p = 0.0003 ) , age ( p = 0.00 ) , marital status ( p = 0.00 ) , education ( p = 0.00 ) , and employment ( p = 0.00 ) .
nevertheless , a significant difference in mean time spent to travel to and from places was observed only with the age of dental health professionals [ p = 0.01 , table 2 ] .
the total mean number of days spent on vigorous - intensity recreational activity was 3.9 1.79 and for moderate
intensity recreational activity was 4.00 1.93 , and the mean minutes were 59.0 42.6 , 46.8 38.3 , respectively .
no demographic variables showed a significant difference with mean days spent on vigorous - intensity recreational activity , nonetheless for moderate - intensity recreational activity , marital status ( p = 0.01 ) , education ( p = 0.0001 ) , and employment ( p = 0.0007 ) were significant . when the mean time was considered for the same ,
significant difference was observed with employment ( for vigorous intensity p = 0.0009 ) and age ( for moderate intensity p = 0.03 ) [ table 2 ] . for the total
mean , mets minutes per week , activity at work , and recreational activity showed a significant difference for marital status and employment where total met scores were high for professionals who were single and those in teaching field .
the total met minutes per week for traveling to and from the place was significant with gender ( p = 0.01 ) , marital status ( p = 0.00 ) , and education ( p = 0.00 ) .
likewise , overall met minutes per week for all physical activity ( at work , commuting , and recreational ) revealed that professionals who were single ( 513.63 588.32 ) and those in teaching field ( 487.05 670.25 ) carried out activity for more minutes [ table 3 ] .
physical activity of study population in mean metabolic equivalents per week in case of mean time ( in minutes ) spent on sedentary behavior , significant association was seen with age ( p = 0.01 ) , education ( p = 0.001 ) , and employment ( p = 0.0004 ) .
it was observed that professionals of age group of 4150 years ( 342.85 135.02 ) , mds ( 340.32 97.64 ) , and those in teaching field ( 363.71 114.18 ) spent more time in sedentary behavior [ table 4 ] .
mean time ( in min ) spent by study population on sedentary behavior when the participants were categorized based on the level of physical activity , it was observed that most of the participants were in moderate activity ( 58.7% ) and was statistically significant with all the variables that are considered in the study .
based on the level of physical activity ( both moderate and severe ) , 60.7% of professionals were considered to be active and 39.3% of them were inactive [ table 5 ] .
according to the who , health has been defined as a state of complete physical , mental , and social well - being and not merely the absence of disease or infirmity .
it is increasingly acknowledged that health is maintained and improved not only through the advancement and use of health science but also through lifestyle choices ( diet , physical activity , and hygiene ) of the individual .
the role of physical environment as a key determinant of health has also been emphasized by the who .
almost 2 million deaths per year are attributable to physical inactivity , thereby making physical activity as the
despite the global concern regarding ncds , increasing obesity and rapid changes in the pattern of work , transport , and recreation ; physical activity surveillance and monitoring are carried out only in few countries . to address this societal issue , action and partnership
are warranted across a broad range of sectors and professions , many of which do not have physical activity as a core element .
health professionals play an important role in guiding patients to adopt healthy lifestyle for prevention of ncd .
mckenna et al . concluded that health professionals who are physically active themselves are three times more likely to regularly promote physical activity in their patients .
although several studies assessed the prevalence of physical activity among general population , there is a dearth of literature among health - care professionals . recognizing this importance , the present study was conducted to determine the prevalence of physical activity among dental health professionals in hyderabad city , telangana , india . in this study ,
gpaq was employed as it is domain specific ( activity at work , commuting , and recreational activity and sedentary behavior ) , quantifies exposure with cross - cultural application .
the validity and reliability are slightly better than other questionnaires ( international physical activity questionnaire ) and physical activity measures ( pedometer ) . in the present study ,
60.7% of the study group were classified as active ( moderate and high ) , with activity at work and commuting activity being the main contributors to total physical activity .
this was comparable with the findings of studies by trinh et al . and guthold et al .
on the other hand , singh and purohit reported physical inactivity among 68% of dental health - care professionals in india . in this study , it was observed that activity at work and recreational activity was carried out mostly by males , whereas females ( 76.7% ) engaged more in commuting activity .
it was also noted that with increasing age , there was a decrease in physical activity with more sedentary behavior , confirming the report of previous work by mcgrady et al .
similar results observed in a study done by frantz and ngambare , participants below the age of 35 years had higher physical activity promoting practices ( 51% ) than any other age group and males ( 46% ) had high physical activity promoting practices compared to the females ( 20% ) .
the current study showed that only 16.6% of participants carried out vigorous - intensity physical activity at work and was not influenced by the variables considered in the study ; on the other hand , the majority of the participants ( 51.1% ) carried out moderate - intensity activity at work and all the variables considerably influenced except for gender . on the other hand , kunene and taukobong found that nearly two - fifth ( 38.5% ) engaged in vigorous and almost half ( 49.5% ) in moderate work - related physical activities for at least 10 min continuously .
it was observed that the mean number of days spent by the participants in the age group of 3140 years were less when compared to other age groups .
further participants with bds degree were more active than mds professionals , and this might be due to time and age factor . although professionals in private practice and both did the activity at work on more days ( 5.78 1.20 ) , the mean time spent was more with those in teaching field ( 77.3 103.4 ) .
the main reasons for this decreased physical activity may be due to the working hours of the clinics and few dentists who work in dental colleges during the day and then continue with their private practice in the evening .
when commuting activity was considered , all the variables had influenced only for the mean number of days spent on activity and not for the mean minutes spent except for the variable age .
professionals who were single , professionals with bachelor 's degree , and those in teaching field had spent more mean days and time .
it was also observed that most of the respondents did not carry out recreational activity ( vigorous 8.63% , moderate
similar findings were observed in a study done by kunene and taukobong , very few engaged in vigorous ( 23.9% ) and moderate ( 37.6% ) sport and moderate , fitness , or recreation physical activities . in a study by kunene and taukobong
, age and gender showed a significant relation with activity at work and recreational activity , and the time spent sitting and reclining among participants was significantly related to age .
however , in our study , age and gender did not influence activity but age showed a significant relation with sedentary behavior . when total mean mets minutes per week was accounted , professionals of age group of 2130 years , professionals who were single , those with bachelor 's degree and those in teaching field performed activity at work , commuting , and recreational activity for more minutes with overall high met minutes per week and least sedentary behavior .
regular physical activity is recommended for all , including dental health professionals as it is one of the keys to counteract the current epidemic of obesity and ncd and musculoskeletal work - related disorders .
, in his 2-year follow - up study among male health professionals , concluded that both sedentary and nonsedentary activities represent separate domains , each with independent risks for overweight . to increase the rates and quality of future physical activity counseling delivered by doctors , lobelo et al .
concluded that medical schools need to increase the proportion of students adopting and maintaining regular physical activity habits .
thus , developing higher physical activity levels in the budding years may limit the in - progress epidemic of childhood , adolescent obesity and resultant adult obesity , and ncd , by establishing the foundations , skills , and attitudes needed for good lifelong habits .
( it 's a justification for the finding of the study which is required to be mentioned ) however , we acknowledge that our study has few limitations , as it measures reported activity rather than actual activity , thus dental health professionals may be prone to overestimate their exercise habits precisely because they know the benefits of physical activity and what they should be doing and within the confines of the present study general population could not be included .
the prevalence of physical activity ( 60.7% ) was high among dental health professionals in this study , with activity at work and commuting activity being the main contributors to total physical activity .
activity at work and recreational activity were carried out mostly by males , whereas females engaged more in commuting activity and it was also observed that there was a decrease in physical activity with increasing age .
all the demographic variables were shown to influence the activity at work and commuting activity . when recreational activity was considered , majority of the respondents had less mean mets per week on comparison to activity at work and commuting activity as they did not carry out recreational activity .
this higher prevalence of physical activity in the study group may be because the respondents were all health professionals and their education , income , and occupation likely led them to engage in healthier behaviors .
since the personal habits of professionals influence their patients , healthy lifestyle should be encouraged , and further efforts should be made to promote activity among those who are physically inactive .
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 : to assess and compare physical activity based on age , gender , marital status , education , and employment among dental health professionals in hyderabad city , india.materials and methods : a cross - sectional questionnaire study was conducted among dentists and they were personally interviewed by a single trained interviewer . the frequency ( in days ) and time ( in minutes ) spent in doing vigorous- and moderate - intensity activity in a typical week in three domains and sedentary behavior
were assessed using 16-item global physical activity questionnaire .
metabolic equivalents ( mets ) were used to express the intensity of physical activities.results:overall , 60.7% of the 313 respondents were physically active , with activity at work and commuting activity were the main contributors of physical activity . with increasing age , there was a decrease in physical activity with more sedentary behavior , professionals in age group of 2130 years , who were single , those with bachelor 's degree and those in teaching field performed activity at work , commuting , and recreational activity for more minutes with overall high met minutes per week and least sedentary behavior.conclusion:the prevalence of physical activity was high among dental health professionals . |
over the last few years , the involvement of the proteasome pathway in ligand - dependent nuclear receptor degradation has been established .
this pathway entails first a polyubiquitination of the substrate , catalyzed by three factors ( e1 , e2 and e3 ) , followed by its proteolysis by the proteasome .
recently , the role of the csn complex that regulates the activity of a class of e3 ubiquitin ligases , the cullin ring ubiquitin ligase superfamily , was demonstrated . the cullin subunits of these ubiquitin ligases are modified by the conjugation of an ubiquitin - like protein , nedd8 .
recent data demonstrate that csn , via its subunit csn5/jab1 , dennedylates cullin , thus increasing the activity of the e3 ligases [ wee et al . ,
several lines of evidence suggest that csn is involved in ligand - dependant nuclear receptor degradation .
first , estrogen receptor ( er ) degradation is dependant on the neddylation pathway [ fan et al . , 2003 ] ; second , csn5/jab1 interacts with both the progesterone receptor and the coactivator src1 and is itself a coactivator of the nuclear receptors [ fan et al . , 2003 ] ; third , jab1/csn5 increases hormone - induced er degradation [ fan et al . ,
the nature of the ligand affects er degradation differently : estradiol and the pure antagonist fulvestrant induce er degradation by the proteasome , whereas the mixed antagonist tamoxifen stabilizes er [ wijayaratne and mcdonnell , 2001 ] .
thus , degradation may play an important role in er function and/or the action of its antagonists .
er undergoes post translational modifications such as phosphorylation [ lannigan , 2003 ] , acetylation [ wang et al . ,
at least two curcumin - sensitive kinases , ckii and pkd , which co - purify with csn , could contribute to this activity [ uhle et al .
the inhibition of er degradation by curcumin and the co - immunoprecipitation of jab1/csn5 with er in the presence of curcumin , suggest that this kinase activity could participate in targeting er for degradation .
in addition , the inhibition by curcumin of the interaction of er with its dna target , points towards a role of this complex in transcription activation [ callige et al . ,
the discovery that e2 and e3 factors and proteasome subunits associate with the transcriptional machinery , reinforces the hypothesis that the degradation of nuclear receptors could be linked to transcriptional activation and may be necessary for efficient transcriptional activity [ nawaz and o'malley , 2004 ] . here
we discuss er degradation pathways in the presence of different ligands and the role of this degradation in er function .
the demonstration that the nedd8 pathway is required for proteasome mediated degradation of er , suggests that the e3 ligases involved belong to the cullin ring ubiquitin ligase superfamily . within this family , mdm2 and e6-ap , were identified as er coactivators [ nawaz et al . , 1999 ; saji et al . ,
mdm2 is also involved in the degradation of glucocorticoid and androgen receptors [ kinyamu and archer , 2003 ; lin et al . ,
er could also be a substrate for brca1/bard1 , another potential e3 ligase , which is recruited by phospho - pol ii and is involved in the degradation of both chromatin proteins and active rna polymerase ii [ starita and parvin , 2003 ] .
one could speculate its involvement in er degradation since this degradation is concomitant with transcriptional activation .
however brca1/bard1 belongs to the hect e3 ligase family , and there is no evidence that the need8 pathway regulates its activity .
transcription activation by er involves a number of co - regulators , in particular coactivators of the p160 family ( src1/src2/src3 ) .
the activity of src3 ( the major er coactivator in breast cancer cell lines ) is regulated by phosphorylation [ wu et al . ,
hormone - induced transcription requires the dissociation of a corepressor ( n - cor / smrt ) from er and its replacement by a coactivator from the p160 family .
tblr1 , a protein that shares homology with tbl1 ( transducin-like1 ) , selectively mediates corepressor / coactivator exchange upon ligand binding to nuclear receptors [ perissi et al . , 2004 ] .
several observations converge towards a link between the recruitment of er cofactors and ligand - dependent degradation by the proteasome .
suppression of src3/aib1 by sirna leads to er stabilization in presence of estradiol [ shao et al . ,
2004 ] and src3/aib1 itself is degraded by the proteasome in a hormone - dependent process [ perissi et al . ,
nuclear receptor co - factors such as tbl1 and tblr1 ( originally identified as components of an n - cor complex ) , besides their er-cofactor exchange activity , serve as specific adaptors for the recruitment of the conjugating/19s proteasome complex and thus participate in co - factor dynamics at the promoter during the transcription initiation process [ perissi et al . ,
inhibition of crm1-dependent nuclear export by leptomycin b blocks estrogen - dependent , but not fulvestrant - induced degradation of er. this demonstrates that the two ligands induce er degradation through different pathways , taking place in different cellular compartments . in the presence of estradiol er
is degraded by a cytoplasmic fraction of the proteasome , whereas in the presence of fulvestrant it is degraded in the nucleus [ callige et al . ,
these findings reinforce observations demonstrating rapid sequestration of the er-fulvestrant complex in a salt - insoluble , nuclear compartment [ giamarchi et al . , 2002 ] and
reduced mobility of the er-fulvestrant complex relative to the estradiol - er complex in the nucleus [ stenoien et al . ,
the fact that fulvestrant is unable to promote either chromatin remodeling over the tff1 estrogen - regulated promoter [ giamarchi et al . ,
2002 ] , or binding of er to its dna target within the same promoter [ reid et al . , 2003 ] , indicates that the nuclear sub - compartment in which the fulvestrant - er complex is degraded , must be distinct from the sub - compartment in which transcription takes place .
the nature of the nuclear compartment in which this degradation takes place remains unknown . to understand better the mechanism of action of the pure antiestrogen fulvestrant , several issues
need to be elucidated : what is the nature of the compartment in which er is sequestered ?
does fulvestrant play a direct role in such targeting , e.g. , by directly recruiting a cofactor that changes the fate of er-fulvestrant complex ?
do corepressors or coactivators play a role in this process ( as suggested by the fact that suppression of src3/aib1 by sirna leads to er stabilization in presence of estradiol but not of fulvestrant ) [ shao et al . ,
the pathways involved in er degradation differ , depending on the ligand ( hormone or full antagonist , such as fulvestrant ) .
a phosphorylation event catalysed by a curcumin - sensitive kinase , that could be ckii , is necessary for both estradiol- and fulvestrant - induced er degradation [ callige et al . ,
2005 ] , making it unlikely that this phosphorylation plays a role in the targeting of er to different degradation - compartments .
the exact role of src3/aib1 in each step of the hormone - induced er degradation process remains to be determined .
the fact that er-oh - tamoxifen complex ( that can not recruit coactivators ) is not released from target promoters [ wijayaratne and mcdonnell , 2001 ] , along with the observations that src3 is co - degraded with er [ shao et al . ,
2004 ] and that suppression of src3 expression abrogates er degradation [ lonard et al . ,
a possible role of the csn in src3/aib1 phosphorylation and degradation should be investigated .
figure 1 presents a model summarizing the different steps that may be involved in estradiol - induced er degradation and the possible links with transcription activation .
er-hormone complex interacts with a coactivator ( src3/aib1 in breast cancer cells ) and with csn5/jab1 .
er and/or src3/aib1 are phosphorylated by a curcumin - sensitive kinase , possibly associated with the csn , leading to the binding of er to promoters of estrogen regulated genes .
curcumin prevents such binding either because er or src3/aib1 phosphorylation is required for dna binding , or because phosphorylation is required for a stable interaction between er and src3/aib1 . upon transcription initiation ,
exchange factors such as tbl1 or tblr1 should facilitate recruitment of coactivators by er and of ubiquitin ligase factors at the promoter .
er and/or src3/aib1 may be tagged for degradation as a result of a post - translational modification and/or a polyubiquitination .
leptomycin b inhibits er degradation and increases hormone dependent transcription , suggesting that the modifications ( putative tagging and/or polyubiquitination ) can be reversed and the complex recycled . the csn could be involved at that step through the recruitment of a deubiquitinating enzyme . in the cytoplasm ,
a first set of results suggests that er degradation is required to sustain transcription [ lonard et al . , 2000 ;
reid et al . , 2003 ] , yet another set concludes that er degradation has no general direct effect on transcription [ alarid et al . , 2003 ; callige et al . , 2005 ; fan et al . , 2003 ] .
it should be noted that the experiments suggesting that er degradation is required to sustain transcription , used treatment times with proteasome inhibitors or drb , for longer than 12 hours [ lonard et al . , 2000 ;
such a long treatment time may have pleiotropic effects on the cell and affect multiple components of the transcription machinery .
in contrast , the experiments suggesting that er degradation has no influence on transcription , used much shorter treatment times ( less than 3 hours ) , minimizing general effects of the inhibitors used .
er degradation seems rather to be the consequence of transcription initiation , as shown by the inhibitory effect of curcumin , rather than transcription per se , since drb that blocks transcription does not prevent hormone - dependent er degradation [ alarid et al .
in addition , the nature of the transcription factors recruited , along with er , as well as the architecture of each promoter seem to be important , suggesting that the effect of proteasome inhibitors on transcription results from the degradation of co - regulators , rather than from that of er [ callige et al . , 2005 ; fan et al . , 2003 ] . whatever the role er degradation plays at the molecular level of transcription activation , ligand induced er degradation may be of importance at the physiological level .
estradiol forms a stable complex with er. this hormone - receptor complex should sustain transcription for extended periods of time .
degradation of the er-hormone complex at each round of transcription could be a way for the cell to fine tune transcription activation in response to rapid changes in hormone concentration .
| in this perspective we consider new aspects of ligand - induced estrogen receptor ( er ) degradation .
what are the possible roles of csn5/jab1 and the csn complex in this process ?
we compare hormone ( estrogen ) or pure antagonist ( fulvestrant ) induced degradation of er and review the effects of kinase - inhibitors and crm1-dependent nuclear export on er degradation and transcription activation . a model for er action integrating these new actors
is proposed and the relation between hormone - induced er degradation and transcription - activation is discussed . |
in many countries , occupational accidents have impaired the health and safety of workers in various professions . in 1998 , france reported 1.35 million occupational accidents among 14.5 million workers , which was a 3% increase in the number of accidents reported in 1997 .
of these accidents , 52% caused injuries ( 1 , 2 ) , and they also cause social and economic problems , disability , and loss of work time , increased healthcare costs , and other adverse consequences ( 37 ) . according to the international labor organization ( ilo ) , there are more than 15 million work - related accidents annually on a worldwide basis , and about one million people die each year due to occupational accidents ( 8) . the mean rate of fatal occupational accidents in the world is 14 such accidents per 100,000 workers , with europe reporting 5.89 fatal accidents per 100,000 workers ( 9 ) , and the u.s . reporting 3.2 fatal accidents per 100,000 workers ( 10 ) .
however , according to the bureau of labor statistics in iran , 43% of accidents are accompanies with injuries ( 11 ) . according to statistics released by the social security organization in iran in 2003
, 268 people died in 14,114 accidents that occurred at the workshops ( 12 ) .
accordingly , a study in semnan between 2002 and 2006 , the rate of occurrence of fatal accidents or other injuries at work was reported to be 3.8 per 100,000 workers ( 13 ) . electrical burns account for a significant portion of accidents that lead to death .
it has been reported that 42% of events that led to death were due to work - related electrocutions and contact with electrical lines between 1992 and 2002 ( 14 ) .
iran is not exempt from this trend , and each year , many citizens incur electrical injuries due to equipment failures and unauthorized use of power sources .
thus , incidents involving injuries due to electrical shocks during the five - year period from 2005 through 2009 were analyzed to study the main causes of such incidents among the citizens served by the western tehran province electricity distribution company .
the purpose of the study was to determine how to prevent such incidents and their consequences , which range from minor injuries to death .
so , in addition to providing detailed information concerning the magnitude of this problem , the results of this study can be a pioneering effort in regional planning to keep citizens safe .
the overall aim of the study was to investigate accidents involving electrical shocks and related factors for the citizens served by the western tehran province electricity distribution company during the specified time period .
this study was conducted using a descriptive - analytical approach in 2010 . in this study , accidents involving electrical shocks to citizens in the specified area were investigated in detail .
the study population consisted of all citizens at risk of exposure to electrical shocks in western tehran province .
the statistical samples were selected from reports of citizens accidents that were filed in the service areas , which included savejbolagh , shahriyar , eastern karaj , qods city , southern karaj , western karaj , malard , and mehrshahr from 2005 to 2009 .
data collection was performed using information recorded buy the western tehran province electricity distribution company .
the variables that were studied included the year of occurrence , comparison of the numbers of accidents in the nine areas covered , the causes of the accidents , the consequences of the accidents , the gender of the people involved , and the times that the accidents occurred .
the data recorded by the electricity distribution companies in west of tehran were used in this study . after obtaining the necessary permits to get information from the offices that register accidents that involve electricity distribution companies , the required data were extracted for each region .
after the data were collected , they were analyzed using spss ( statistical package for social sciences ) , version 16 .
the results showed that highest accident rates occurred in 2005 and in the first half of 2009 .
also , the data indicated that the maximum rate of accidents occurred in the last 10 days of the months ( table 1 ) .
the data indicated that there were 11 cases of fatal electric shock , with the three deaths in the qods city area in 2009 being the largest number recorded in any of the nine specific areas ( table 1 ) .
our investigation indicated that about 36.7% of accidents were caused by exposures to electricity transmission networks during in buildings ( 10% ) and during unauthorized construction operations of residential houses in the areas of medium voltage ( mv ) and lowest voltage ( lw ) lines ( 26.7% ) .
most of the accident victims were men ( 27 out of 30 or 90% ) .
the results of the analysis of the eight regions over about five - year period indicated that most of the accidents occurred in the malard area ( 14 out of the 30 incidents ) and the next highest number ( six accidents ) was in the qods city region .
our study showed that most of the accidents , i.e. , 46% , occurred in the summer .
the mean age of the accident victims was determined to be 35 , with accidents occurring among people in the age range of 20 to 70 .
the analysis of the data based on level of education indicated that most of the accidents ( 24 of 30 , 80% ) involved people at the lower education levels ( diploma and lower ) .. however , there were only five accidents involving people with an associate degree , and there was only one accident that involved a person with a b.s .
the data indicated that 36.7% of the accidents ( 26.7 + 10 ) occurred during the construction of illegal houses in private areas where there were medium - voltage ( between 1 to 33 kv ) power lines .
the results showed that there was an increasing trend of such accidents over the period of the study .
however , due to the lack of national statistics concerning accidents involving electrical shocks , it was not possible to reach any conclusions concerning the occurrence of such accidents nationwide .
however , some studies have reported an upward trend in accidents at near of power plants ( 14 ) .
( year ) conducted a study of the electricity distribution company in alborz province to assess the rate of occupational accidents , and they noted that there was an increasing trend four such accidents over an eight - year period ( 15 ) .
thus , considering the results of rahmani et al.s study and the results of our study , both of which indicated increasing trends in electrical accidents , effective management is in order to improve the safety of the electricity distribution company in west of tehran province is required .
mahmudi suggested that merely having knowledge and awareness does not lead to good performance ; he stated that attitudes also must change , and he said that the structure of people s beliefs must be based on deep scientific principles to function properly ( 16 ) .
the highest accident rate occurred in 2008 and 2009 , and the main causes of these accidents performing construction operations in the power network area and the exposure of buildings in the area to lowest - voltage ( lower than 1 kv ) lines .
also , the control chart that was plotted based on unsafe behavior measurements ( 16 ) showed that unsafe behavior and unsafe conditions lead to more frequent electrical accidents involving civilians .
the results of our study indicated that 36.7% of accidents resulted in death and that the victims were burned in 33.3% of the accidents .
the results of our study were in good agreement with the results of naqvi knjyn and rahbar s study , which reported that the citizens mortality rate was 38% ( 14 ) .
in tirasci et al.s study , electrocution was reported as the second highest cause of death in the construction industry in the united states and turkey ( 17 ) .
rahmani et al.s study on occupational accidents in the electricity distribution company in west of tehran indicated that 75% of such accidents occurred due to the negligence of workers and that most of the incidents resulted in workers being burned ( 15 ) .
rahmani et al.s study and the findings of this study both indicated that there was a very high burn rate in the accidents that have occurred in the west of tehran .
lack of standardization of power transmission lines in this company could be one explanation for the high rate of burns among citizens .
therefore , it is recommended the relevant authorities take proper actions by providing solutions to reduce the accidents and the associated burns that occur in the company .
the results showed that most accident victims were male ( 90% ) , and this finding was consistent with the results of sheikhazadi et al.s study conducted in tehran in 2009 ( 18 ) and naqvi knjyn and rahbar s study ( 14 ) .
the high rate of electrocution accidents in men is likely due to their dominance in power - related workplaces .
thus , it is important for managers and supervisors to emphasize relevant training to make employees and citizens more aware dangers associated with power lines .
such measures could reduce the incidents of electrocution , given that 50% of the accidents in the southern region in 2008 and 50% of the accidents in the western region in 2009 were related to the placement of buildings in the regions where there were medium voltage power lines .
the results of this study were consistent with rahbar and naqvi knjyn s study in which they reported 50% of accidents involving the electrocution of citizens were related to the placement of buildings in the areas where there were medium voltage power lines .
therefore , it is recommended that the relevant officials and authorities of the electricity distribution companies in west of tehran take effective measures to improve the transmission lines and make the electrical networks safer and more secure .
naqvi knjyn and rahbar also reported that most of the accidents in tehran occurred in the summer ( 14 ) .
the results of rahmani et al.s study and sadeghian et al.s study regarding accidents in the electricity distribution company in western tehran , alborz province showed that most of the incidents occurred in the summer ( 15 , 19 ) .
because of the importance of protecting people lives , it is recommended that protective preventive instructions be developed and that competent centers conduct follow - up assessments of their use and effectiveness to reduce the number of electrocution accidents in the summer .
another explanation could be the prevalence of young people in the stealing electrical equipment and instruments in tehran . in studies conducted by darveniza et al .
( 20 ) , the highest rates of accidents in different occupations occur among young people .
the results of rahmani et al.s study also showed that the highest rate of occupational accidents in the electricity distribution company in tehran occurred among young adults ( mean age of 36 ) ( 15 ) , while , in the u.s .
, the greatest rate of occupational accidents has been reported to occur among people with a mean age of 45 ( 21 ) , which was not consistent with the results of our study .
the difference in the various results may be due to differences in the systems that govern the communities and to differences in cultural and social conditions of the communities that were studied . in this study ,
the highest rate of accidents occurred among people with education levels of diploma and lower .
rahmani et al.s results also showed that 53% of occupational accidents in the electricity distribution company in tehran occurred among those with education levels below high school .
apparently , they concluded that the low level of education leads to a lack of understanding and full awareness regarding the dangers associated with electrical equipment , and our findings were in complete agreement with their findings ( 15 ) .
human error and expansion of electricity network frontage , the lack of safety training , failure to use personal protective equipment , supervisory and equipment efficiency , and the lack of availability of technical and safety instructions were the most significant reasons for accidents .
thus , some actions were evaluated for their effectiveness in reducing the occurrence of such accidents , and the actions included technical and safety training before starting work , the time spent working in a given day , increasing the frequency of safety inspections , recording and reporting of minimum events , and keeping the network up to date and in good condition ( 14 ) .
our investigation of the causes of electrical accidents and the frequency of their occurrence clearly indicated that there is a need for emphasizing the development of a culture of safety in different areas of various communities , especially among employees engaged in occupations related to electricity and construction and education to school children to reduce the accident rate .
thus , the following recommendations should be implemented : 1 ) facilitation and expedition for the process of people transition from space of distribution network to other areas , 2 ) identification of high - risk areas as the responsibility of all personnel , especially authorities who have responsibility for regions safety , 3 ) implementation of immediate measures for emergency cases in which modifications are required , 4 ) increasing public awareness about the dangers associated with power lines and electricity networks by providing educational pamphlets and safety messages via radio broadcasts . | background : electrical burns account for a significant percentage of fatal accidents .
each year , a number of consumers in iran suffer from electrical injuries due to technical problems , equipment failures , and the unauthorized use of electricity .
the aim of this study was to examine the root causes of accidents that involved electricity in the district served by the western tehran province electricity distribution company.methods:this was a descriptive study in which incidents involving electricity - related injuries were investigated among customers served by the western tehran province electricity distribution company .
therefore , we collected and analyzed incident reports filed by citizens from 2005 through the first half of 2009 in the distribution company s coverage area , including savejbolagh , shahriyar , eastern karaj , qods city , southern karaj , western karaj , malard , and mehrshahr .
the reported events were analyzed using spss software.results:exposure of electricity lines and unauthorized construction of residential houses in areas where there were medium- and low - voltage lines were responsible for 37% of the injuries .
the findings showed that the highest rate of accidents occurred in 2008 and the first half of 2009 .
the highest rate of accidents occurred among people with a mean age of 35.conclusion:the results from investigating the causes of electrical accidents emphasized the necessity of developing a culture of safety in communities , especially among employees who are engaged in occupations related to electricity , construction workers , and school children to reduce the rate of such accidents . |
extragonadal occurrence is rare , and it is usually found in the anterior mediastinum , retroperitoneum , and sacrococcygeal region .
primary retroperitoneal teratoma is unusual in patients above the age of 30 years ; only 10% have been reported to occur after that age .
teratomas confined to the adrenal gland are exceptional ; only 3 cases have been reported in the english - language literature .
we report 2 cases of mature teratomas of the adrenal gland that were laparoscopically excised .
an 8-year - old boy was seen for lumbar pain after a fall . upon physical examination ,
excretory urogram revealed a mass in the upper pole of the right kidney ( figure 1 ) .
abdominal computed tomography showed an 8-cm heterogenous right adrenal lesion with calcifications ( figure 2 ) .
laparoscopic transperitoneal exploration was carried out , and a complete right adrenal resection was performed .
no bleeding occurred during surgery , and the patient was discharged home after 48 hours .
the tumor was found to be a mature cystic teratoma located in the right adrenal .
three years after surgery , the patient remained asymptomatic and was free of tumor recurrence .
a 61-year - old obese woman was incidentally diagnosed with a left adrenal mass during spinal surgery
. computed tomographic scan revealed an 8-cm solid left adrenal lesion ; no calcifications were noted .
the patient underwent percutaneous drainage of the collection and was discharged on postoperative day 10 .
pathologic examination of the tissue confirmed a mature cystic teratoma that measured 8 cm in diameter and weighed 54 g. after 12 months , a small access port - site hernia was diagnosed .
an 8-year - old boy was seen for lumbar pain after a fall . upon physical examination ,
excretory urogram revealed a mass in the upper pole of the right kidney ( figure 1 ) .
abdominal computed tomography showed an 8-cm heterogenous right adrenal lesion with calcifications ( figure 2 ) .
laparoscopic transperitoneal exploration was carried out , and a complete right adrenal resection was performed .
no bleeding occurred during surgery , and the patient was discharged home after 48 hours .
the tumor was found to be a mature cystic teratoma located in the right adrenal .
three years after surgery , the patient remained asymptomatic and was free of tumor recurrence .
a 61-year - old obese woman was incidentally diagnosed with a left adrenal mass during spinal surgery .
computed tomographic scan revealed an 8-cm solid left adrenal lesion ; no calcifications were noted .
the patient underwent percutaneous drainage of the collection and was discharged on postoperative day 10 .
pathologic examination of the tissue confirmed a mature cystic teratoma that measured 8 cm in diameter and weighed 54 g. after 12 months , a small access port - site hernia was diagnosed .
retroperitoneal teratomas are extremely rare . generally , the exact location is difficult to define .
differentiation of a retroperitoneal teratoma in the para - adrenal area from a true adrenal teratoma is tricky . to our knowledge
, only 3 cases of para - adrenal teratoma and adrenal
teratoma have been described .
clinical presentation at diagnosis is usually vague with unspecific lumbar and abdominal pain . on computed tomographic scans
magnetic resonance t2 weighed images show teratoma as a highlighted intensity around the tumor components . according to lam et al ,
of 149 consecutive laparoscopic adrenalectomies performed in the last 10 years at our institution , only 2 ( 1.34% ) were adrenal teratomas .
pathologic criteria for benign lesions were met in both cases : absence of malignant or immature elements in the tumor , absence of other similar lesions in other parts of the body , normal serum levels of afp and hcg , and absence of recurrence in the long - term follow - up .
we prefer transperitoneal laparoscopic adrenalectomy for adrenal tumors , because it is a minimally invasive , well - tolerated , highly effective technique .
laparoscopic transperitoneal adrenalectomy is a feasible and effective technique that enables excellent oncologic results . to our knowledge | background : teratoma is a germ - cell tumor that commonly affects the gonads .
its components originate in the ectoderm , endoderm , and mesoderm .
extragonadal occurrence is rare .
teratomas confined to the adrenal gland are exceptional ; only 3 cases have been reported in the english - language literature .
we report 2 cases of mature teratomas of the adrenal gland that were laparoscopically excised.methods:two patients ( ages 8 and 61 years ) were diagnosed with adrenal teratoma at our institution .
radiological examination showed a solid 8-cm adrenal lesion in both cases .
hormonal assessment was normal .
both patients underwent laparoscopic transperitoneal adrenalectomy.results:surgical time was 120 minutes and 50 minutes , respectively .
one patient was discharged on postoperative day 2 , and the other remained hospitalized until day 10 .
the latter patient required percutaneous drainage of a retroperitoneal collection .
both tumors were identified as mature cystic teratomas .
no evidence was present of recurring disease in either patient.conclusions:adrenal teratoma is rare .
laparoscopic transperitoneal adrenalectomy is a feasible , effective technique that enables excellent oncologic results . to our knowledge
, this is the first report of laparoscopic adrenalectomy for pure adrenal teratoma . |
non - lichenoid cutaneous reactions secondary to imatinib mesylate are the most common non - hematologic adverse reactions associated with its use.lichenoid drug eruption usually occurs on the photo - exposed parts of the skin .
non - lichenoid cutaneous reactions secondary to imatinib mesylate are the most common non - hematologic adverse reactions associated with its use .
imatinib mesylate ( i m ) is an anticancer drug judiciously designed to selectively inhibit certain protein tyrosinase kinase implicated in oncogenesis .
i m has been used to treat chronic myeloid leukemia ( cml ) , gastrointestinal stromal tumors ( gist ) , and dermatofibrosarcoma protuberans .
cutaneous reactions to i m have been reported to occur in 9.5 - 69% of patients in different series .
non - lichenoid cutaneous reactions due to i m have been well - documented in the literature and are the commonest non - hematologic adverse reactions associated with its use .
a case of a generalized lichenoid drug eruption ( lde ) associated with imatinib mesylate therapy has been described here for its rarity and unusual clinical presentation . in view of the fact that the clinical usage of i m is growing
it is thus important to realize the potential of i m to produce lichenoid drug reaction and to differentiate this entity from idiopathic lichen planus .
reports of im - associated lichenoid drug eruption in the english language literature , described in the pubmed and medline database , have also been reviewed .
a 63-year - old man was referred to the dermatology department with a 3-week history of acute onset , raised , itchy , purple - colored fairly generalized skin lesions over his body .
two months before the onset of the skin lesions the patient was put on monotherapy with imatinib mesylate ( i m ) ( 400 mg / day ) for the treatment of cml .
considering the skin eruption , i m had been discontinued ten days prior to his first dermatological consultation . according to the patient , stoppage of the drug led to mild improvement of the skin lesions .
cutaneous examination revealed numerous , well - defined , violaceous , discrete and coalescing papules and plaques over the limbs , abdomen , chest , and the back .
there was fine scaling but no evident vesiculation , oozing or crusting [ figures 1 and 2 ] .
( a and b ) numerous violaceous , coalescing papules and plaques exhibiting koebnerisation and fine scaling ( a and b ) close - up showing purple - colored coalescing papules and plaques with fine scaling lesional punch biopsy specimens were obtained from two different sites .
histopathological examination [ figures 3 and 4 ] of both the specimens showed similar features .
epidermis showed hyperkeratosis with focal parakeratosis , irregular acanthosis and focal wedge - shaped hypergranulosis .
focal basal cell degeneration and pigment incontinence were found . an upper dermal band - like infiltrate comprising mononuclear cells and eosinophils was present at the dermoepidermal junction .
serological tests for human immunodeficiency virus ( hiv ) and hepatitis b and c were negative . ( a )
hyperkeratosis , irregular acanthosis , and focal wedge - shaped hypergranulosis , upper dermal band - like cellular infiltrate at the dermoepidermal junction and a few colloid bodies .
( b ) focal basal cell degeneration and melanin incontinence ( h and e , 400 ) .
( c ) eosinophil in the dermis ( marked by arrow ) ( h and e , 1000 ) ( a ) focal parakeratosis , ( marked by arrows ) irregular acanthosis , wedge - shaped hypergranulosis , focal basal cell degeneration , pigment incontinence , and upper dermal band - like infiltrate ( h and e , 100 ) .
( b and c ) prominent parakeratosis ( marked by arrows ) ( h and e , 400 ) based on the clinical presentation and histopathological examination , a diagnosis of generalized lde associated with i m was made . according to the naranjo probability scale for adverse drug reaction ,
the patient was treated with topical clobetasol propionate ( 0.1% ) cream application over the active skin lesions as well as oral antihistamines . within a couple of weeks ,
almost all the lesions healed with residual post - inflammatory hyperpigmentation [ figure 5 ] and new lesions stopped appearing . since the type of the adverse cutaneous drug reaction was not life- threatening , i m was gradually re - introduced to achieve a dose of 400 mg / day by hemato - oncologists .
after re - introduction of imatinib , he had a few new lesions of lp over the next couple of months .
however , he was being maintained on intermittent topical steroid application over the active lesions with excellent control of the skin condition .
non - lichenoid dermatoses associated with i m therapy may include : maculopapular eruption , edema , stevens - johnson syndrome , toxic epidermal necrolysis , acute generalized exanthematous pustulosis , vasculitis and mycosis fungoides - like eruption .
other less commonly described side effects of i m include , hypopigmentation , pityriasis rosea , psoriasis , neutrophilic eccrine hidradenitis , sweet 's syndrome , erythema nodosum , epstein - barr virus - positive cutaneous b - cell lymphoproliferative disease , follicular mucinosis and pseudolymphoma - like drug eruptions amongst others . on the other hand ,
only a few reports of im - associated lichenoid drug eruption have been described over the last decade .
lichenoid drug eruption is an interface dermatitis induced by ingestion , contact or inhalation of a variety of drugs and has a latent period that ranges from a few weeks to several months .
the latent period between the start of treatment and the appearance of lde may be shortened if the individual has had earlier exposure to the agent .
lde have been described with a wide gamut of medications . as newer agents continue to be introduced and prescribed , the list of the offending drugs for lde is ever expanding .
these may commonly include , angiotensin converting enzyme inhibitors , thiazide diuretics , gold , antimalarials , penicillamine , nonsteroidal anti - inflammatory agents , dental amalgams , sulfasalazine , -blockers , and proton - pump inhibitors , to name afew .
clinically , these eruptions are often look like those of lichen planus ( lp ) .
lde commonly presents as skin lesions comprising papules or plaques , usually on the photo exposed parts of the skin .
lesions of lde are often larger in size , less monomorphic and more prone to be eczematous and associated with desquamation in contrast to that of lp .
. patients with lde are often taking more than one medication , which may make the recognition of the causative agent tricky .
histopathologically , a number of findings are more typical of lde and comprise focal parakeratosis , presence of eosinophils and plasma cells , and a deeper perivascular and periadnexal infiltrate .
however , one interesting and unusual finding in the present case was the presence of lde in both photo exposed and covered parts of the body . it is worth mentioning that , in the setting of cml , im - associated oral lde must be differentiated from idiopathic lichen planus , paraneoplastic pemphigus , and graft - versus - host disease .
specific causes of imatinib induced skin lesions remain elusive . due to its fairly low molecular weight ,
the dose dependence of adverse reactions favors the assumption that imatinib related cutaneous reactions are mediated by changes in tyrosinase kinase signaling rather than immunologic mechanism .
it has been also proposed that the lesions in oral lde may be closely correlated with the altered expression of epidermal markers caused by imatinib .
imatinib , lichenoid drug eruption , lichenoid eruption and cutaneous lichenoid eruption. table 1 summarizes reported cases of im - associated lde in the english language literature as found in these databases .
the severity of lde due to i m varied from mild to severe and the extent of involvement varied from localized to extensive .
profile of patients of lichenoid drug eruption associated with imatinib mesylate therapy some of the reported patients had only mucosal lesions some had only cutaneous lesions and the remainder had both lesions .
all the patients took imatinib mesylate in the dose of 400 mg / day or above .
this observation probably supports the view that the im - associated lde are dose dependent rather than immunogenic in nature .
most common indication of i m usage was cml . in a few cases i m was used to treat gist .
the time interval between the adverse effects appeared and the initiation of i m ranged from one month , to 15 months . in some cases such drug eruptions have led to temporary or permanent discontinuation of i m .
the use of a systemic or topical corticosteroid and a gradual increase in the i m dose may be a useful and practical approach to permit the continuation of treatment with i m .
has shown that acitretin may be successfully used to treat im - associated lde , enabling the continuation of the effective i m dosage . in the present patient , temporary discontinuation of i
m improved the condition . on restarting and gradually increasing the dose of i m to 400 mg / day , only a few lesions of lp reappeared .
it is important to understand the potential of i m to produce lde and to differentiate this entity from its idiopathic counterpart .
moreover , early recognition of the morphological pattern of such drug eruption is crucial to prevent the subsequent discontinuation of i m , which has radically changed the treatment and prognosis of cml and gist .
generalized lichenoid drug eruption may rarely be associated with imatinib mesylate therapy.imatinib mesylate induced lichenoid drug eruption may involve both the photo exposed and covered parts of the body .
imatinib mesylate induced lichenoid drug eruption may involve both the photo exposed and covered parts of the body . | imatinib mesylate ( i m ) , an anticancer drug , has been widely used to treat chronic myeloid leukemia ( cml ) , gastrointestinal stromal tumors ( gist ) , and dermato - fibrosarcoma protuberans .
cutaneous reactions to i m have been reported to occur in varying number of patients in different case series .
non - lichenoid cutaneous reactions secondary to i m have been well - documented in the literature and are the commonest non - hematologic adverse reactions associated with its use .
lichenoid drug eruption ( lde ) associated with i m therapy has rarely been reported in the literature . a case of a generalized lde associated with i
m therapy has been described here for its rarity and interesting clinical presentation . as the clinical usage of i
m is increasing , one might expect an increasing number of similar patients in the future .
it is thus important to realize the potential of i m to produce lde and to differentiate this entity from idiopathic lichen planus . in the present article , the reports of im - associated lde , described in the pubmed and medline database ( in english language literature ) , have also been reviewed . |
in older adults , performance of many habitual daily activities and tasks requires moving and controlling the center of gravity ( defined as the vertical projection of the center of mass ) to various positions within the base of support .
however , effective displacement of the center of gravity during either self - initiated voluntary movements or externally triggered perturbations requires the capacity to remain within the limits of stability , which is described as the maximum distance a person can intentionally displace his or her center of gravity from a midline vertical position in any direction without losing balance and stepping or grasping for support.14 thus , a constricted limits of stability boundary or uncontrolled center of gravity movement can impair functional balance of older adults and constrain safe performance of their daily activities such as reaching , bending , walking , gait initiation , and moving from sit - to - stand.5 current literature suggests that aging is associated with diminished limits of stability capacity69 due to multiple factors , including reduced ankle muscle strength10 and poor cutaneous mechanoreceptor function in the soles of the feet , thus exposing older adults to more instability in functional daily activities and a higher risk of falling.11,12 therefore , from the perspectives of injury prevention and/or clinical rehabilitation of physical function , developing exercise intervention strategies that maximize the center of gravity sway envelope ( ie , the area an individual can move his or her center of gravity with control within the base of support)4 and subsequently expand the limits of stability boundary becomes functionally important for improving balance and mobility and , critically , for reducing the risk of falling among the elderly , which is a significant public health problem worldwide.13,14 because of its functional significance and implications for risk of falling , limits of stability should be integrated into exercise balance training programs that target fall prevention in older adults .
however , among the many types of group- and evidence - based exercise programs1518 designed to prevent falls , few have a specific , tailored focus on integrating training elements that would maximize limits of stability .
given that constrained limits of stability can lead to an increased propensity to fall in older adults,1,19 fall prevention programs that explicitly integrate therapeutic balance training of limits of stability are needed . taking a therapeutic approach ,
li et al20,21 developed the tai ji quan : moving for better balance ( tjqmbb ) program that specifically includes an element of training on limits of stability .
integrating tai ji yin - yang theory22 and theories of motor control and clinical practice,1,2,23 tjqmbb uses an innovative strategy of embracing a complementary interplay between stability and instability ( dualities of yin - yang ) during training to achieve balance equilibrium.20 more specifically , training focuses on eliciting both reactive and anticipatory responses to self - induced , deliberately controlled and coordinated tai ji quan movements aimed at stimulating postural adjustments to progressively increase the sway envelope .
the primary purpose of this study , therefore , was to determine whether a year - long training intervention with tjqmbb , given its specific focus on training limits of sway , could enhance limits of stability among community - dwelling older adults .
it was hypothesized that tjqmbb would enhance indicators of limits of stability . in light of the functional significance of the limits of stability , a secondary purpose was to examine concomitant changes between limits of stability and physical performance resulting from the intervention .
the study design involved a single - group pre- and post - intervention with repeated measures , conducted between may 2012 and january 2014 .
participants in this study were a subset of community - dwelling older adults recruited for a large community - based dissemination project through promotions ( newsletters , flyers , sign - up sheets ) at local senior centers in lane county , or , usa . the research protocol was approved by the institutional review board of oregon research institute , and an informed consent was obtained from all participants .
those who met the following criteria were eligible for participation : ( 1 ) 65 years of age or over ; ( 2 ) physically mobile ( objectively assessed by the ability to walk at least one block with or without an assistive device ) ; ( 3 ) showing no severe cognitive deficits , as defined by the mini - mental state evaluation ( 23);24 and ( 4 ) having medical clearance from a health care provider .
individuals who responded to the promotions were subsequently contacted and scheduled for an in - office visit where the details of the study were explained . following a consent process ,
baseline data were collected at a research laboratory prior to the beginning of the tjqmbb program with follow - ups at 24 and 48 weeks . trained research staff collected demographic and study outcome data . the tjqmbb program , with the conceptual basis and technical detail fully described elsewhere,20 is a balance training modality based on applications of tai ji quan with a specific focus on training limits of stability and sensory integration .
eligible participants participated in a full 60-minute tjqmbb class session taught by a trained instructor twice per week for 48 consecutive weeks . in terms of the training protocol , each session began with tai ji quan - based movement warm - up exercises ( 510 minutes ) , followed by teaching and practicing the core components ( described in the following section ) ( 4045 minutes ) , and ending with a simple set of breathing exercises ( 35 minutes ) .
the training protocol involves a core set of adapted yang style22 tai ji quan - based forms with built - in practice variations and a subset of mini therapeutic movements aimed at stimulating and integrating musculoskeletal and sensory systems via performance of self - initiated tai ji quan movements .
exercise sessions were structured from an easy - to - difficult learning - and - practice progression with an emphasis on within - session practice integration of forms , variations of the forms , and mini movements . both seated and standing exercises were implemented , with seated exercises emphasizing weight shift , trunk rotation , transitioning from sit - to - stand and stand - to - sit , and standing exercises focusing on the features described in the following section . during each session
, participants practiced choreographed tai ji quan forms / movements ( eg , part wild horse s mane ) while engaging in controlled circular ankle sways ( ie , lateral , anterior posterior , medial
lateral together ) that moved the center of gravity progressively toward and near the edge of the base of support with planted feet ; trunk - driven rotational ( laterally and posteriorly ) weight shift ; plantar / dorsiflexion during transitioning between moves ; reactive and proactive ( anticipatory ) postural adjustments in response to self - induced perturbed actions on either fixed support or
change - in - support;25 and active and coordinated eye head hand movements . throughout the training and practice , emphasis was placed on ( 1 ) an interplay between stability ( static movements ) and instability ( self - induced movement perturbations ) in performing each form / move ; ( 2 ) functional ankle - based training by mimicking reaching , tilting , stepping , and turning tasks ; and ( 3 ) a variable approach that stressed practice variations in movement speeds , range of joint motion , movement directions and patterns , and base of foot support .
the primary outcome was limits of stability , which consisted of three indicators : ( 1 ) endpoint excursion ; ( 2 ) movement velocity ; and ( 3 ) directional control , assessed by computerized dynamic posturography ( balance master system ; neurocom , clackamas , or , usa ) .
endpoint excursion assesses the limits of self - initiated movements as participants shift or lean their center of gravity toward the theoretical limit ( 100% ) in eight prespecified target directions without loss of balance ( scores expressed as a percentage ; % los ) .
movement velocity assesses the average speed of the center of gravity movement from the center to the eight targets ( scores expressed as degrees / second ) .
directional control provides a measure of movement accuracy by comparing the amount of movement toward the target to the amount of extraneous movement ( scores expressed as % of 100 ) .
reliability of limits of stability has been established26 or shown in previous studies.21,27 in this study , 24-week test retest reliability ( correlation ) ranged from 0.520.64 ( 0.57 for endpoint excursion , 0.52 for movement velocity , and 0.64 for directional control ) .
secondary measures included physical performance measures of the : ( 1 ) timed up and go test28 and ( 2 ) 50-foot speed walk , adopted from the physical performance test.29 timed up and go measured the time ( in seconds ) taken to rise from a chair , walk 10 feet ( 3 m ) , return , and sit down , with a lower time indicating better mobility . the 50-foot speed walk measured the time ( in seconds ) it took participants to walk as quickly as possible 25 feet out and 25 feet back .
the 6-month test retest reliability was 0.82 for timed up and go and 0.91 for the 50-foot walk test , respectively .
intervention outcomes were analyzed on a sample size of 145 participants who provided at least two data points ( baseline , 24-week , and/or 48-week follow - up ) .
descriptive statistics ( mean , standard deviation ) were performed to describe the study sample characteristics .
coefficient of variation was calculated for each variable across time as the ratio of standard deviation to the mean , multiplied by 100 . minimal detectable change ( mdc )
scores were calculated for each of the limits of stability indicators to determine whether the change score was at or above the minimal level of detectible change .
mdc is based on the standard error of measurement and calculated at a 95% confidence interval ( mdc95 ) using the formula of :
standard error of measurementn1.96,(1 ) where the standard error of measurement is derived from the square root of the mean square error term in the repeated measures analysis of variance and n is the number of measurements used .
intervention effect on the primary and secondary outcomes was analyzed via latent growth curve analysis . in the latent curve analysis ,
the primary interest was on the following estimates : ( 1 ) slope means on each of three limits of stability indicators and ( 2 ) a slope - to - slope correlation between each of the limits of stability indicators and the two physical performance measures .
given the single - group intervention design , change at 48 weeks from baseline was estimated for the indicators of endpoint excursion and directional control using the pooled estimates from prior studies.21,27 assuming an alpha level of 0.05 with 80% power to detect 7% los points difference in endpoint excursion and 8% of 100 points in directional control with an anticipated dropout rate of 30% for the 48-week intervention , a minimal sample size of 70 participants for the tjqmbb was planned .
all descriptive analyses including mdc scores were performed using spss version 17 ( spss inc . , chicago , il , usa ) and microsoft office excel software for windows ( microsoft corporation , redmond , wa , usa ) .
changes in the latent means of the limits of stability indicators and correlations of changes between limits of stability and physical performance measures were performed using mplus software ( v 6 ; muthn & muthn , los angeles , ca , usa).30
the study design involved a single - group pre- and post - intervention with repeated measures , conducted between may 2012 and january 2014 .
participants in this study were a subset of community - dwelling older adults recruited for a large community - based dissemination project through promotions ( newsletters , flyers , sign - up sheets ) at local senior centers in lane county , or , usa . the research protocol was approved by the institutional review board of oregon research institute , and an informed consent was obtained from all participants .
those who met the following criteria were eligible for participation : ( 1 ) 65 years of age or over ; ( 2 ) physically mobile ( objectively assessed by the ability to walk at least one block with or without an assistive device ) ; ( 3 ) showing no severe cognitive deficits , as defined by the mini - mental state evaluation ( 23);24 and ( 4 ) having medical clearance from a health care provider . individuals who responded to the promotions were subsequently contacted and scheduled for an in - office visit where the details of the study were explained . following a consent process ,
baseline data were collected at a research laboratory prior to the beginning of the tjqmbb program with follow - ups at 24 and 48 weeks . trained research staff collected demographic and study outcome data .
the tjqmbb program , with the conceptual basis and technical detail fully described elsewhere,20 is a balance training modality based on applications of tai ji quan with a specific focus on training limits of stability and sensory integration .
eligible participants participated in a full 60-minute tjqmbb class session taught by a trained instructor twice per week for 48 consecutive weeks . in terms of the training protocol , each session began with tai ji quan - based movement warm - up exercises ( 510 minutes ) , followed by teaching and practicing the core components ( described in the following section ) ( 4045 minutes ) , and ending with a simple set of breathing exercises ( 35 minutes ) .
the training protocol involves a core set of adapted yang style22 tai ji quan - based forms with built - in practice variations and a subset of mini therapeutic movements aimed at stimulating and integrating musculoskeletal and sensory systems via performance of self - initiated tai ji quan movements .
exercise sessions were structured from an easy - to - difficult learning - and - practice progression with an emphasis on within - session practice integration of forms , variations of the forms , and mini movements . both seated and standing exercises were implemented , with seated exercises emphasizing weight shift , trunk rotation , transitioning from sit - to - stand and stand - to - sit , and standing exercises focusing on the features described in the following section . during each session
, participants practiced choreographed tai ji quan forms / movements ( eg , part wild horse s mane ) while engaging in controlled circular ankle sways ( ie , lateral , anterior posterior , medial
lateral together ) that moved the center of gravity progressively toward and near the edge of the base of support with planted feet ; trunk - driven rotational ( laterally and posteriorly ) weight shift ; plantar / dorsiflexion during transitioning between moves ; reactive and proactive ( anticipatory ) postural adjustments in response to self - induced perturbed actions on either fixed support or
change - in - support;25 and active and coordinated eye head hand movements . throughout the training and practice , emphasis was placed on ( 1 ) an interplay between stability ( static movements ) and instability ( self - induced movement perturbations ) in performing each form / move ; ( 2 ) functional ankle - based training by mimicking reaching , tilting , stepping , and turning tasks ; and ( 3 ) a variable approach that stressed practice variations in movement speeds , range of joint motion , movement directions and patterns , and base of foot support .
the primary outcome was limits of stability , which consisted of three indicators : ( 1 ) endpoint excursion ; ( 2 ) movement velocity ; and ( 3 ) directional control , assessed by computerized dynamic posturography ( balance master system ; neurocom , clackamas , or , usa ) .
endpoint excursion assesses the limits of self - initiated movements as participants shift or lean their center of gravity toward the theoretical limit ( 100% ) in eight prespecified target directions without loss of balance ( scores expressed as a percentage ; % los ) .
movement velocity assesses the average speed of the center of gravity movement from the center to the eight targets ( scores expressed as degrees / second ) .
directional control provides a measure of movement accuracy by comparing the amount of movement toward the target to the amount of extraneous movement ( scores expressed as % of 100 ) .
reliability of limits of stability has been established26 or shown in previous studies.21,27 in this study , 24-week test retest reliability ( correlation ) ranged from 0.520.64 ( 0.57 for endpoint excursion , 0.52 for movement velocity , and 0.64 for directional control ) .
secondary measures included physical performance measures of the : ( 1 ) timed up and go test28 and ( 2 ) 50-foot speed walk , adopted from the physical performance test.29 timed up and go measured the time ( in seconds ) taken to rise from a chair , walk 10 feet ( 3 m ) , return , and sit down , with a lower time indicating better mobility .
the 50-foot speed walk measured the time ( in seconds ) it took participants to walk as quickly as possible 25 feet out and 25 feet back .
the 6-month test retest reliability was 0.82 for timed up and go and 0.91 for the 50-foot walk test , respectively .
intervention outcomes were analyzed on a sample size of 145 participants who provided at least two data points ( baseline , 24-week , and/or 48-week follow - up ) .
descriptive statistics ( mean , standard deviation ) were performed to describe the study sample characteristics .
coefficient of variation was calculated for each variable across time as the ratio of standard deviation to the mean , multiplied by 100 .
minimal detectable change ( mdc ) scores were calculated for each of the limits of stability indicators to determine whether the change score was at or above the minimal level of detectible change .
mdc is based on the standard error of measurement and calculated at a 95% confidence interval ( mdc95 ) using the formula of :
standard error of measurementn1.96,(1 ) where the standard error of measurement is derived from the square root of the mean square error term in the repeated measures analysis of variance and n is the number of measurements used .
intervention effect on the primary and secondary outcomes was analyzed via latent growth curve analysis . in the latent curve analysis ,
the primary interest was on the following estimates : ( 1 ) slope means on each of three limits of stability indicators and ( 2 ) a slope - to - slope correlation between each of the limits of stability indicators and the two physical performance measures . given the single - group intervention design , change at 48 weeks from baseline was estimated for the indicators of endpoint excursion and directional control using the pooled estimates from prior studies.21,27 assuming an alpha level of 0.05 with 80% power to detect 7% los points difference in endpoint excursion and 8% of 100 points in directional control with an anticipated dropout rate of 30% for the 48-week intervention , a minimal sample size of 70 participants for the tjqmbb was planned .
all descriptive analyses including mdc scores were performed using spss version 17 ( spss inc . , chicago , il , usa ) and microsoft office excel software for windows ( microsoft corporation , redmond , wa , usa ) .
changes in the latent means of the limits of stability indicators and correlations of changes between limits of stability and physical performance measures were performed using mplus software ( v 6 ; muthn & muthn , los angeles , ca , usa).30
the major reasons for being determined ineligible were age ( n=13 ) , time conflict ( n=3 ) , or significant change in health ( n=2 ) .
in addition , six eligible participants ( 4% ) were removed because they attended only one session and provided no follow - up data .
the mean age at enrollment was approximately 75 years , 79% of the participants were women , and approximately 92% were caucasian . in all , 124 participants ( 85% ) reported having two or more chronic conditions , 70 ( 48% ) were taking two or more prescribed medications , and 73 ( 50% ) reported having fallen at least once in the previous 6 months , with about 25% of those having fallen two or more times .
a total of 98 participants ( 68% ) reported having some level of fear of falling .
of the total 145 participants , 21 ( 14% ) dropped out of the study during the 48-week intervention period , due primarily to reasons such as deterioration in medical conditions ( n=14 ) , change in health status ( n=5 ) , or relocation ( n=2 ) .
there were , however , no significant differences in baseline demographics and functional characteristics between the participants who completed the intervention and those who dropped out of the study ( completers : n=124 versus dropouts : n=21 ) .
the average number of class sessions attended per participant for the intervention ( a total of 96 sessions ) was 66 sessions ( range : 596 sessions ; median : 73 sessions ) for the whole sample ( n=145 , including dropouts [ n=21 ] ) compared to 73 sessions ( range : 2896 sessions ; median : 78 sessions ) for those who completed the 48-week intervention ( n=124 ) .
the percentage attendance for the intervention was 69% ( median : 76% ) for the whole sample ( n=145 ) and 76% ( median : 81% ) for the completers ( n=124 ) .
table 2 provides descriptive statistics ( mean , standard deviation ) for the three primary and two secondary outcome variables across the three measurement time points .
the observed means for each of the three limits of stability show a linear improvement over time .
change from baseline values were 17.21% los ( 32% ) for endpoint execution , 1.71 degrees / second ( 68% ) for movement velocity , and 12.41% of 100 ( 19% ) for directional control compared to mdc95 values , which were 16.88% los for endpoint excursion , 1.63 degrees / second for movement velocity , and 12.14% of 100 for directional control , respectively .
results of the latent growth analysis are presented in table 3 , which further indicates that there was a significant rate of change ( as reflected in latent linear slope means ) in the indicators of endpoint excursion , movement velocity , and directional control over the 48-week intervention period .
the analyses also show a significant small - to - moderate negative correlation ( indicating improvement ) between rate of change in each of the limits of stability indicators and the two physical performance measures over time ( table 3 )
. there were no significant adverse events attributable to the intervention observed during the intervention period .
three falls were reported during warm - up sessions , but the participants sustained no injuries and continued the classes .
the mean age at enrollment was approximately 75 years , 79% of the participants were women , and approximately 92% were caucasian . in all , 124 participants ( 85% ) reported having two or more chronic conditions , 70 ( 48% ) were taking two or more prescribed medications , and 73 ( 50% ) reported having fallen at least once in the previous 6 months , with about 25% of those having fallen two or more times .
a total of 98 participants ( 68% ) reported having some level of fear of falling .
of the total 145 participants , 21 ( 14% ) dropped out of the study during the 48-week intervention period , due primarily to reasons such as deterioration in medical conditions ( n=14 ) , change in health status ( n=5 ) , or relocation ( n=2 ) .
there were , however , no significant differences in baseline demographics and functional characteristics between the participants who completed the intervention and those who dropped out of the study ( completers : n=124 versus dropouts : n=21 ) . the average number of class sessions attended per participant for the intervention ( a total of 96 sessions ) was 66 sessions ( range : 596 sessions ; median : 73 sessions ) for the whole sample ( n=145 , including dropouts [ n=21 ] ) compared to 73 sessions ( range : 2896 sessions ; median : 78 sessions ) for those who completed the 48-week intervention ( n=124 ) .
the percentage attendance for the intervention was 69% ( median : 76% ) for the whole sample ( n=145 ) and 76% ( median : 81% ) for the completers ( n=124 ) .
table 2 provides descriptive statistics ( mean , standard deviation ) for the three primary and two secondary outcome variables across the three measurement time points .
the observed means for each of the three limits of stability show a linear improvement over time . change from baseline values were 17.21% los ( 32% ) for endpoint execution , 1.71 degrees / second ( 68% ) for movement velocity , and 12.41% of 100 ( 19% ) for directional control compared to mdc95 values , which were 16.88% los for endpoint excursion , 1.63 degrees / second for movement velocity , and 12.14% of 100 for directional control , respectively .
results of the latent growth analysis are presented in table 3 , which further indicates that there was a significant rate of change ( as reflected in latent linear slope means ) in the indicators of endpoint excursion , movement velocity , and directional control over the 48-week intervention period .
the analyses also show a significant small - to - moderate negative correlation ( indicating improvement ) between rate of change in each of the limits of stability indicators and the two physical performance measures over time ( table 3 ) .
there were no significant adverse events attributable to the intervention observed during the intervention period .
three falls were reported during warm - up sessions , but the participants sustained no injuries and continued the classes .
this is the very first study to evaluate the effect of a specifically tailored tai ji quan training program on improving limits of stability , an important prerequisite for performing many of the functional activities of daily living in older adults .
the results showed that 48 weeks of tjqmbb training significantly improved older adult limits of stability as reflected in increases in the indicators of movement excursion , velocity , and control , and that increased limits of stability were concomitantly associated with improved physical performance .
thus , with no significant adverse events during the study period , it appears that the intervention is an appropriate one for this at - risk population . while cross - sectional studies that showed that older adult practitioners of tai ji quan tended to exhibit better stability limits compared to nonpractitioners31,32 provided early nonintervention data on the potential of tai ji quan to enhance limits of stability , more recent intervention studies from the author s group that integrated limits of sway training demonstrated the benefit of this focused tai ji quan program for improving limits of stability in older adults and patients with parkinson s disease.21,27 in contrast , other tai ji quan - based studies , ranging from 4 to 12 months of intervention,33,34 have not shown significant improvements in postural stability measures in older adults .
the tjqmbb program differs from the programs in these other studies in a clinically important way in that this approach transforms traditional martial arts - based training into a therapeutically oriented training paradigm that specifically emphasizes the practice of deliberate , voluntarily controlled tai ji quan postural actions .
these actions include manipulation of the center of gravity movements , variations in the base of support , and adaptation training with reactive and proactive actions to self - induced perturbations , with the purpose of increasing the sway envelope and thereby expanding the limits of stability , with the ultimate goal of reducing the risk of falling .
although there are currently no guidelines for gauging clinically meaningful change in the measures of limits of stability , this 48-week , twice - per - week training schedule intervention appears to have provided a substantive change over time in the measures used in this study : 32% on endpoint excursion , 68% on movement velocity , and 19% on directional control .
improvements in these limits of stability indicators support the potential for the tjqmbb program to address problems of improper weight shifts that are observed in elderly people residing in long - term care.35 consistent with findings from a cross - sectional study,5 the results demonstrate that improved measures on limits of stability through this tai ji quan training in older adults was sufficiently sensitive to elicit change in clinical measures of balance and mobility , suggesting that training with tjqmbb offers the potential to improve functional activities and activities of daily living , including sit - to - stand , walking , turning , and stand - to - sit tasks .
the author s intervention approach linking martial - arts - based tai ji quan to clinical and functional training of balance in older adults offers some practical implications . from an applied perspective
, the training approach used in this study can be readily introduced in various settings .
for example , as an effective balance training modality , tjqmbb can be readily adopted in diverse communities for the purpose of preventing falls36,37 and in health care settings to assist patients with balance disorders and fall risk problems.21 uniquely different from all other existing tai ji quan - based programs , tjqmbb also has a specific , tailored clinical focus in that training incorporates therapeutic approaches such as closed and open kinetic chain movements;38 motor control mechanisms through manipulation of movement magnitude , speed , direction , and base of support ; and training of functional activities that take into account interactions between individuals , tasks at hand , and environmental constraints imposed on the tasks.23 these specific training features thus make the program amenable and applicable to clinical training and rehabilitation that address neuromuscular and movement disorders in patients with balance and gait problems and who are at high risk of falls .
the mechanism by which the training protocol improves the limits of stability indicators in this study was not explored and therefore remains unclear .
limits of stability is known to be influenced by a number of physiological and biomechanical factors,4,23 including lower extremity strength , range of ankle motion , and cutaneous mechanoreceptors in the feet,2 acting either alone or in combination . through integration of tai ji quan and physical therapy , the protocol in this study
puts great emphasis on controlled weight shifting and ankle movement sway , alternation between a narrow stance and a wide stance to continually change the base of support , rotational trunk - driven movements , and dorsiflexion and plantar flexion during movement transitioning .
these therapeutically tailored training features may have made an impact on important sensorimotor elements that contribute to enhanced limits of stability .
therefore , it is plausible that improvement in the limits of stability performance may be attributed to unmeasured improvements in flexion / extension ankle torques , the range of ankle motion , movement recovery strategies , or sensory integration features that are inherent in tjqmbb training . some of these mechanisms of action may be revealed by evaluating the kinematics of body movement or conducting kinetic analysis of these ankle swing - based static and dynamic movement exercises , thus offering a better understanding of the movement strategies and forces contributing to the intervention - induced change in limits of stability .
its design does not include a control condition , making it difficult to draw a firm conclusion regarding the true intervention effect of tjqmbb on limits of stability .
therefore , the results should be corroborated by future clinical randomized controlled trials . given the importance of plantarflexor muscle strength in limits of stability,10
another limitation is the lack of a measure of ankle muscle strength , which precludes the opportunity to examine the role of ankle strength in the observed improved limits of stability outcomes .
finally , the study used theoretical limits of stability values derived from computerized dynamic posturography that correspond to the maximum range in which center of gravity can be moved safely without changing the base of support.26 however , the capacity of stability limits may vary depending on the task performed;23 therefore , consideration of performance - based or functional limits of stability resembling real - world situations may be needed in future assessments.39
the tjqmbb program , a transformed protocol of traditional tai ji quan training approaches , was found to improve limits of stability in older adults , and the improved limits of stability were associated with improved functional performance . within the limitations of the current study , the combined results provide promising preliminary data that suggest the potential utility of tjqmbb for increasing limits of stability in older adults , thereby enhancing functional performance of habitual daily activities . | backgroundlimits of stability , defined as the ability to maintain the center of gravity within the boundary of the base of support , is critically important for older adults in performing their activities of daily living .
however , few exercise programs specifically tailored to enhance limits of stability exist .
the primary purpose of this study was to determine whether a therapeutically designed intervention , tai ji quan : moving for better balance ( tjqmbb ) , could improve limits of stability in older adults .
a secondary purpose was to examine concomitant change in limits of stability and physical performance as a result of the intervention.methodsa single - group design was used in which 145 community - dwelling older adults ( average age : 75 years ) were enrolled in tjqmbb classes , participating twice weekly for 48 weeks .
primary outcome measures were three indicators of limits of stability ( los ) ( endpoint excursion , movement velocity , and directional control ) , with secondary measures of physical performance being timed up and go and 50-foot speed walk ( in seconds ) , which were assessed at baseline , 24 weeks , and 48 weeks .
changes in the repeated measures of outcome variables were analyzed via latent curve analysis.resultsat 48 weeks , a significant rate of change ( improvement ) over time was observed in the three limits of stability indicators ( endpoint execution : 8.30% los , p<0.001 ; movement velocity : 0.86 degrees / second , p<0.001 ; directional control : 6.79% of 100 , p<0.001 ) ; all reached a threshold of real change as judged by the minimal detectable change values .
improvements in the three limits of stability measures were concomitantly correlated with improved ( reduced times ) performance scores in the timed up and go ( 0.30 , 0.45 , and 0.55 , respectively ) and 50-foot walk ( 0.33 , 0.49 , and 0.41 , respectively).conclusionin this single - group study , community - dwelling older adults trained through tjqmbb significantly improved their limits of stability , providing preliminary support for the use of tjqmbb as a therapeutic modality for enhancing functional activities in older adults . |
this is in contrast to the regular cytotoxic chemotherapy drugs that either damage the deoxyribonucleic acid or inhibit the microtubules and kill or inhibit the rapidly dividing cancer cells .
maximum tolerated doses ( mtd ) of these drugs are designed to kill as many tumor cells as possible .
such doses require prolonged periods of break between successive therapies so as to allow the normal tissues to recover and also to allow recovery from the harmful side - effects .
though the cure rate is high , relapses are commonly seen in more aggressive tumors . in the past decade , the concept that smaller individual doses other than the mtd could be used emerged ; which could be more effective with lesser toxic effects .
this concept of regular chemotherapy scheduling in low doses , below the mtd with no prolonged drug - free breaks is called mc .
this type of chemotherapy inhibits tumor growth by anti - angiogenic mechanisms and also reduces the undesirable side - effects .
few studies have shown that in patients with cancer that are either refractory to treatment or which have relapsed after conventional chemotherapy , mc can induce satisfactory tumor stabilization , also the duration of clinical benefit is longer with this type of low dose scheduling than the conventional chemotherapy with high doses . the conventional chemotherapy targets the proliferating tumor cells , whereas in the low dosage metronomic therapy the target is the endothelial cell of the growing vasculature of a tumor .
thus , mc is also called as anti - angiogenic chemotherapy as coined by browder et al .
this low dose mc is one of the most efficient anti - angiogenic chemotherapy and also has beneficial immunologic effects .
this type of treatment leads to a long term asymptomatic control of the disease by inducing angiogenic dormancy .
many clinical trials have shown favorable results in patients who were untreated or had earlier received conventional chemotherapy in various types of cancer .
few studies have shown that no improved patient survival was seen in patients on mc , but mc does induce tumor stabilization and the duration of clinical benefit can be longer than the benefit obtained with the conventional chemotherapy .
most of these studies have assessed clinical parameters in assessing response of this low dose anti - angiogenic chemotherapy . with this background
, we undertook this retrospective analysis to evaluate whether anatomic imaging modality like computed tomography ( ct ) scan or a functional imaging modality like positron emission tomography ( pet ) should be used for assessment of treatment response in patients on mc .
all patients on metronomic therapy who were referred to our department for a pet ct study prior to start of the treatment and for a response evaluation after 12 - 14 weeks , between may 08 and january 12 were included in this retrospective evaluation .
a total of 43 patients of various cancers , on metronomic therapy who underwent pet ct were assessed by ct and pet using new response evaluation criteria in solid tumors ( recist 1.1 ) and pet response criteria in solid tumors ( percist 1.0 ) criteria .
there were 22 patients of breast cancer , 7 of musculoskeletal cancer ( soft tissue sarcoma-2 , primitive neuroectodermal tumor [ pnet]-5 , osteogenic sarcoma-1 ) , 4 of head and neck cancer ( nasopharyngeal-3 , tongue-1 ) , 2 of non hodgkins lymphoma , 2 of esophageal and 1 each of gall bladder , ovarian cancer , hemangiopericytoma , ganglioneuroblastoma and 1 a paraganglioma [ table 1 ] .
patient characteristics all patients were asked to fast for 4 - 6 h prior to the study and blood glucose levels were checked and confirmed to be < 150 mg / dl .
the studies were performed 60 - 90 min following intravenous administration of 5 mbq / kg of f - fluorodeoxyglucose ( f - fdg ) .
imaging was performed on a discovery st pet - ct system ( general electric medical systems ) .
it combines a 16 slice ct scanner with a dedicated pet scanner ( bismuth germanium oxide crystal ) .
ct was performed over 5 - 8 bed positions from the skull base to the mid - thigh ; using multislice ( 16 slice ) ct component of the system .
ct parameters included 140 kv , 110 - 210 ma , 0.8 s / rotation , pitch of 1.75:1 , field of view ( fov ) 50 cm , length of scan 1.0 - 1.6 m , 0.625 spatial resolution and slice thickness of 3.75 mm . intravenous and oral contrast was not routinely administered in all patients unless there was a specific indication and request to do so .
this was followed immediately by acquisition of pet data in the same anatomic locations with 15.4 cm axial fov acquired in 3d mode with 3 min / bed position .
the images were reconstructed using a standard vendor provided reconstruction algorithm which incorporated ordered subset expectation maximization .
image fusion was performed using co - ordinate based fusion software and subsequently reviewed at a workstation that provided multiplanar reformatted images and displayed pet images , ct images and pet - ct fusion images .
the images were evaluated by experienced radiologist and nuclear medicine physician blinded to each other 's results .
the ct findings were analyzed using the recist 1.1 and the pet findings by percist 1.0 criteria respectively . any area with intensity greater than background that could not be identified as physiological activity or which on ct correlation did not fit into benign ( infective / inflammatory / degenerative )
the response criteria as per recist 1.1 was as follows - complete response ( cr ) was disappearance of all target lesions , partial response ( pr ) as 30% or more decrease in sum of diameters of target lesions , progressive disease ( pd ) as 20% or more increase in sum of diameters of target lesions and also an absolute increase of at least 5 mm and/or appearance of one or more new lesions , stable disease ( sd ) who did not qualify for either sufficient shrinkage to qualify for pr nor sufficient increase to qualify for pd .
complete resolution of fdg uptake within the measurable target lesion that is less than the mean liver activity and indistinguishable from surrounding background , with appearance of no new lesion , was labeled as complete metabolic response ( cmr ) .
reduction of minimum of 30% of standardized uptake value lean ( sulpeak ) in the target volume in the same lesion as the baseline measurement was grouped under partial metabolic response ( pmr ) .
progressive metabolic disease ( pmd ) was a 30% increase in the sulpeak of the fdg uptake or appearance of fdg avid new lesions that was morphologically typical of cancer .
stable metabolic disease ( smd ) was disease , which did not qualify for cmr , pmr or pmd .
the sul was calculated by a 1.2 cm diameter volume region of interest ( roi ) placed on the hottest site of the tumor .
it was also determined whether the sulpeak of the tumor was higher than 1.5 times the liver sul mean + 2 sds ( in a 3 cm - diameter spheric roi in the normal right lobe of liver ) .
the responses were graded as follows : pd-1 , sd-2 , pr-3 , cr-4 and non - classifiable 0 for recist and pmd-1 , smd-2 , pmr-3 , cmr-4 and non - classifiable-0 for percist .
a total of 43 patients of various cancers , on metronomic therapy who underwent pet ct were assessed by ct and pet using new response evaluation criteria in solid tumors ( recist 1.1 ) and pet response criteria in solid tumors ( percist 1.0 ) criteria .
there were 22 patients of breast cancer , 7 of musculoskeletal cancer ( soft tissue sarcoma-2 , primitive neuroectodermal tumor [ pnet]-5 , osteogenic sarcoma-1 ) , 4 of head and neck cancer ( nasopharyngeal-3 , tongue-1 ) , 2 of non hodgkins lymphoma , 2 of esophageal and 1 each of gall bladder , ovarian cancer , hemangiopericytoma , ganglioneuroblastoma and 1 a paraganglioma [ table 1 ] .
all patients were asked to fast for 4 - 6 h prior to the study and blood glucose levels were checked and confirmed to be < 150 mg / dl .
the studies were performed 60 - 90 min following intravenous administration of 5 mbq / kg of f - fluorodeoxyglucose ( f - fdg ) .
imaging was performed on a discovery st pet - ct system ( general electric medical systems ) .
it combines a 16 slice ct scanner with a dedicated pet scanner ( bismuth germanium oxide crystal ) .
ct was performed over 5 - 8 bed positions from the skull base to the mid - thigh ; using multislice ( 16 slice ) ct component of the system .
ct parameters included 140 kv , 110 - 210 ma , 0.8 s / rotation , pitch of 1.75:1 , field of view ( fov ) 50 cm , length of scan 1.0 - 1.6 m , 0.625 spatial resolution and slice thickness of 3.75 mm .
intravenous and oral contrast was not routinely administered in all patients unless there was a specific indication and request to do so .
this was followed immediately by acquisition of pet data in the same anatomic locations with 15.4 cm axial fov acquired in 3d mode with 3 min / bed position .
the images were reconstructed using a standard vendor provided reconstruction algorithm which incorporated ordered subset expectation maximization .
image fusion was performed using co - ordinate based fusion software and subsequently reviewed at a workstation that provided multiplanar reformatted images and displayed pet images , ct images and pet - ct fusion images .
the images were evaluated by experienced radiologist and nuclear medicine physician blinded to each other 's results .
the ct findings were analyzed using the recist 1.1 and the pet findings by percist 1.0 criteria respectively . any area with intensity greater than background that could not be identified as physiological activity or which on ct correlation did not fit into benign ( infective / inflammatory / degenerative )
the response criteria as per recist 1.1 was as follows - complete response ( cr ) was disappearance of all target lesions , partial response ( pr ) as 30% or more decrease in sum of diameters of target lesions , progressive disease ( pd ) as 20% or more increase in sum of diameters of target lesions and also an absolute increase of at least 5 mm and/or appearance of one or more new lesions , stable disease ( sd ) who did not qualify for either sufficient shrinkage to qualify for pr nor sufficient increase to qualify for pd .
complete resolution of fdg uptake within the measurable target lesion that is less than the mean liver activity and indistinguishable from surrounding background , with appearance of no new lesion , was labeled as complete metabolic response ( cmr ) .
reduction of minimum of 30% of standardized uptake value lean ( sulpeak ) in the target volume in the same lesion as the baseline measurement was grouped under partial metabolic response ( pmr ) .
progressive metabolic disease ( pmd ) was a 30% increase in the sulpeak of the fdg uptake or appearance of fdg avid new lesions that was morphologically typical of cancer .
stable metabolic disease ( smd ) was disease , which did not qualify for cmr , pmr or pmd .
the sul was calculated by a 1.2 cm diameter volume region of interest ( roi ) placed on the hottest site of the tumor .
it was also determined whether the sulpeak of the tumor was higher than 1.5 times the liver sul mean + 2 sds ( in a 3 cm - diameter spheric roi in the normal right lobe of liver ) .
the responses were graded as follows : pd-1 , sd-2 , pr-3 , cr-4 and non - classifiable 0 for recist and pmd-1 , smd-2 , pmr-3 , cmr-4 and non - classifiable-0 for percist . these values were statistically compared using wilcoxon signed - rank test .
there was concordance in 75% of patients and discordance in 25% of patients [ table 2 ] . in patients with concordant result , 2 patients showed pr / pmr , 8 patients showed sd / smd and 22 patients showed pd / pmd , as assessed by recist 1.1 for morphologic criteria and percist for metabolic criteria , respectively .
pr / pmr was seen in two patients of breast cancer as there was decrease in size and metabolic activity of the lesions by > 30% [ figure 1a c ] . a case of carcinoma
( a ) maximum intensity projection image shows left internal mammary node ( block arrow ) and left chest wall deposit ( arrow ) .
subsequent scan shows complete resolution of the internal mammary node and partial metabolic response in the chest wall deposit ( arrow ) .
( b ) transaxial images of hypermetabolic internal mammary node ( arrows ) with complete metabolic and morphologic regression .
( c ) transaxial images of hypermetabolic left chest wall deposit ( block arrow ) with residual disease ( arrow ) sd / smd was seen in 8 patients as there was no significant change in size and metabolic activity of the lesions to classify for either pr / pmr or pd / pmd .
out of the 22 patients who showed pd / pmd by both the morphologic and metabolic criteria , 17 showed pd by virtue of new lesions , which were hypermetabolic [ figure 2a and b ] . in 5 patients , there was increase in sum total of the diameter and sulpeak of the target lesions by > 20% and > 30% respectively .
( a ) transaxial images show new hypermetabolic liver lesion in lower panel ( arrows ) suggesting progressive disease .
( b ) new hypermetabolic nodules in the left lung in the lower panel ( arrow ) , suggesting progressive disease by new response evaluation criteria in solid tumors and pet response criteria in solid tumors in 11 patients ( 25% ) there was discordance in the result assessed by morphologic ( recist 1.1 ) and metabolic criteria ( percist ) [ table 3 ] .
totally 5 patients had sd by recist 1.1 , of which 4 showed pmd by percist . in all these 4 patients
, there was an increase in the metabolism ( change in sulpeak between 32 and > 100% ) with no significant change morphologically . on follow - up , 2 of them
had pd both morphologically and metabolically on subsequent follow - up and 1 patient expired due to disease progression . in the fourth patient ,
there was pmr decrease in metabolism of liver lesions by 61.7% with no change morphologically [ figure 3 ] .
results of patients with discordant result transaxial computed tomography ( ct ) and fused positron emission tomography ct images of a patient with carcinoma breast [ patient no .
the lower panel shows mild decrease in the uptake visually , with no significant morphologic change
. stable disease ( sd ) and partial metabolic response ( pmr)-discordant result in 3 patients , the disease was non - pd ( all three had sclerotic bone lesions ) . out of these , 1 showed cmr as the sclerotic sternal lesion was non - metabolic .
, there was an increase in metabolism with no morphologic change and hence showed a pmd . on follow - up imaging , both these patients had metabolic and morphologic progression of disease . in 1 patient of breast cancer [ table 3 , patient
9 ] , there was increase in number of sclerotic lesions in the vertebrae and hence showed pd as per recist 1.1 whereas there was no metabolic activity in these lesions and showed cmr as per percist . in this particular case , it is likely that pet may have missed the lesions as it is known that sclerotic lesions in patients with breast cancer may not be hypermetabolic .
however there is no follow - up imaging that is available , but the patient remains asymptomatic . in a 12-year - old girl , a case of ganglioneuroblastoma [ table 3 , patient no . 10 ] , a new hypermetabolic marrow lesion in the ischium was detected on pet ( pmd ) and not seen on ct with other lesions showing no morphologic change ( pr ) . in another patient of pnet [ table 3 , patient
11 ] , a sacral marrow lesion was not detected on ct but was picked up by pet- thus the disease was cr by recist and pmd by percist . on subsequent
follow up imaging when the patient was on metronomic treatment , there was sclerosis ( healing response ) in the sacral marrow lesion and was then evident on ct [ figure 4 ] .
transaxial computed tomography ( ct ) and fused positron emission tomography ( pet ) ct images of a patient with primitive neuroectodermal tumor [ patient no .
the upper panel shows no lesion in the sacrum on the ct image with a hypermetabolic marrow lesion in the left sacral ala in the fused pet ct image ( arrow ) ( complete response and progressive metabolic disease - discordant result ) .
the lower panel is follow up study showing minimal sclerosis in the left sacral ala in the ct image with no hypermetabolism in the fused pet ct image ( arrow ) suggesting response pet upstaged the disease in 81% of patients ( 9/11 ) and down staged the disease in 19% of patients ( 2/11 ) .
wilcoxon signed - rank test was used to compare the response evaluation using recist and percist , using the above - mentioned grades . using a 2-tailed hypothesis , the p < 0.05 ( z-0.0445 , p-0.9681 ) and was thus not significant .
wilcoxon signed - rank test was used to compare the response evaluation using recist and percist , using the above - mentioned grades . using a 2-tailed hypothesis , the p < 0.05 ( z-0.0445 , p-0.9681 ) and was thus not significant .
though recist is a widely applied tumor response criteria being dependent on morphological changes , it has limitations . as the assessment done by pet based criteria like percist uses metabolic changes which are closely related to changes occurring in a malignant tissue ; it would be a more accurate modality for assessing response .
furthermore pet can detect metabolic changes when there are no or minimal morphological changes as seen in our study , particularly in detection of metastatic marrow lesions .
our results show that in patients with discordant result , pet appropriately assessed the response as was evident by follow - up imaging in most patients .
pet showed morphologic progression ( pmd ) in 8 patients ; 4 out of these on follow - up imaging had both morphologic and metabolic progression of disease ( patient no . 2 , 3 , 7 , 8) .
one patient ( patient no 1 ) expired due to disease progression . in one patient ( patient no 4 ) , the increased metabolism persisted .
an important observation made in this study is that , in two patients [ patient no .
on subsequent follow - up imaging one of these patients [ patient no 11 ] , showed morphologic and metabolic resolution of the disease on metronomic therapy ( mc ) .
this indicates that though mc is used in a palliative setting with effective pain palliation and tumor stabilization ; it may also have a curative effect in few patients .
this was a retrospective observational study and a statistical difference between the response evaluation by the two methods could not be achieved .
this can also be attributed to the low sample size and moreover there were 32 ties ( concordance ) and only 11 patients showed differences in response .
it is well - known that metabolic response depicted by fdg pet - ct well precedes the anatomic response and has been well - demonstrated in few studies .
the pet response is depicted by a decrease in the glycolytic metabolism of a tumor unlike anatomic response criteria which are based on size of the tumor which lags weeks and months behind the metabolic response .
many newer cancer therapies including metronomic therapy are mainly cytostatic and achieving a long standing sd is the more beneficial outcome .
such effects have been seen in patients of gastrointestinal stromal tumors treated with tyrosine kinase inhibitors where though the shrinkage in size of tumor is less the survival of patients is improved with sd .
major limitations of this study are the retrospective nature of the data collection and heterogeneous group of patient population ( the response may vary depending on the type of cancer ) . a better evaluation in a prospective trial setting is warranted .
also this study does not assess clinical outcomes , but was undertaken to look at better response assessing modality . in conclusion , as this type of metronomic treatment brings about tumor or angiogenic dormancy it is natural that the treatment response criteria based on morphologic characteristics will not be evident soon after therapy and thus using functional imaging criteria like percist would be the best to monitor the treatment response .
our study shows that metabolic response is a more appropriate way of assessing the response to metronomic therapy .
tumorostatic treatment regimens like metronomic therapy where alteration in morphological features would take time to manifest , assessment of metabolic response using fdg pet would be more appropriate .
we thus recommend that pet should the imaging modality of choice in response assessment of patients of metronomic therapy .
though metronomic therapy is used as a palliative therapy , if response evaluation by pet shows cr , this form of treatment has the potential to become a mode of treatment rather than palliation in some tumors and this has to be evaluated with larger , homogenous patient population in a prospective mode . | introduction : metronomic chemotherapy ( mc ) is a novel therapeutic variation for resistant cancers , wherein chemotherapeutic drugs are administrated in low doses with no prolonged drug - free break .
it lessens the level of toxicity , is better tolerated and enhances the quality of life .
this retrospective analysis was undertaken to evaluate whether anatomical ( computed tomography [ ct ] ) or functional ( positron emission tomography [ pet ] ) imaging be used for response assessment in patients on mc.materials and methods : a total of 16 males and 27 females with age range of 12 - 83 years on mc who underwent pet / ct were assessed by new response evaluation criteria in solid tumors ( recist 1.1 ) and pet response criteria in solid tumors ( percist 1.0).results : concordance between recist 1.1 and percist was seen in 32 ( 75% ) patients .
there was discordance in 11 ( 25% ) patients . in patients with discordance ,
the results were confirmed by follow - up imaging .
pet upstaged the disease in 81% of patients ( 9/11 ) and down - staged the disease in 19% of patients ( 2/11).conclusions : metabolic response accurately identified the disease status as assessed by clinical or imaging follow - up .
alteration in morphology takes time to manifest , which is demonstrated by ct or magnetic resonance ; whereas in mc which brings about tumor dormancy , assessing metabolic response by pet would be more appropriate .
mc is usually given in palliative setting but in few cases complete metabolic response was demonstrated in our study . in such a scenario
this form of treatment has the potential to become an adjunct mode of treatment in some tumors .
this needs to be evaluated with larger , homogenous patient population in a prospective mode . |
humans ( indeed all biological multicellular organisms ) are made of multiscale hierarchy of structures ranging from subcellular structures ( 10 m ) to cells ( 10 m ) , basic functional units ( the smallest aggregation of diverse cells that behaves like the parent organ 10 m ) , organs ( 1010 m ) , and bodies ( 10 m ) .
in addition to the local scale variation , biological structures are also characterized by shape .
for example , the blood vessels are tubular objects interconnected into a complex network and have a range of structural scale ( 5 m diameter capillaries to 3 cm aorta ) .
this large - scale range presents two major problems ; one is just making the measurements , and the other is the exponential increase of component numbers with decreasing scale .
quantitative analysis of such systems , for example , blood vessel trees and networks of neurological dendrites and axons , seem to be best measured from 3d images of those structures . with the remarkable increase in the volume imaged by , and resolution of , modern day imagers , the practical problem now is the extraction of this multiscale data from those large , detailed image data sets .
thus , there is a need for an automatic tool which mines data across both space and scale to capture local information about the objects which describes the feature and now becomes associated with its appropriate position and scale . without prior information for a scale description of the image content , an image has to be studied at all scales .
the basis for design of an automatic tool for such description could be derived from human perception model .
the human eye comprises a large number of individual receptors ( over 150 million rods and cones ) .
such imager has no prior information about input , and therefore , it is designed to extract the information by applying sampling apertures at a wide range of sizes simultaneously [ 1 , 2 ] .
since the information from a single individual sensor is almost meaningless , the sampling should be done not by the individual rods and cones in a human eye or detectors in imagers but by the sensor neighborhoods .
such sensor neighborhoods implement fundamental multiscale perception at different scales simultaneously but at this point no memory or analysis is involved yet .
the very first level of analysis starts when grouping and storing the information from the local neighborhoods into meaningful sets ( so - called blurring signal ) and establishing interconnections between neighborhoods .
since a priori size of the features in the object ( signal ) is unknown , the blurring is used by both humans and machines for adaptive multiscale representation of meaningful signal features of a different size . within the next step when we extract an object , certain properties have been already attributed to it , and the rest of the information is now considered as nonobject and often called noise or
as both noise and object are always parts of the perception process , there is no way to separate the noise from the object if models of the object and of the noise are absent . to distinguish noise from object ,
we need to model the image content . that could be achieved by using certain mathematical operators , feature detectors , interacting with the data followed by analysis of the results of feature extraction .
since objects in images comprise features with varying size , it is quite logical to perform feature extraction at the scale which matches the local feature size , its native scale .
a recipe for automatic feature extraction in multiscale framework can be given as follows : ( i ) build multiscale representation by smoothing the image at each scale ; ( ii ) choose an appropriate feature detector to compute local structural properties ( e.g. , gradients , curvatures , flows ) ; ( iii ) compute local extrema of a feature detector response function ; ( iv ) find the strongest local response of the structural properties which is considered as feature identification .
to build the multiscale representation of the image , the proper aperture ( windowing ) function as an operator should be chosen .
formalism for the scale - space representation was introduced by witkin and further developed by koenderink .
the idea of this approach is to generate a one parameter family of smoothed images i(x , y ; t ) obtained by convolving the original image i0(x , y ) with a gaussian kernel g(x , y ; t ) of size t
( 1)i(x , y;t)=i0(x , y)g(x , y;t),where g(x , y;t)=12te(x2+y2)/2 t .
in case of gaussian kernels ,
the kernel size t is called variance , which is related to standard deviation as t = . the parameter t in this family represents the scale at which the finer image structures are still perceptible whereas the spatial structures with size smaller than t= will be smoothed out as shown in figure 1 . as pointed out by koenderink and hummel , this family of smoothed images may also be derived as the solutions of the diffusion equation
( 2)i(x , y;t)t=[c(x , y;t)i(x , y;t)]=c(x , y;t)i(x , y;t)+c(x , y;t)i(x , y;t ) .
if c(x , y ; t ) is constant , it reduces to the isotropic diffusion equation i(x , y ; t)/t = ci(x , y ; t ) , and the linear spatial scale - space representation can be generated using gaussian ( continuous ) or binomial ( discrete ) kernels [ 24 , 610 ] .
data sampled in the temporal domain ( e.g. , the movie frames are samples taken at regular intervals ) can also be scaled in similar to the spatial domain fashion . to treat a multiscale context over the temporal domain ,
perona and malik extended the scale - space concept to nonlinear scale - spaces based on nonlinear diffusion formulation with nonconstant diffusion coefficient c(x , y ; t ) ; ( see ( 2 ) ) .
comprehensive overview of nonlinear scale - spaces based on parabolic partial differential equations could be found in [ 15 , 16 ] . for such nonlinear cases , the bessel scale - space can be built .
recently , wavelets and their applications to signal and image processing have attracted attention of the scientists in many fields .
a very good collection of papers on the wavelet theory and its applications can be found in the book by heil and walnut .
the relationship between the wavelets and the scale spaces was demonstrated by mallat and hwang . in [ 2022 ] , methods for generating scale - space representations based on wavelets were suggested , and the results of application of wavelets were very promising . as an extension of the gaussian and wavelet approaches , wang and lee proposed the scale - space representation derived from b - splines . even though different kernel functions have been proposed to generate the scale - space representations ,
the gaussian kernels remain the best candidates so far [ 110 ] . such uniqueness of the gaussian kernels was shown by babaud et al . and is based on a priori scale - space constraint formulated by witkin , koenderink , and yuille and poggio : no new feature points ( no spurious detail ) should be created with increasing scale .
[ 25 , 26 ] extended constraints and formulated the mathematical requirements known as axioms for an uncommitted visual front - end which should be satisfied for the systems without any a priori knowledge about inputs : ( i ) linearity : no feedback from the system ; ( ii ) spatial shift invariance : no preferred spatial or temporal location ; ( iii ) isotropy : no preferred spatial or temporal orientation ; ( iv ) scale invariance : no preferred size or scale of the aperture .
another motivation to use the gaussian scale spaces has some support from neurophysiological and psychological experiments which has shown that receptive field profiles in the mammalian retina and visual cortex can be well modeled by sums of gaussian and gaussian derivative components [ 2931 ] .
all these properties make the linear gaussian scale spaces the best choice for development of the automatic unsupervised systems for multiscale signal and image analysis , when there is no in advanced information available concerning preferable scales .
the recipe which allows adaptively choosing the proper local scale parameter at every geometrical location was suggested by lindeberg [ 32 , 33 ] .
this recipe comprises a two - step procedure : ( i ) convolving the original image i0(x , y ) with a gaussian kernel g(x , y ; ) of size ; ( see ( 1 ) ) and ( ii ) analyzing a response r(x , y ; ) function from derivatives or some ( possibly nonlinear ) combination of derivatives of that convolution
( 3)r(x , y;)=m+ni(x , y;)xmyn ,
where m and n are orders of derivatives .
the strongest response of such function ( with respect to ) over scales then indicates the proper gaussian scale probe ( gaussian observation kernel ) probe with width corresponding to object feature size obj has been found . due to the commutative properties of the convolution and taking derivative operations , the order of operations in the procedure above
could be changed to convolving the original image i0(x , y ) with the operators constructed from the derivatives of gaussian ( so called gaussian derivatives ) .
the distinguishing characteristic of such operators is a combination of the opposing properties , localization , and optimal response to noise [ 3537 ] . to demonstrate this principle , we modeled the original image with an object with gaussian intensity profile of kernel size obj = 5 and convolved this image with the first- ( figure 2 ) and second- ( figure 3 ) order gaussian derivatives with kernel sizes varying over the range probe = 115 ( in the figures only probe = 1 , 5 , 10 are shown ) .
then , we measured intensity values of response functions ( see ( 3 ) ) , across the object and extracted the strongest responses for all scales of the gaussian probe .
the strongest responses to both first and second gaussian derivatives with kernel probe varied over the range probe = 115 and convolved with the original image with feature size obj = 5 are shown in figure 4 .
these plots demonstrate the property of the response functions such that it has the strongest response when gaussian probe size approaches the object size .
unfortunately , the amplitude of gaussian derivative operators tends to decrease with increasing scale due to the fact that with increasing scale , the response is increasingly smoothed .
this gives more preference to smaller scales . to compensate such increase and , thus , improve accuracy of the automatic scale selection , lindeberg [ 10 , 32 , 34 ] suggested using the so - called -parameterized normalized derivatives
( 4)m+numvn=(m+n)m+nxmyn .
this method of scale selection allows feature detectors to find such points in the image that the -normalized operator response has an extremum with respect to both position and scale . in the case of tubular - like structures in an image , ridge detection with automatic scale selection
can be done using a second derivative of gaussian kernel function [ 38 , 39 ] .
depending on values of the -parameter , the detected object features can be quite different .
analyzing the influence of the -parameter on feature detection with automatic scale selection , lorenz et al .
chose to be 1.5 which worked well for variety of intensity line profiles ( e.g. , gaussian , bar- , triangle - like ) .
however , for elongated structures with bar - like intensity profiles ( such intensity profiles can be found in high - quality imagers with narrow point spread functions [ 41 , 42 ] or imagers that use deconvolution preprocessing algorithms [ 43 , 44 ] ) , the ridge detector creates false responses at small scales ( basically these are edge responses at small scales ) as depicted in figure 5 .
the line intensity profiles demonstrating the problem are shown in figure 6 . since in automatic approaches
there is no the preferred scale ( all scales should be treated equally ) in an image , a number of solutions have been proposed to avoid or suppress these false responses in scale space .
koller et al . suggested applying a nonlinear operator that combines the response of two edge - detectors on both sides of a hypothetical ridge .
lorenz et al . used an edge - indicator to suppress the response to edges .
lindeberg used a hybrid approach taking the useful properties from both the scale - space height ridge and the second derivative scale - space ridge . while studying the problem of the influence of the -parameter on feature detection with automatic scale selection , majer [ 38 , 39 ] derived the g - normalization parameter value from the statistical approach based on a white noise sampling model . in these studies
, he concluded that the ridges generated by a second - order gaussian derivative operator do not suffer from the false responses to edges if the value = 1.25 is used .
early vision perception occurs at all scale simultaneously and can be modeled by generating the image scale - space representation that introduces an additional variable , spatial scale size [ 14 ] . at this point ,
as soon as the local scales are established , the early analysis of an image starts with analysis of intensity variations and directions by means of spatial derivatives to reveal local image structure .
for example , for elongated objects , the derivative value along the object is close to zero whereas the derivatives across the object are large negative values and their ratio is close to unity . following the scale - space ideas developed so far , a complete hierarchical set of scaled differential operators has to be used .
the gaussian derivative operators described earlier in this paper constitute the natural differential operations on a given scale .
thus , a set of gaussian derivatives and their combinations could be used for very complex object models and analysis .
some well - known combinations of the high - order derivatives have special names like hessian , laplacian , and so forth and are used to build special functions for identification of certain shape patterns in images .
such mathematical functions model human and machine perception and are ultimately used in unsupervised object tracking systems [ 1 , 2 , 35 , 36 ] . for this purpose ,
the hessian - based multiscale object enhancement filters were developed [ 4 , 25 , 4648 ] . in these anisotropic filters ,
the pixel intensity transformation is locally governed by the objectness measure functions which are built using the combination of local hessian eigenvalues and calculated in multiscale framework [ 45 , 4954 ] .
the responses of the filters based on these functions are computed at different scales and are expected to have a maximum value at a scale corresponding to the width of the object [ 32 , 54 ] .
such selectivity to the object shape along with capability to adaptively choose the optimal scale allows these filters to extract the looked - for objects at their native local scales . as noted by others [ 32 , 55 ] ,
the hessian matrix h(f ) ( or simply hessian ) is the square matrix composed of second - order partial derivatives of some scalar - valued multivariable function f(x1 , x2 , , xn ) , and it describes the local curvatures of this function f(x1 , x2 , , xn ) .
assuming continuity of the second - order derivatives , the mixed derivatives do not depend on the order of differentiation ( e.g. , f/x1x2 = f/x2x1 , etc . ) .
the hessian is then a symmetric matrix which for a 3d image i(x , y , z ) can be written a 3 3 matrix ( see ( 5 ) )
( 5)h(i)=[ixxixyixziyxiyyiyzizxizyizz ] .
in ( 5 ) ,
ixx = ( /xx)i(x , y , z ) , ixy = ( /xy)i(x , y , z ) , ixy = iyx , and so on . due to symmetric property of the hessian , for 3d images only , six out of nine values have to be calculated .
let the eigenvalues of the hessian h(i ) be 1 , 2 , and 3 with their corresponding eigenvectors e1 , e2 , and e3 . if the eigenvalues ordered as 1 > 2
> 3 , then the eigenvector e1 gives the direction of the maximum of the second derivative .
following the scale - space ideas described earlier , the partial second derivatives of the image i(x , y , z ) in the hessian h(i ) have to be replaced by the -parameterized normalized gaussian derivatives ( see ( 4 ) ) , convolved with the image which results in that now the eigenvalues 1 , 2 , and 3 become adjusted to the local size of the tubular object in an image .
various algorithms for multiscale tubular object tracking and enhancement were developed depending on the way hessian eigenvalues are combined in the objectness measure function .
for instance , sato et al . [ 4951 ] suggested the objectness measure function which used only two out of three values of hessian eigenvalues
( 6)f(1;c)={exp ( 122(1c)2 ) 10 , c0exp ( 122(2c)2 ) 1>0 , c00 c=0 .
in ( 6 ) , 1 0 and 2 3 0 ( for a bright line on dark background ) , c = min ( 2 , 3 ) , and 1 < 2 .
however , it did not have a parameter to control noise ( background ) suppression .
extended the objectness measure function so that it includes the combination of all three hessian eigenvalues and a factor with a parameter which controls noise suppression ( see more details later in this paper ) .
that function reflects shape and scale of the objects , has a single maximum on the center of the vessels segments , and has bell - shape close to gaussian with width proportional to object size .
de facto become a basis for building even more sophisticated hybrid filters . manniesing et al .
used this response function to develop an effective denoising filter , where the image intensity transformation is based on anisotropic diffusion governed by the
. such an approach , which incorporates the shape pattern analysis along with multiscale data representation , would give us an extremely powerful tool to model artificial system learning .
for neuron network reconstruction from 3d confocal microscope images , the tubularity measure function was used to design a statistical learning system for training a classifier and generating the probability that a given structure belongs to the tubular - like object [ 56 , 57 ] . in pulmonology ,
the algorithms based on tube detectors were effectively used for airway and lung vascular tree reconstruction from 3d ct images [ 58 , 59 ] .
if properly normalized , the multiscale tube detectors could be used to build various cost and propagation functions required in the level set and fast marching segmentation algorithms [ 60 , 61 ] . to take the full advantage of the power of the multiscale shape detector filter in object tracking algorithms applied for a large variety of medical applications , in this work we focus on the process of automation of this filter .
the filter itself has many control parameters which can be separated into several groups : brightness measure ( objects are bright relative to background ) ; objectness measure ( shape description ) , scale description ( range plus scale step function ) , and background noise suppression parameter ( frobenius norm scale factor ) .
parameters in all groups , except the last one , describe general properties of the object itself so they do not depend on the imaging system characteristics . since in vascular studies the object brightness , tubularity , and range of diameters
hence , the only parameter which prevents the algorithm to be fully automated is control of the noise suppression .
this parameter depends on the acquisition system and imaging conditions ; therefore , it has to be experimentally found for each image set .
we present our development of the multiscale hessian - based tubular object - tracking filter with automatic selection of the parameter used for suppression of background noise . that finalizes the automation of the filter . in our approach ,
the information required for the parameter calculation is acquired from the image being processed thus it automatically takes into account all the individual properties of the particular image such as voxel size and noise level .
this allows for increased automation as well as parallel processing thereby greatly decreasing processing time .
for our studies , we used both gray - scale images numerically derived and acquired by scanners .
tubular objects with various widths were placed amid different background : gaussian random noise , nontubular objects , background with noise , and polynomial varying intensity . for simulations of images degraded by noise
, we used the c++ classes contributed to the itk insight - journal by lehmann .
these noise simulation classes are implemented with multithread support and are based on the mersenne twister uniform pseudorandom number generator which has a period ( 21 ) , 632-dimensional equidistribution , and up to 32-bit precision .
thus , this generator could be considered as a true random which results in that generated noise does not produce any
the noise was generated with various standard deviations sd = 25.0 , 50.0 , 100.0 , and mean m = 0.0 .
we also used biomedical images of the heart acquired by the micro - ct scanner , cerebellar climbing fibers [ 6567 ] , and hippocampal ca3 interneuron .
specimen h61 ( coronary artery branch within a human heart wall ) was a methyl methacrylate cast prepared as described previously .
a cast of that coronary arterial tree was scanned with an isotropic voxel size of 0.018 mm and 500 500 541 voxel ct image volume .
the custom - made micro - ct scanners generate images up to 2048 2048 1000 isotropic voxels down to 4 m on a side .
to be able to process large images using the developed algorithms , we built a specialized server with four 64 bit amd opteron 8350 quad core 2.0 ghz cpus and 128 gb memory .
the server is located in a server room and it is accessible in multiuser mode through the local network using remote clients . for our software development , we used the library of c++ classes from the national library of medicine insight segmentation and registration toolkit ( itk ) [ 6971 ] .
the library was compiled with multithread support based on the posix thread ( pthreads ) model using 64 bit c++ compiler gcc 4.3.2 [ 7375 ] and installed on 64 bit debian linux 2.6.26 [ 76 , 77 ] .
the developed multiscale shape detector filter is based on the objectness measure function suggested by frangi et al .
after thoroughly conducted studies and tests , we found that the c++ classes contributed by antiga satisfy our purposes the best ; therefore , our further developments are based on those classes .
let k be the eigenvalue of the hessian matrix at voxel x ordered such that |1
| |2 | |3| ( we drop the dependency to x ) . in the case of the ideal bright tubular structure , the voxels should satisfy the following relation for eigenvalues |1 | 0 ; |1 | |2| ; 2 3 and for bright objects both 2 and 3 must be negative .
proposed to use the eigenvalues to define vesselness measure (x ) as below :
( 7)(x)={0 if 2>0 or 3>0,(1exp ( ra22a2))exp ( rb22b2)(1exp ( s22c2 ) ) ,
where a , b , and c are the parameters that control the sensitivity of the filter to the measures ra , rb , and s. the measures have the following meaning .
ra = |2 | /|3| is used to distinguish between plate - like and line - like patterns .
the measures ra and rb are gray intensity level invariant and capture only the topological information of the objects in the image .
the choice of the control parameters a and b defines the object pattern to be studied .
for example , if tubular - like objects are chosen , the parameters are fixed to a = 0.5 , b = 0.5 .
the parameter c ( see ( 7 ) ) , controls background noise suppression in the hessian - based object enhancement filter .
the frobenius hessian matrix norm is chosen as a measure s = sqrt(1 + 2 + 3 ) to distinguish background noisy pixels .
since the parameter c strongly depends on the individual properties of particular image such as voxel size and noise level , for every new study , the optimal parameter value should be experimentally found again by trial .
the very wide search range of the optimal parameter value in concert with a highly time - expensive calculation to derive the hessian taken in multiscale framework makes this algorithm very labor - inefficient especially for large 3d biomedical images with high resolution .
for example , one trial run to process the 16 bit 500 500 514 gray - scale micro - ct image on our server took about an hour ; thus , the interactive search for the parameter c might take hours for the user who operates the program interactively . the method for automating the selection of the parameter for suppression of background noise
uses a scalar function ( nondirectional ) of the image voxels the laplacian of the image .
the laplacian is a well known operator in image processing which is easy to calculate [ 8183 ] .
ultimately , the laplacian calculates the trace of hessian matrix or , equivalently , the sum of its eigenvalues ( 1 + 2 + 3 ) making it invariant with respect to a change of tensor basis .
the schematic description of complete algorithm is below . for each voxel in the image , calculate the laplacian . in the calculated laplacian array , find the maximum value of laplacian , that is , ( 1+2+3)max .
take one tenth of that maximum value of laplacian , that is , ( 1+2+3)max /10 .
assign the calculated value to parameter c.
in this approach , the information required for parameter calculation is acquired solely from the image being processed ; thus , it automatically takes into account all the individual properties of the particular image such as a voxel size and noise level .
there are two measures commonly used for objective evaluation of the perceptual quality of images : mean square error ( mse ) and peak signal to noise ratio ( psnr ) [ 8490 ] .
consider two images being compared x = { xi | i = 1,2 , , n } and y = { yi | i = 1,2 , , n } , where n is the number of points ( pixels ) in the data sets and xi and yi are intensity levels in the images .
let x be an ideal image and y be a degraded image .
the mse measure is then defined as mse = ( 1/n)i=1(xi yi ) and psnr is , respectively , psnr = 10 log10(l / mse ) where l is the dynamic range of allowable pixel intensities . as follows from the definitions above , the lower the values of mse , the lower the error and the higher the psnr , the better quality of processed image .
the mse and psnr measures were developed using c++ classes from the itk library and contribution .
in manual mode , the control parameters have to be provided by the operator before running the code .
if the result is not acceptable , the operator has to change parameters and rerun the code again .
the mse and psnr measures were used for objective quality evaluation of the processed image . as an
ideal image , there was used a modeled image comprised three tube - like objects with gaussian intensity profiles of different width .
then , the ideal image was degraded by the random gaussian noise with standard deviation sd = 100.0 and mean m = 0.0 and processed in manual mode by the algorithm .
the background suppression parameter value was sampled over a wide range 10500 to surely cover prospective optimal control parameter .
it can be seen that both measures indicate the optimal value is about c = 200 which corresponds to mse value 0.00214 and psnr value 26.69 .
if run in automatic mode , the algorithm finds even more precise the optimal value c = 194.3 which corresponds to mse value 0.00203 and psnr value 26.93 .
these results demonstrate capability of the developed algorithm in automatically finding the optimal control parameter for background suppression .
to test efficiency of the algorithm in fully automatic mode , we used the modeled images described above .
the control parameters were chosen and fixed : brightness on , objectness measure tubular , scale range
130 , scale steps 20 , step function logarithmic , noise suppression mode
first , we processed the images with curved tubular objects with various widths which were placed amid nontubular objects .
the images were degraded by gaussian random noise with sd = 25.0 , 50.0 , and m = 0.0 .
as can be seen from figure 8 , the algorithm automatically finds the optimal parameters and successfully tracks tubular objects .
we also tested performance of the algorithm for neurological confocal microscopy image processing affected by tiling , shading , gaussian noise , nonlinear background , and so forth ( see , e.g. , figure 9 ) . the modeled images were programmed so to present three parallel tubular objects aligned in the z - direction with different diameters and distance between objects along with gray - scale distribution described by gaussian intensity profiles .
the polynomial background was added with and without gaussian random noise ( figure 10 ) . in figure 11 ,
there are depicted intensity profiles across the tubular objects with gaussian intensity profiles in the processed images with various polynomial noisy backgrounds added sd = 0.0 , 25.0 , 50.0 , and mean m = 0.0 . as could be seen
, the algorithm effectively suppresses the background and successfully extracts the tubular images . to evaluate time efficiency of our automatic method , we processed the 500 500 514 ( 16 bits per voxel ) micro - ct image with 0.018 cubic mm size in multithreaded mode using a linux - based server with the 16 processors and 128 gb memory as described above . first , we ran the automatic algorithm to find the parameter c. then , we ran nonautomatic ( the found parameter was manually plugged into a program ) version .
the amount of cpu time spent for a single run in non - automatic mode is 58 minutes whereas for automatic mode is 64 minutes , which is just about 10% longer .
if the server is able to allocate enough memory for the run , the time spent by the algorithm in fully automated mode is comparable with time for the nonautomatic mode .
the result of applying our automatic algorithm to the micro - ct image of the coronary arteries in a heart in concert with the nonprocessed image is shown in figure 12 using maximum intensity projection ( mip ) images . before preprocessing ( a ) there are the
blobs of white material due to a contrast medium accumulation in a cardiac chamber . those
blobs are selectively removed by the algorithm as can be seen in right panel ( postprocessing ) . in figure 13 , there are the not processed ( a ) and processed ( b ) mip images of cerebellar climbing fibers .
as expected , the tubular measure filter effectively suppresses the fixed pattern background and noise and delineates the tubular objects .
we also explored the efficiency of the algorithm as an initial filter in the central line extraction pipe line .
the original image sample h61 was processed with the developed filter and then segmented using the region growing connected threshold algorithm [ 6971 ] .
the seed for segmentation was allocated at the beginning of the main tree root thus the side trees were excluded .
afterwards , a center line was extracted using the 3d thinning approach based on the c++ classes submitted to the insight journal by homann .
as could be seen from the figure , the algorithm effectively suppresses background and delineates the tree .
we have presented a method for automation of adaptive nonsupervised system for tracking tubular objects that is based on analysis of local structures performed in multiscale framework .
the designed filter has demonstrated a great potential for complete automation and showed very good performance in both background noise suppression and tubular object tracking .
the developed approach can be used in the reconstruction pipeline right after image deconvolution operation . even though the convolution operator will reconstruct the object features at finer scales
, those features will appear in increased noise environment which in return might require additional postprocessing for noise suppression yet to preserve extracted features .
this filter can be used as a preprocessing filter for vessel enhancement and background noise suppression right before segmentation or immediately in the segmentation algorithms itself , for instance , in the family of segmentation algorithms which require distributed seeds [ 70 , 71 ] . by using the thresholded output of the vessel enhancement filter as a seeder , it can increase the speed efficiency of the segmentation process considerably .
since the response function is built using exponents , with proper normalization this function can be considered as a probability function with values distributed over the interval 0.0 - 1.0 . in this case , after processing , the output image holds voxels with values of probability of the event that a voxel belongs to the object with tubular shape . these probabilities can be used in many ways .
the most traditional way is to rescale it back to a gray - scale image .
although such images do not keep a proper intensity calibration , they still can be used for morphometric analysis .
if calibration is of concern , the probabilities could be converted to a mask for sampling the original micro - ct image from which the calibration could be recovered . since the filter generates the response function with only one maximum across scale space at a scale that is proportional to the diameter of the tubular object and that maximum is located at the center of the object , the probability image is more suitable to construct various cost functions .
the images with cost functions can further be used as the feature image in various image processing pipelines , for instance , such as in flux - driven centerline extraction algorithms [ 92 , 93 ] , level - set and fast - marching segmentation algorithms [ 61 , 6971 ] , and so forth . as the multiscale vessel enhancement filter is very robust against noise
, it can be superior over traditional approaches like , for example , in a filter pipeline segmented image , distance map , and cost function
, since it can directly generate the cost function avoiding steps for producing segmentation and distance map .
in addition , the multiscale space feature can be used to build the cost function in multiscale representation and use it for multiscale vessel tracking as suggested in . | the blood vessels and nerve trees consist of tubular objects interconnected into a complex tree- or web - like structure that has a range of structural scale 5 m diameter capillaries to 3 cm aorta .
this large - scale range presents two major problems ; one is just making the measurements , and the other is the exponential increase of component numbers with decreasing scale . with the remarkable increase in the volume imaged by , and resolution of , modern day 3d imagers ,
it is almost impossible to make manual tracking of the complex multiscale parameters from those large image data sets . in addition
, the manual tracking is quite subjective and unreliable .
we propose a solution for automation of an adaptive nonsupervised system for tracking tubular objects based on multiscale framework and use of hessian - based object shape detector incorporating national library of medicine insight segmentation and registration toolkit ( itk ) image processing libraries . |
deep brain stimulation ( dbs ) is an effective treatment option for a number of neurological disorders , including movement disorders , pain , and epilepsy [ 14 ] . following dbs procedures ,
skin erosion and/or infection can occur , most commonly at the connector site [ 5 , 6 ] .
infection constitutes a serious adverse event because it often leads to removal of the dbs system , with consequent loss of the clinical benefits of stimulation .
also , from an economical perspective , infection greatly adds to the costs of dbs treatment , as this complication often requires hospitalization , prolonged antibiotic therapy , and additional surgical procedures .
currently there are no guidelines that specifically address the issue of hardware - related infection . in a pooled analysis of ten studies concerning hardware - related complications of dbs , which included 922 patients ,
nevertheless , reports of successful conservative treatment with antibiotics alone seem to indicate that removal of the stimulation device is not always necessary and that conservative treatment could be considered as first choice in cases of circumscribed extracranial hardware infections .
the evaluation of the true extent of the extracranial infection can be troublesome in clinical practice and the decision of which patients are candidates for a conservative medical approach in contrast to immediate surgical treatment is usually not sufficiently clear - cut . to address the issue of infection management following dbs surgery , we set to evaluate the potential role of immunoscintigraphy with tc - labelled antigranulocyte antibody fragments ( sulesomab ) in the development of a rational therapeutic approach to infection in patients with parkinson 's disease ( pd ) submitted to dbs .
tc - sulesomab consists of monoclonal murine igg antibody fab ' fragments labeled with technetium-99 m that have affinity for a 42 kda glycoprotein found on the surface of activated granulocytes ( nca-90 surface antigen ) .
immunoscintigraphy with tc - sulesomab has been used in different clinical settings to detect infectious foci , including bone and joint infection , soft tissue infection , and in the investigation of fever of unknown origin [ 8 , 9 ] .
this preliminary study involved eight consecutive pd patients with persistent device - related skin erosion and/or infection .
all patients had been previously submitted to dbs of the subthalamic nuclei ( stn ) in a single center , in the period between november 2002 and october 2008 .
bilateral electrode implantation in the stn was followed by impulse generator ( kinetra , medtronic inc . ) placement in an infraclavicular subcutaneous pouch , in a single - stage procedure performed by the same team of surgeons .
in addition , two of the patients underwent routine ipg replacement surgery in october 2008 and april 2009 , respectively .
after the surgery , routine follow - up visits were scheduled at regular intervals with a team of movement disorders specialists .
in addition , some of the patients underwent partial removal and/or replacement of hardware components in an attempt to control infection .
because of persistent or recurrent wound dehiscence , immunoscintigraphy with tc - sulesomab was subsequently performed between july 2009 and april 2010 . planar scans and spect - ct
were acquired 4 h and 24 h after injection of tc - sulesomab ( immunomedics inc ) using a dual - head gamma camera coupled with a low - power x - ray tube .
depending on the scan results , patients were further subjected to wound debridement alone or in combination with either partial or complete hardware removal .
microbiological examinations were performed from the skin exudates , when available . at followup , three patients repeated the scan in order to determine the feasibility of reimplantation of the stimulation hardware .
patients ' characteristics , including the location of skin erosion and/or infection and the results of any surgical reinterventions preceding the immunoscintigraphy , are described in table 1 .
the results of the tc - sulesomab immunoscintigraphy and the subsequent therapeutic procedures are summarized in table 2 , as well as patient outcome and followup .
of the six patients who developed hardware - related erosion and/or infection after the initial dbs surgery , five patients presented with wound dehiscence of the retroauricular incision , which in most cases occurred in isolation ( patients 24 ) . in the remainder of cases , the retroauricular wound dehiscence occurred in association with dehiscence at distinct locations , namely , the left frontal incision ( patient 1 ) and the skin along the extension cables pathway in the neck ( patient 5 ) .
two additional patients presented with subclavicular wound dehiscence that developed after routine ipg replacement due to end of battery life .
patient 7 initially developed a hematoma at the ipg implantation site that was surgically addressed before persistent infection developed .
in addition , patient 8 subsequently presented with wound dehiscence of the left frontal incision .
staphylococcus aureus was isolated from the wound exudate in most cases . in most patients , the results of the immunoscintigraphy matched the exact location of the wound dehiscence , with a focal pattern of uptake underlying the area of skin infection ( figures 1(a ) and 1(b ) )
firstly , both patients 1 and 5 showed an increased tracer uptake that was more diffuse than could be expected from clinical inspection of the wounds ; specifically , these patients ' scans showed a diffuse tracer uptake that included the frontal areas , albeit the absence of clinical signs of infection at these locations ( figure 1(c ) ) .
the presence of infection at these clinically unapparent locations was confirmed during surgery . as described in the following paragraph , these findings have had an impact on subsequent treatment strategies .
another exception was patient 4 , whose scan did not show areas of increased uptake ; of relevance , this was the only documented case with a negative microbiological examination of the wound serous exudate .
finally , patient 8 had unexpected intracranial uptake of the tracer ( figure 1(d ) ) , which also influenced the course of treatment decisively .
the two patients who presented with a diffuse tracer uptake ( patients 1 and 5 ) underwent a complete removal of the stimulation system , and with this approach the skin infections healed properly .
it should be mentioned that both these patients had already undergone surgical removal of extracranial dbs components ( ipg and extension cables ) prior to the immunoscintigraphy , a procedure that had been ineffective .
therefore , in both patients with diffuse tracer uptake at multiple locations along the trajectory of extracranial hardware components , only complete removal of the stimulation system was effective in treating the hardware - related infection .
patient 6 had an abnormal focal tracer uptake in the left frontal area ( corresponding to the burr hole site ) , and partial removal of the stimulation system including the left electrode was performed ; the decision to remove the electrode was based on the fact that it was in direct contact with the pus , as could be observed during surgery .
although the right electrode was initially spared , it was later removed due to persistence of infection . of note ,
the right electrode followed an extracranial trajectory to the left and was thus under the area of the original infection . because patient 8 presented with intracranial tracer uptake , a complete removal of the stimulation system was undertaken , even though the patient had no clinical symptoms or signs of encephalitis , and cranial ct could not demonstrate areas of contrast enhancement suggestive of intracranial infection .
patients 2 and 3 initially underwent a more conservative surgical approach , owing to the fact that wound dehiscence and tracer uptake were both localized to the retroauricular incision .
wound debridement was the treatment option for patient 2 , based on the fact that no purulent exudate could be identified during surgery performed after prolonged antibiotic therapy despite the presence of tracer uptake at that location .
patient 3 had previously removed the ipg and extension cables and also underwent wound debridement , in addition to removal of the electrode protective caps , which contained a purulent exudate .
however , due to persistence of infection , this patient later underwent bilateral electrode removal . the only patient with isolated subclavicular wound dehiscence ( patient 7 ) had a focal tracer uptake on the chest wall and underwent removal of the ipg and extension cables , which proved to be a successful strategy .
finally , the only patient with a negative scan ( patient 4 ) underwent wound debridement alone .
later , due to persistent skin erosion , in the absence of a purulent drainage or positive cultures , contralateral transposition of the extracranial dbs components was performed .
patients 2 , 3 , and 5 repeated the immunoscintigraphy scans in order to evaluate the feasibility of a reimplantation surgery .
while patient 2 had a normal scan and underwent reimplantation of the ipg device , patients 3 and 5 maintained foci of increased tc - labeled sulesomab uptake , and reimplantation procedures were not attempted .
hardware infection in the context of dbs occurs at a rate of approximately 115% , depending on the series .
it usually represents a major management concern , often leading to the loss of the stimulation device .
several studies have analyzed the role of potential host risk factors in the development of skin complications following dbs implantation and failed to demonstrate a significant association [ 6 , 1013 ] .
nevertheless , because the rate of skin complications is higher in patients with pd than in patients with other diagnoses , it has been suggested that skin alterations in relation to pd itself could contribute to the occurrence of this adverse event [ 11 , 12 ] .
also , a straight scalp incision and a period of externalization of the electrodes in dual - stage procedures have been suggested to increase the infection rate , although the latter has not been confirmed in other studies [ 10 , 12 , 13 ] .
the utilization of kinetra devices has also been implicated as a risk factor for the development of skin complications , due to its larger volume when compared to other types of ipg [ 11 , 13 ] .
strategies to reduce the incidence of this complication have focused mainly on the development of smaller hardware components , reduction of operative times , and avoidance of temporary electrode externalization .
it has also been suggested that local antibiotic application in addition to intravenous prophylaxis could lower the rate of infection after deep brain stimulator implantation . while our knowledge on what factors associate with the development of skin complications does not progress to the point they can be prevented
, it is important to focus on strategies that help clinicians decide the best treatment approach for each individual patient .
several authors consider that a conservative treatment trial with antibiotics may be warranted in certain cases [ 7 , 14 ] while others find an early surgical approach more appropriate .
nevertheless , the management of skin complications following dbs is still controversial , and no specific guidelines have been issued . in most published
series , infection is finally treated with complete hardware removal , despite initial attempts at more localized treatment .
the major issue about infection management is being able to identify which patients should undergo hardware removal and to what extent .
the most consensual approach is that all hardware components that are in direct contact with the infection should be removed and that complete hardware removal should be used in patients with disseminated or multiple infectious foci [ 13 , 16 ] .
while generally agreeing with this strategy , there are still a large proportion of patients who fail the attempts to preserve the brain electrodes . of relevance ,
our immunoscintigraphy results demonstrate that infection can be present in areas that look clinically intact ( i.e. , without skin erythema and swelling , erosion , or purulent discharge ) due to the subcutaneous dissemination of infection from the primary site . in two of our patients
, there was an unexpected tracer uptake in the frontal areas , despite the fact that the scalp wounds were clinically intact .
significantly , infection at these apparently intact locations was confirmed during surgery by the presence of pus . by allowing the accurate determination of the subcutaneous extent of the infection , which may not be clinically evident , tc - sulesomab scans could indeed influence the treatment strategy of post - dbs infection , with a diffuse or frontal uptake indicating the necessity of a complete removal of the stimulation system at presentation , thus avoiding conservative strategies that will most likely fail , in an unsuccessful attempt to partially preserve the hardware .
the same strategy must apply to patients with intracranial tracer uptake ( which was also an unexpected finding in our series ) , who should undergo immediate removal of dbs entirely . in those cases of circumscribed infection with a focal tracer uptake , hardware - related infection
could potentially be treated with a more conservative approach . in particular , infections around the ipg or the extension cables could be successfully treated with electrode preservation .
the accurate determination of which hardware components are in direct contact with the infection can be accomplished during surgery , which explains the recommendation that the surgical treatment of hardware infection should depend on surgical findings .
but the fact that the tc - sulesomab immunoscintigraphy can help determine the actual extent of the infection in the preoperative stage can turn out to be very helpful in planning the surgical approach , as it can demonstrate all areas of involvement by the infectious process before the surgical procedure .
the results of a follow - up tc - sulesomab immunoscintigraphy could also facilitate the decision of whether and when a reimplantation procedure would be appropriate
. in other words , a normal follow - up scan can help identify which patients are more likely to be safely reimplanted at a later time .
in contrast , patients who persistently demonstrate an abnormal uptake should not be considered for a reimplantation procedure , as the risk of recurrent infection is presumably high . to our knowledge
, there is no data reporting tc - sulesomab immunoscintigraphy efficacy in the context of dbs .
our findings indicate that the scintigraphy correlates well with the presence of infection in this particular clinical setting , since all patients with a positive bacterial culture of the skin exudate had an abnormal scan , and the only patient with a negative culture had a normal scan .
also , the location of the foci of infection , as demonstrated by the presence of pus during the surgical procedures , matched the location of the tracer uptake , even in those cases with clinically unapparent infection .
this indicates that tc - sulesomab immunoscintigraphy is a reliable method to detect infection in the context of dbs , thus contributing to the assessment of patients with hardware - related infection . a final statement on the availability of the tc - sulesomab immunoscintigraphy
should be made this imaging modality should be available at every hospital that has access to nuclear medicine imaging , specifically a spect - ct scanner .
although the costs of the scintigraphy are still considerable ( approximately 850 us dollars ) , it is likely cost effective . by allowing a better distinction between patients who should have immediate removal of the whole system and those who could undergo a more conservative approach , it has the potential to reduce the number of hospital admissions , surgical interventions , and prolonged antibiotic treatments that most patients endure while attempting to preserve the stimulation device .
although only preliminary data is available in a small number of patients and more extensive followup is desirable , tc - sulesomab immunoscintigraphy seems to correlate well with the presence and extent of infection and therefore has a potential role in the management of skin complications following dbs surgery .
it is particularly helpful in discriminating between patients who should remove dbs entirely at presentation and those in whom a conservative approach is more likely to succeed , thus complementing clinical and surgical assessments .
also , tc - sulesomab immunoscintigraphy has the potential to be very useful in the determination of which patients should be considered for reimplantation surgery . finally , we propose an algorithm for the management of infection following dbs , based on the different patterns of tracer uptake in the tc - sulesomab immunoscintigraphy ( figure 2 ) . | infection constitutes a serious adverse event in patients submitted to deep brain stimulation , often leading to removal of the device .
we set to evaluate the potential role of immunoscintigraphy with 99mtc - labelled antigranulocyte antibody fragments ( 99mtc - sulesomab ) in the management of infection following dbs .
99mtc - sulesomab immunoscintigraphy seems to correlate well with the presence and extent of infection , thus contributing to differentiate between patients who should remove the hardware entirely at presentation and those who could undergo a more conservative approach .
also , 99mtc - sulesomab immunoscintigraphy has a role in determining the most appropriate timing for reimplantation .
finally , we propose an algorithm for the management of infection following dbs surgery , based on the results of the 99mtc - sulesomab immunoscintigraphy . |
the ability to grip is one of the most important functions of the hand , and grip strength can be used to reflect overall muscular strength .
several studies have shown that grip strength is inversely associated with all - cause mortality , functional impairments , frailty markers , cognitive impairments , physical disabilities , and nutritional status.[2 - 4 ] one example of using grip strength is the assessment of ' sarcopenia ' , which is characterized by progressive and generalized loss of skeletal muscle mass and strength with decreased physical performance .
although lower extremity muscle strength is more relevant than upper extremity in terms of gait and physical function , grip strength has been widely used for assessing sarcopenia because it correlates well with lower extremity muscle strength . furthermore , grip strength measurement is simple and cost - effective .
grip strength has been found to be associated with numerous factors such as demographics ( age , gender ) , body construct ( height , weight , bone mineral density [ bmd ] , hand size , upper arm circumference , hand dominance ) , socioeconomic variables ( occupation , social status , lifestyle ) and physical and psychosocial variables .
furthermore , grip strengths significantly differ between ethnicities.[6 - 18 ] although there are a few studies that have assessed normative hand grip strength in koreans , few studies have looked at comprehensively those factors that potentially affect grip strength in a population - based cohort regarding elderly koreans .
the aim of this study was to evaluate potential factors influencing grip strength in elderly koreans .
this study was conducted as a substudy of the korean longitudinal study on health and aging ( klosha ) .
the klosha was designed as a population - based prospective cohort study of health , aging , and common geriatric diseases in the elderly korean population , conducted from september 2005 to august 2006 , and involved residents of seongnam city , a satellite of metropolitan seoul , south korea .
the institutional review board of the authors ' hospital approved this cohort - based study , and written informed consent was obtained from every participant .
candidates were randomly drawn from a roster of patients 65 years or older using a computer - generated list of resident registration numbers .
we invited 1,118 individuals to participate in this study by letter and telephone , of which 696 agreed to participate ( a response rate of 62.3% ) .
a specially trained nurse recorded demographic data and conducted an interview , asking patients questions regarding a current or past medical history of illness or surgery .
several questionnaires such as general health status , upper and lower extremity functions , cognitive function , depression etc . , were used at the point of enrollment .
we excluded participants who had a history of trauma in the hand or wrist or prior surgery on the upper extremity .
we also excluded those with symptoms of shoulder or elbow pain , and patients with rheumatoid arthritis .
finally , 266 participants ( 143 males , 123 females ) were included in the analysis .
the mean age of subjects was 71.7 4.4 years ( range , 65 - 87 years ) in men , 70.7 4.5 years ( range , 65 - 86 years ) in women .
we evaluated grip strengths of the dominant hand in a standardized manner . in a neutral position of the arm , forearm , and wrist , 2 consecutive attempts with 1-minute intervalswere each measured in kilograms with a jamar 5030j1 hydraulic dynamometer ( sammons preston , bolingbrook , il , usa ) .
factors assessed for potential association with grip strength were ; 1 ) demographics such as age and gender , 2 ) body constructs such as height , body mass index ( bmi ) , and bmd , 3 ) upper extremity functional status using disabilities of the arm , shoulder and hand ( dash ) scores , and 4 ) mental health status using a depression scale ( geriatric depression scale korean version , gds - k ) and the short form-36 ( sf36 ) mental health score.[23 - 25 ] height and body weight were measured by standard methods .
bmi was calculated as weight in kilograms divided by the square of height in meters .
the bmd ( g / cm ) of the lumbar spine ( l2-l4 ) and proximal femur was measured by dual - energy x - ray absorptiometry ( dxa ; model dpx , ge - lunar corp . , madison , wi , usa ) .
all scans performed on the same machine by the same operator were analyzed with the same software .
we used femur neck bmd for statistical analysis ; marin et al . reported that handgrip strength and lean mass were the variables that were best explained by femoral neck bmd .
upper extremity functional status was assessed by using the korean version of the dash scores .
dash is a self - administered , upper extremity - specific questionnaire and contains 30 items : 21 on physical function , 5 on symptoms , and 4 on social role functions .
the dash has been widely used for evaluations of upper extremity functions , and is also well known for its reliability and validity .
the dash provides overall scores ranged from 0 and 100 ( a best possible score of 0 and a worst possible score of 100 ) .
mental health status was assessed using a depression scale ( gds - k ) and the sf36 mental health score .
the gds - k , which is a standardized self - questionnaire scores ranging from 0 to 15 ( a score of 5 or more indicates depression ) were used to specifically assess the influence of depression .
the sf36 is commonly used to represent broad aspects of patient - reported general health status , especially regarding musculoskeletal disorders .
the thirty - six items in the questionnaire are grouped on eight health subscales , and these 8 scales can be combined into two summary measures that provide overall estimates of physical health ( physical component summary , pcs ) and mental health ( mental component summary , mcs ) .
comparison of variables between male and female was tested by using student t - test .
univariate analyses were performed initially between grip strength and potential factors , and then variables that were significant at p < 0.1 in the univariate analyses as well as those factors deemed clinically important were included in the multivariate analyses .
multiple linear regression analyses with stepwise variable selection were used to assess the relative contributions of factors that influence the grip strength .
multiple linear regression models assess the abilities of explanatory variables to account for variations in response variables while accounting for any confounding between explanatory variables , and produce a statistic called adjusted r , which reflects the percentage of overall variability in the dependant variable that can be explained by the explanatory variables included in a multiple linear regression model .
, chicago , il , usa ) and statistical significance was accepted for p values of < 0.05 .
this study was conducted as a substudy of the korean longitudinal study on health and aging ( klosha ) .
the klosha was designed as a population - based prospective cohort study of health , aging , and common geriatric diseases in the elderly korean population , conducted from september 2005 to august 2006 , and involved residents of seongnam city , a satellite of metropolitan seoul , south korea .
the institutional review board of the authors ' hospital approved this cohort - based study , and written informed consent was obtained from every participant .
candidates were randomly drawn from a roster of patients 65 years or older using a computer - generated list of resident registration numbers .
we invited 1,118 individuals to participate in this study by letter and telephone , of which 696 agreed to participate ( a response rate of 62.3% ) .
a specially trained nurse recorded demographic data and conducted an interview , asking patients questions regarding a current or past medical history of illness or surgery .
several questionnaires such as general health status , upper and lower extremity functions , cognitive function , depression etc . , were used at the point of enrollment .
we excluded participants who had a history of trauma in the hand or wrist or prior surgery on the upper extremity .
we also excluded those with symptoms of shoulder or elbow pain , and patients with rheumatoid arthritis .
finally , 266 participants ( 143 males , 123 females ) were included in the analysis .
the mean age of subjects was 71.7 4.4 years ( range , 65 - 87 years ) in men , 70.7 4.5 years ( range , 65 - 86 years ) in women .
we evaluated grip strengths of the dominant hand in a standardized manner . in a neutral position of the arm , forearm , and wrist , 2 consecutive attempts with 1-minute intervalswere each measured in kilograms with a jamar 5030j1 hydraulic dynamometer ( sammons preston , bolingbrook , il , usa ) . for statistical analysis
factors assessed for potential association with grip strength were ; 1 ) demographics such as age and gender , 2 ) body constructs such as height , body mass index ( bmi ) , and bmd , 3 ) upper extremity functional status using disabilities of the arm , shoulder and hand ( dash ) scores , and 4 ) mental health status using a depression scale ( geriatric depression scale korean version , gds - k ) and the short form-36 ( sf36 ) mental health score.[23 - 25 ] height and body weight were measured by standard methods .
bmi was calculated as weight in kilograms divided by the square of height in meters .
the bmd ( g / cm ) of the lumbar spine ( l2-l4 ) and proximal femur was measured by dual - energy x - ray absorptiometry ( dxa ; model dpx , ge - lunar corp . ,
all scans performed on the same machine by the same operator were analyzed with the same software .
we used femur neck bmd for statistical analysis ; marin et al . reported that handgrip strength and lean mass were the variables that were best explained by femoral neck bmd .
upper extremity functional status was assessed by using the korean version of the dash scores .
dash is a self - administered , upper extremity - specific questionnaire and contains 30 items : 21 on physical function , 5 on symptoms , and 4 on social role functions .
the dash has been widely used for evaluations of upper extremity functions , and is also well known for its reliability and validity .
the dash provides overall scores ranged from 0 and 100 ( a best possible score of 0 and a worst possible score of 100 ) .
mental health status was assessed using a depression scale ( gds - k ) and the sf36 mental health score .
the gds - k , which is a standardized self - questionnaire scores ranging from 0 to 15 ( a score of 5 or more indicates depression ) were used to specifically assess the influence of depression .
the sf36 is commonly used to represent broad aspects of patient - reported general health status , especially regarding musculoskeletal disorders .
the thirty - six items in the questionnaire are grouped on eight health subscales , and these 8 scales can be combined into two summary measures that provide overall estimates of physical health ( physical component summary , pcs ) and mental health ( mental component summary , mcs ) .
comparison of variables between male and female was tested by using student t - test .
univariate analyses were performed initially between grip strength and potential factors , and then variables that were significant at p < 0.1 in the univariate analyses as well as those factors deemed clinically important were included in the multivariate analyses .
multiple linear regression analyses with stepwise variable selection were used to assess the relative contributions of factors that influence the grip strength .
multiple linear regression models assess the abilities of explanatory variables to account for variations in response variables while accounting for any confounding between explanatory variables , and produce a statistic called adjusted r , which reflects the percentage of overall variability in the dependant variable that can be explained by the explanatory variables included in a multiple linear regression model .
, chicago , il , usa ) and statistical significance was accepted for p values of < 0.05 .
the mean grip strength of dominant hand aged over 65 years was 27.7 7.3 kg in men , and 14.9 5.3 kg in women , and the difference was significant ( p < 0.001 ) .
in addition , grip strength decreased continuously with further aging in both men and women , as expected ( table 1 ) . demographics and other variables assessed for the study subjects are summarized in table 2 . there were significant differences in height , weight , bmd , dash , gsd - k , and sf36-mcs scores between men and women ( table 2 ) .
as many variables were found to differ between men and women , univariate and multivariate analyses were performed with separating the subjects into both sexes .
univariate analyses demonstrated that age , height , weight , log bmi , bmd , dash , sf36-mcs , and gds - k were significantly associated with grip strength of the dominant hand in men . for women , age ,
height , weight , and bmd were found to be significantly associated with grip strength ( table 3 ) . in multivariate regression analyses ,
grip strength was found to be independently associated with age , height and bmi in men , and a model including these factors accounted for 21.3% ( adjusted r = 0.213 , p < 0.001 ) of the observed variability of grip strength . in women ,
grip strength was independently associated with age and height , and a model including these factors accounted for 19.7% ( adjusted r = 0.197 , p < 0.001 ) of the observed variability of grip strength ( table 4 ) .
grip strength has been used as a measure of function in various health - related conditions .
although grip strength is known to be affected by both physical and psychological factors , few studies have looked at those factors comprehensively in a population - based cohort regarding elderly koreans .
this study demonstrates that in elderly koreans , grip strength is mainly influenced by age and height in both men and women , and additionally by bmi in men but not in women .
bmd or self - reported physical or mental health status was not found to be associated grip strength in elderly koreans .
there is a consensus that increasing age is inversely related with grip strength , and height correlates with grip strength .
however , there are contradictory findings regarding association between grip strength and body weight or bmi .
. found that among arthopometric data , grip strength was correlated best with height in both gender , and weight was associated in men , but not in women in caucasians .
however , silventoinen et al . reported that grip strength is positively associated with height , weight , and bmi in both sexes in a study of one million swedish people .
reported that there is age - related changes in body composition , particularly increases in fat and central fat deposition and decreases in lean mass , which may result in low skeletal muscle mass and obesity ( sarcopenic obesity ) .
therefore , one can have a high bmi with weak muscle strength due to sarcopenic obesity .
jang reported the prevalence of sarcopenic obesity is 35% in men and 48% in women in elderly koreans .
high prevalence of sarcopenic obesity in women may explain why bmi was not found to be associated with grip strength in women in our study .
it has been suggested that muscle strength may be positively related with higher bmd.[34 - 36 ] bayramolu et al .
verified that the isometric strength of hip abductors had a significant correlation with femoral neck bmd in postmenopausal sedentary women with and without osteoporosis .
marin et al . also found that grip strength in postmenopausal women was the one that best associated with bmd in all analyzed bone sites . in our study , however , grip strength was not associated with bmds . in this study , self - reported upper extremity functional status or mental health status
was not found to be associated with grip strength . as we used self - administered questionnaires , we are not able to correlate grip strength with objective test results of physical and mental status .
watson et al . investigated the relationship between grip strength and depression using the center for epidemiologic studies depression ( ces - d ) scale , pain anxiety symptoms scale , and pain catastrophizing scale .
they reported that depression scores were minimally associated with decreased hand grip strength and that psychological factors influenced disability ( patient - reported health status ) more than function ( performance - based measures ) . in contrast , rantanen et al .
demonstrated a stronger association between grip strength and depression using the ces - d scale .
these authors examined grip strength data of 924 community - dwelling korean people aged over 65 years without dementia , and the mean grip strengths were 29 kg in men and 17 kg in women .
grip strengths in koreans seem to be weaker compared to those in caucasians ( table 5 ) .
previous studies found ethnic differences in grip strength in association with anthropometric differences,[41 - 43 ] and also withmuscle characteristics .
first , our study focused on elderly koreans aged over 65 years , but included only small number of participants aged over 80 's ( 3.5% in men , 4.1% in women ) , which may not represent true elderly population .
second , although we attempted to assess factors influencing grip strength comprehensively , we did not evaluate other factors such as hand size , upper arm circumference , occupation , lifestyle , medications , nutritional status , and other objective physical and mental status measures .
lastly , this is a cross - sectional study , therefore associations found between grip strength and variables may not be causal .
a longitudinal study with follow - up grip strength evaluations is required to generalize the findings of this study .
in conclusion , we found in elderly koreans without a known history of upper extremity disorder or surgery , grip strength is independently associated with age and height in both gender , and with bmi in men .
bmd or self - reported physical or mental health status was not found to be associated with grip strength .
this information may be helpful in future studies using grip strength as a measure of function in elderly koreans . | objectivesgrip strength has been used as a measure of function in various health - related conditions .
although grip strength is known to be affected by both physical and psychological factors , few studies have looked at those factors comprehensively in a population - based cohort regarding elderly koreans .
the aim of this study was to evaluate potential factors influencing grip strength in elderly koreans.methodswe evaluated dominant hand grip strengths in 143 men and 123 women older than 65 years who participated in a population - based cohort study , the korean longitudinal study on health and aging ( klosha ) .
individuals who had a history of surgery for musculoskeletal disease or trauma in the upper extremity were excluded .
factors assessed for potential association with grip strength were ; 1 ) demographics such as age and gender , 2 ) body constructs such as height , body mass index ( bmi ) , and bone mineral density ( bmd ) , 3 ) upper extremity functional status using disabilities of the arm , shoulder and hand ( dash ) scores , and 4 ) mental health status using a depression scale and the short form-36 ( sf36 ) mental health score .
multivariate analyses were performed in order to identify factors independently associated with grip strength.resultsgrip strengths of dominant hands in elderly koreans were found to generally decrease with aging , and were significantly different between men and women , as expected .
multivariate analyses indicated that grip strength was independently associated with age , height and bmi in men ( r2 = 21.3% ) , and age and height ( r2 = 19.7% ) in women .
bmd , upper extremity functional status , or mental health status were not found to be associated with grip strength.conclusionsthis study demonstrates that in elderly koreans , grip strength is mainly influenced by age and height in both men and women , and additionally by bmi in men .
bmd or self - reported physical or mental health status was not found to influence grip strength in elderly koreans .
this information may be helpful in future studies using grip strength as a measure of function in elderly koreans . |
asbestosis is one of the most frequently studied diseases associated with the inhalation of asbestos fibers .
the exposure to asbestos is known to cause inflammation , fibrous scarring in the lung , and cancer [ 1 , 2 ] .
it has been suggested that , in addition to reactive oxygen species ( ros ) , the reactive nitric species ( rns ) , such as nitric oxide ( no ) , may also be involved in the development of asbestos - related diseases [ 36 ] .
nitric oxide is a free radical which readily reacts with ros , such as superoxide anion , producing peroxynitrite , which can generate hydroxyl radicals [ 711 ] . in the living organisms
no is synthesized by nitric oxide synthases ( noss ) , which catalyse the conversion of amino acid l - arginine and molecular oxygen into no and l - citrulline [ 11 , 12 ] .
three isoforms of nos have been identified : neuronal nos ( nnos or nos1 ) , inducible nos ( inos or nos2 ) , and endothelial nos ( enos or nos3 ) .
inducible nos is often present at sites of chronic inflammation [ 5 , 11 ] .
nitric oxide generation is thought to be an important mediator of cell injury and inflammation caused by asbestos .
studies indicate that asbestos fibers may upregulate the activity of inos and thus the production of no by alveolar macrophages and pulmonary epithelial cells [ 5 , 6 , 1315 ] .
several studies described the association between no production and the adverse effects of asbestos [ 36 , 13 ] .
reported an association between the magnitude of no production by lung epithelial cells and dna oxidation at in vitro exposure to asbestos .
treatment of human lung epithelial cells with asbestos was shown to result in the synthesis of inos mrna and increased intracellular levels of nitrate , a stable oxidation product of no . in addition , a temporal correlation was demonstrated between no production by alveolar macrophages and neutrophil infiltration into lung after inhalation of asbestos .
the findings of these studies suggest that no may play an important role in the initiation and progression of asbestosis .
however , the studies in inos knockout mice indicate that inos - derived no exerts a dual role in acute asbestos - induced lung injury as inos deficiency resulted in an exacerbated inflammatory response but improved oxidant - promoted lung tissue damage .
several types of polymorphisms have been identified in the promoter region of the inos gene , among them the tandem repeat number polymorphism of a ccttt pentanucleotide ( ( ccttt)n ) [ 17 , 18 ] .
the ccttt pentanucleotide repeats within the promotor region of the human inos gene have been associated with the biochemical promoter activity .
several studies have suggested an association between inflammatory diseases or cancer and the number of the ccttt repeats [ 1821 ] .
however , to our knowledge and according to the literature available , this polymorphism has not been studied so far in association with asbestosis . in this study we investigated whether inos ( ccttt)n polymorphism is associated with an increased risk of asbestosis in workers exposed to asbestos .
the study population comprised 262 subjects who were diagnosed with asbestosis as an occupational disease and 265 subjects with no asbestos disease as the control group .
all the subjects were selected from a cohort of 2080 workers who were occupationally exposed to asbestos in the asbestos cement manufacturing plant of salonit anhovo , slovenia , and presented at the state board for recognition of occupational asbestos diseases in the period between 1 january 1998 and 31 december 2003 . to obtain the information about smoking , all cases and controls were interviewed by the same investigator , using a standardized questionnaire [ 23 , 24 ] .
for each subject included in the present study , the cumulative asbestos exposure data were obtained from the previous study .
the diagnosis of asbestosis or no asbestos - related disease was confirmed by two groups of experienced occupational experts of the state board for recognition of occupational asbestos diseases at the clinical institute of occupational medicine .
each group of experts consisted of an occupational physician , a radiologist , and a pulmonologist skilled in diagnosis of asbestos - related diseases .
the diagnosis was based on the helsinki criteria for diagnosis and attribution of asbestos diseases and on the american thoracic society recommendations .
high - resolution computer tomography ( hrct ) was used for radiological diagnosis of asbestosis . in
genotyping of the ( ccttt)n in the inos gene promoter was performed by the amplification of the corresponding region of the promoter by pcr using a primer pair with one primer being fluorescently labelled .
the amplified fragments were analysed on the abi prism 310 genetic analyser ( applied biosystems ) , and the number of polymorphic repeats was determined with genemapper analysis software ( applied biosystems , norwalk , usa ) as previously reported .
first , the statistical analysis included the descriptive statistics , t - test , and test .
next , univariate logistic regression was performed to estimate the risk of asbestosis in relation to individual variables ( inos , cumulative asbestos exposure , gender , age , and smoking ) .
the subjects with asbestosis were on average 61 and the controls 57 years old ( t = 5.18 , p = .000 ) . comparison of smoking between the cases and the controls showed no statistically significant difference ( = 0.01 , p = .919 ) .
approximately forty - five percent of subjects ( 117 cases and 120 controls ) were ever smokers .
the average cumulative asbestos exposure was significantly higher in subjects with asbestosis compared to those without asbestos - related disease ( 37.67 fibres / cm - years for cases and 11.23 fibres / cm - years for controls ; = 4.78 , p = .000 ) .
genotyping of the ( ccttt)n in the inos gene promoter was successful in 258 cases and 259 controls .
thirteen alleles for the inos microsatellite ranging from 166 to 226 base pears ( bp ) ( 6 to 18 ccttt repeats ) were observed ( table 1 ) .
the alleles with 11 ccttt repeats were defined as short alleles ( s ) and alleles with 12 repeats as long alleles ( l ) ( table 1 ) .
accordingly , the subjects were classified into three groups of genotypes : ss , sl , and ll .
inducible nitric oxide synthase ll genotype was observed in 132 cases and 121 controls , sl genotype in 83 cases and 96 controls , while ss genotype was found in 43 cases and 42 controls ( table 2 ) .
when combining genotypes , no significant difference was found in the frequency of the inos ll genotype versus the frequency of combined sl and ss genotypes ( table 2 ) . no association has been observed between asbestosis and smoking ( ever / never ) ( or = 0.98 , 95% ci = 0.691.39 ) .
cumulative asbestos exposure was logarithmically transformed , leading to an or of asbestosis of 3.21 ( 95% ci = 2.434.23 ) .
determining the associations between inos genotypes and asbestosis , the or of asbestosis was 1.07 ( 95% ci = 0.651.74 ) for the ll genotype versus ss genotype , 1.26 ( 95% ci = 0.861.85 ) for the ll genotype compared to sl genotype , 0.84 ( 95% ci = 0.501.42 ) for the sl genotype versus ss genotype , and 1.20 ( 95% ci = 0.851.69 ) for the ll genotype compared to the combined sl and ss genotypes .
the risk of asbestosis for the inos ll genotype versus the combined sl and ss genotypes remained practically unchanged after adjustment by gender , age , smoking , and cumulative asbestos exposure ( table 3 ) .
it has been suggested that inos - derived no may play an important role in the development of asbestos - related diseases [ 5 , 6 , 1315 ] . in a nested case - control
study the authors investigated the influence of the inos ( ccttt)n polymorphism on the risk of developing asbestosis .
a slightly ( 20% ) higher risk of asbestosis for the inos ll genotype compared to the combined sl and ss genotypes and almost 30% for the ll versus sl genotype has been detected .
this finding , although borderline significant , could be in agreement with the observations of some other studies which demonstrated an association between a disease and longer forms of ( ccttt)n repeat in the inos promoter region [ 18 , 20 , 21 ] .
these results could be explained by a higher promoter activity of the inos gene which has been shown to increase with the ( ccttt)n repeat number in an in vitro study and consequently increased production of no .
since no is a free radical and its production has been associated with pulmonary damage , inflammation , and disease progression caused by asbestos [ 5 , 6 ] , our finding could be considered as biologically plausible .
all the cases and controls who participated in this study were recruited in a small geographical area with an ethnically homogenous population .
therefore , no bias was introduced by genetic heterogeneity . although the board of experts used the hrct as a widely recommended method for the diagnosis of asbestosis [ 25 , 26 ] in all subjects , there is still a slight possibility that some subjects might have been recruited as controls when they had already developed asbestosis , which could be diagnosed only by the histopathological examination . in that case
considering that our study included cases who had inos ll genotype more frequently than the controls , the association between asbestosis and inos ll genotype might have been slightly stronger in that case . in conclusion , to our knowledge , this is the first study investigating the association between asbestosis and inos genetic polymorphism .
considering that the results of the current study are borderline significant , further studies including a larger number of subjects are needed to clarify this association . | asbestos , a known occupational pollutant , may upregulate the activity of inducible nitric oxide synthase ( inos ) and thus the production of nitric oxide ( no ) .
this study investigated whether inos ( ccttt)n polymorphism is associated with an increased asbestosis risk in exposed workers .
the study cohort consisted of 262 cases with asbestosis and 265 controls with no asbestos - related disease .
for each subject the cumulative asbestos exposure data were available .
the number of ccttt repeats was determined following pcr amplification of the inos promoter region .
logistic regression was performed to estimate asbestosis risk .
the or of asbestosis was 1.20 ( 95% ci = 0.851.69 ) for the ll genotype compared to the combined sl and ss genotypes and 1.26 ( 95% ci = 0.861.85 ) for the ll genotype compared to the sl genotype .
the results of this study are borderline significant and suggest a possible role of inos ( ccttt)n polymorphism in the risk of asbestosis ; however , further studies are needed . |
we conducted a prospective , single - center observational study over 2 time periods ( july to november 2013 and february to april 2014 ) .
semistructured interviews were conducted with consecutive eligible acs patients admitted to a metropolitan tertiary hospital located in melbourne , australia .
the study was approved by ethics committees at monash university and the alfred hospital , and all participants gave informed consent . between may and august 2013 , a fully paid mass media campaign across television and radio was conducted in the melbourne metropolitan area , with digital advertising run nationally across the whole of 2013 .
this paid media activity was built on an intermittent campaign strategy conducted between 2009 and 2013 , including paid and pro bono advertising on television , radio , and websites .
other pro bono community - based activities , such as stories in community newspapers and promotional materials distributed to community groups and hospitals , also took place in some parts of melbourne .
a campaign - specific website was established to provide comprehensive information about the warning signs of heart attack and the appropriate actions to take.15 patients were identified from reviewing the daily cardiology admission lists for acs patients : st - elevation myocardial infarction ( stemi ) , non - stemi ( nstemi ) , and unstable angina ( ua ) . to be eligible , patients had to have a confirmed primary diagnosis of out - of - hospital acs defined as a rise in a cardiac biomarker ( troponin i ) above the 99th percentile upper reference limit ( url ) and/or clinical symptoms consistent with myocardial ischemia , with 1 of the following : new or presumed new significant st - segment t - wave changes or new left bundle branch block on the ecg ; development of pathological q waves on the ecg ; imaging evidence of new loss of viable myocardium or new regional wall motion abnormality ; or identification of an intracoronary thrombus by angiography.16 other eligibility criteria included : aged between 35 and 75 years ; competent to provide consent ; english - speaking ; community dwelling resident ( ie , not residential care ) ; and medically stable and able to participate in a 30-minute interview .
data were collected from an audit of the patient s medical record and from semistructured interviews . to examine the representativeness of the final sample , deidentified demographic ( age , sex ) and clinical ( acs subtype ) data were collected from the medical record for patients who were ineligible and eligible patients who declined to participate . for participating patients , data collected from the medical record , and patient interviews if missing , included ( 1 ) sociodemographic data ( age , sex , marital status , country of birth , level of education , first aid , medical or nursing training , and ems insurance cover ) ; ( 2 ) clinical history ( previous acs , risk factors , and major comorbid conditions ) ; and ( 3 ) current presentation ( time of symptom onset and presentation to hospital , and acs subtype ) .
face - to - face interviews were conducted by using the modified response to symptoms questionnaire ( mrtsq ) , with additional questions to specifically address the awareness and effect of the campaign .
the mrtsq was developed17 and modified18 to collect data about the symptoms ( type , context , antecedents , and appraisal ) and subsequent response ( behavioral , cognitive , and emotional ) .
awareness of the campaign was first tested unprompted ( by asking prior to this hospitalization , had you seen any television commercials and/or advertising about heart attacks ? ) and prompted by showing video stills of campaign advertising ( by asking prior to this hospitalization , had you seen any of the following television commercials and/or advertising ? ) .
the open - ended questions captured ( 1 ) the symptoms experiences in the patient s own words , ( 2 ) how the experience was different to what they expected a heart attack to be like , and ( 3 ) reasons for delay .
the responses to open - ended questions were content analyzed by 2 experience researchers with nursing training .
descriptive statistics were performed to compare the demographic and clinical data of participants and nonparticipants .
descriptive statistics were also used to compare the demographic and clinical data of acs patients who indicated that they were aware of the campaign ( exposed group ) with those of the patients who were not aware of the campaign ( unexposed group ) . mann
whitney and t tests were used to analyze continuous variables , and the test was used for categorical variables .
two time periods were calculated : prehospital delay time ( time interval from symptom onset to arrival at hospital ) and patient delay ( time interval from symptom onset to the decision to seek medical attention ) .
prehospital times were established from medical record review and information given by the patient in the interview .
prompts and detailed questioning were used to aid recall of times.11,14 patients were carefully interviewed to determine the time of onset of the symptom that was the reason they went to the hospital.14 the decision to seek medical attention was defined as the time the patient realized they need to seek medical care and was obtained during the interview and where possible confirmed with times recorded in the medical record ( eg , time ambulance called ) .
the time of arrival at hospital was taken as the time of arrival in the emergency department as recorded in the nursing triage notes .
multivariable logistic regression models were developed to examine the association between campaign awareness and ( 1 ) a patient delay time of 1 hour and ( 2 ) a prehospital delay time of 2 hours.19,20 given the sample size and large number of potential variables , propensity scores were estimated to adjust for the conditional probability that an individual was aware of the campaign.21 the propensity score included variables significantly associated with campaign awareness and prehospital delay time ( tables1 and 2 ) and was used as a covariate in the subsequent multivariable models .
propensity scores and final models were adjusted for variables that are consistently reported as associated with delay times in the literature : patient delay ( level of education,21 recognized symptoms as heart related,20,22 choosing to wait and see if symptoms would go away,18 sudden onset,23 intermittent symptoms18,21,23 and stemi20,23,24 ) and prehospital delay ( recognized symptoms as heart related,2224 choosing to wait and see if symptoms would go away,18 did nt want to alarm or trouble others,18,21 stemi,20,23,24 wanted to see local doctor first23,25 ) .
statistical analysis was conducted in stata and p - values < 0.05 were considered statistically significant .
demographics of the overall sample and a comparison by campaign awareness cabg indicates coronary artery bypass grafting ; ems , emergency medical services ; ihd , ischemic heart disease ; mi , myocardial infarction ; pci , percutaneous coronary intervention .
univariate analysis of factors related to patients delay of 1 hour and prehospital delay of 2 hours cabg indicates coronary artery bypass grafting ; ems , emergency medical services ; ihd , ischemic heart disease ; mi , myocardial infarction ; or , odds ratio ; pci , percutaneous coronary intervention ; stemi , st - elevation myocardial infarction . combined as highly correlated .
we conducted a prospective , single - center observational study over 2 time periods ( july to november 2013 and february to april 2014 ) .
semistructured interviews were conducted with consecutive eligible acs patients admitted to a metropolitan tertiary hospital located in melbourne , australia .
the study was approved by ethics committees at monash university and the alfred hospital , and all participants gave informed consent .
between may and august 2013 , a fully paid mass media campaign across television and radio was conducted in the melbourne metropolitan area , with digital advertising run nationally across the whole of 2013 .
this paid media activity was built on an intermittent campaign strategy conducted between 2009 and 2013 , including paid and pro bono advertising on television , radio , and websites .
other pro bono community - based activities , such as stories in community newspapers and promotional materials distributed to community groups and hospitals , also took place in some parts of melbourne .
a campaign - specific website was established to provide comprehensive information about the warning signs of heart attack and the appropriate actions to take.15
patients were identified from reviewing the daily cardiology admission lists for acs patients : st - elevation myocardial infarction ( stemi ) , non - stemi ( nstemi ) , and unstable angina ( ua ) . to be eligible , patients had to have a confirmed primary diagnosis of out - of - hospital acs defined as a rise in a cardiac biomarker ( troponin i ) above the 99th percentile upper reference limit ( url ) and/or clinical symptoms consistent with myocardial ischemia , with 1 of the following : new or presumed new significant st - segment t - wave changes or new left bundle branch block on the ecg ; development of pathological q waves on the ecg ; imaging evidence of new loss of viable myocardium or new regional wall motion abnormality ; or identification of an intracoronary thrombus by angiography.16 other eligibility criteria included : aged between 35 and 75 years ; competent to provide consent ; english - speaking ; community dwelling resident ( ie , not residential care ) ; and medically stable and able to participate in a 30-minute interview .
data were collected from an audit of the patient s medical record and from semistructured interviews . to examine the representativeness of the final sample , deidentified demographic ( age , sex ) and clinical ( acs subtype ) data were collected from the medical record for patients who were ineligible and eligible patients who declined to participate . for participating patients ,
data collected from the medical record , and patient interviews if missing , included ( 1 ) sociodemographic data ( age , sex , marital status , country of birth , level of education , first aid , medical or nursing training , and ems insurance cover ) ; ( 2 ) clinical history ( previous acs , risk factors , and major comorbid conditions ) ; and ( 3 ) current presentation ( time of symptom onset and presentation to hospital , and acs subtype ) . face - to - face interviews were conducted by using the modified response to symptoms questionnaire ( mrtsq ) , with additional questions to specifically address the awareness and effect of the campaign .
the mrtsq was developed17 and modified18 to collect data about the symptoms ( type , context , antecedents , and appraisal ) and subsequent response ( behavioral , cognitive , and emotional ) .
awareness of the campaign was first tested unprompted ( by asking prior to this hospitalization , had you seen any television commercials and/or advertising about heart attacks ? ) and prompted by showing video stills of campaign advertising ( by asking prior to this hospitalization , had you seen any of the following television commercials and/or advertising ? ) .
the open - ended questions captured ( 1 ) the symptoms experiences in the patient s own words , ( 2 ) how the experience was different to what they expected a heart attack to be like , and ( 3 ) reasons for delay .
the responses to open - ended questions were content analyzed by 2 experience researchers with nursing training .
descriptive statistics were performed to compare the demographic and clinical data of participants and nonparticipants .
descriptive statistics were also used to compare the demographic and clinical data of acs patients who indicated that they were aware of the campaign ( exposed group ) with those of the patients who were not aware of the campaign ( unexposed group ) . mann
whitney and t tests were used to analyze continuous variables , and the test was used for categorical variables .
two time periods were calculated : prehospital delay time ( time interval from symptom onset to arrival at hospital ) and patient delay ( time interval from symptom onset to the decision to seek medical attention ) .
prehospital times were established from medical record review and information given by the patient in the interview .
prompts and detailed questioning were used to aid recall of times.11,14 patients were carefully interviewed to determine the time of onset of the symptom that was the reason they went to the hospital.14 the decision to seek medical attention was defined as the time the patient realized they need to seek medical care and was obtained during the interview and where possible confirmed with times recorded in the medical record ( eg , time ambulance called ) .
the time of arrival at hospital was taken as the time of arrival in the emergency department as recorded in the nursing triage notes .
multivariable logistic regression models were developed to examine the association between campaign awareness and ( 1 ) a patient delay time of 1 hour and ( 2 ) a prehospital delay time of 2 hours.19,20 given the sample size and large number of potential variables , propensity scores were estimated to adjust for the conditional probability that an individual was aware of the campaign.21 the propensity score included variables significantly associated with campaign awareness and prehospital delay time ( tables1 and 2 ) and was used as a covariate in the subsequent multivariable models .
propensity scores and final models were adjusted for variables that are consistently reported as associated with delay times in the literature : patient delay ( level of education,21 recognized symptoms as heart related,20,22 choosing to wait and see if symptoms would go away,18 sudden onset,23 intermittent symptoms18,21,23 and stemi20,23,24 ) and prehospital delay ( recognized symptoms as heart related,2224 choosing to wait and see if symptoms would go away,18 did nt want to alarm or trouble others,18,21 stemi,20,23,24 wanted to see local doctor first23,25 ) .
statistical analysis was conducted in stata and p - values < 0.05 were considered statistically significant .
demographics of the overall sample and a comparison by campaign awareness cabg indicates coronary artery bypass grafting ; ems , emergency medical services ; ihd , ischemic heart disease ; mi , myocardial infarction ; pci , percutaneous coronary intervention .
univariate analysis of factors related to patients delay of 1 hour and prehospital delay of 2 hours cabg indicates coronary artery bypass grafting ; ems , emergency medical services ; ihd , ischemic heart disease ; mi , myocardial infarction ; or , odds ratio ; pci , percutaneous coronary intervention ; stemi , st - elevation myocardial infarction . combined as highly correlated .
over the 2 study periods , there were 323 acs patients admitted and screened for inclusion in the study .
of these , 214 ( 66% ) were eligible : 199 ( 93% ) consented and were interviewed and 15 declined to participate .
reasons for exclusion in screened patients included age outside 35 to 75 years ( n=77 ) , non english speaking ( n=15 ) , medically unstable ( n=8 ) , inpatient event ( n=5 ) , cognitive impairment ( n=3 ) , and previously interviewed and readmitted ( n=1 ) . although the study intentionally included a younger subset of patients ( aged 35 to 75 years ) , there was no difference in acs subtypes or in the proportion of males between those interviewed and those excluded .
the median age of the patients interviewed was 62 years , and 68% were males ( table1 ) . just over half of the patients ( 56% ) were born in australia ( 67% born in an english - speaking country ) , with the majority ( 80% ) currently residing in the city of melbourne .
most of those interviewed had received a high school level or higher education ( 70% ) , and almost half had previously undergone either first - aid ( 44% ) or nursing ( 2% ) training .
patients presented with nstemi ( 44% ) , ua ( 35% ) , and stemi ( 21% ) .
overall , two - thirds of patients ( n=127 ) recalled seeing or hearing the campaign advertising either unprompted or after viewing screen shots of the advertising ( figure1 ) . over half of those aware of the campaign estimated
they had seen the campaign during the period of paid advertising ( may to august 2013 ) : unprompted=55% and prompted=66% .
those aware of the campaign were more likely to be : younger ( median age 60 years versus 65 years , p=0.04 ) ; born in australia ( 63% versus 43% , p=0.007 ) or an english - speaking country ( 75% versus 54% , p=0.003 ) ; and first aid trained ( 50% versus 35% , p=0.04 ) ( table1 ) .
no other demographics or cardiac risk factors were associated with campaign awareness , including a history of previous acs events or being a melbourne resident . the proportion of acs patients who recalled seeing commercials or advertising about heart attacks unprompted or after viewing screen shots of the advertising ( n=199 ) .
the majority of patients ( 91% ) stated they knew at least 1 symptom or sign of heart attack ( symptom knowledge ) prior to this admission to hospital .
sources of this knowledge included the media ( 47% ) , personal experience ( 29% ) , first - aid training ( 19% ) , family members ( 21% ) , friends ( 11% ) , and local physicians ( 9% ) .
campaign awareness was not associated with increased symptom knowledge ( 91% versus 90% , p=0.80 ) .
however , the majority of those aware stated that the campaign increased their awareness of what is a heart attack ( 63% ) and the signs and symptoms of a heart attack ( 68% ) ( figure2 ) .
patients who stated the campaign influenced their knowledge of what is a heart attack were more likely to attribute their symptoms for this hospitalization to the heart ( 58% versus 41% , p=0.016 ) .
the influence of the campaign material on ( 1 ) awareness of what is a heart attack , ( 2 ) awareness of signs and symptoms , and ( 3 ) actions taken in response to symptoms ( restricted to those aware of the campaign ( n=127 ) .
almost half ( 43% ) of the patients who were aware of the campaign said it influenced their actions in response to symptoms ( figure2 ) .
this was highest among patients who were admitted for a stemi ( 62% ) , compared with nstemi ( 45% ) and ua ( 30% ) .
those who stated the campaign influenced their behavior were more likely to be younger than 65 years ( 44% versus 24% , p=0.02 ) .
the median patient delay time was 90 minutes ( iqr=20 to 661 ) , with almost half of patients ( 47% ) deciding to seek medical attention within 1 hour of symptom onset .
factors significantly associated with a patient delay time of 1 hour at the univariate level are given in table2 .
after adjustment for other important predictors and the propensity score , awareness of the campaign was significantly associated with patient delay time of 1 hour ( adjusted odds ratio [ aor]=2.25 , 95% ci:1.03 to 4.91 , p=0.04 ) ( table3 ) .
models testing the association of campaign awareness and patient delay of 1 hour and prehospital delay of 2 hours or indicates odds ratio ; stemi , st - elevation myocardial infarction . adjusted for propensity score .
the median prehospital delay was 210 minutes ( iqr=100 to 1035 ) , with 34% presenting to hospital within 2 hours of symptom onset .
table2 provides the factors significantly associated with a prehospital delay time of 2 hours . after adjustment , awareness of the campaign
was significantly associated with a prehospital delay time 2 hours ( aor=3.10 , 95% ci : 1.36 to 7.09 , p=0.007 ) ( table3 ) .
overall , 18% of patients visited their general practitioner ( gp ) before presenting to hospital .
patients admitted with unstable angina ( 26% ) or nstemi ( 20% ) were more likely to visit their gp than were patients experiencing stemi ( 2% , p=0.007 ) .
campaign awareness was not associated with lower rates of visiting a gp ( 20% versus 14% , p=0.24 ) , although those aware of the campaign who stated the campaign influenced their behavior were less likely to visit a gp ( 7% versus 22% , p=0.017 ) .
the ems system was used by 53% of patients , although 8% of these patients first attempted transport in a car or drove to a gp who called ems .
ems use was highest among patients admitted with stemi ( 69% ) , compared with nstemi ( 52% ) and ua ( 47% ) .
no difference was seen in ems use by campaign awareness ( 52% versus 57% , p=0.50 ) or in those who stated the campaign influenced their behavior ( 56% versus 53% , p=0.78 ) .
over the 2 study periods , there were 323 acs patients admitted and screened for inclusion in the study .
of these , 214 ( 66% ) were eligible : 199 ( 93% ) consented and were interviewed and 15 declined to participate .
reasons for exclusion in screened patients included age outside 35 to 75 years ( n=77 ) , non english speaking ( n=15 ) , medically unstable ( n=8 ) , inpatient event ( n=5 ) , cognitive impairment ( n=3 ) , and previously interviewed and readmitted ( n=1 ) . although the study intentionally included a younger subset of patients ( aged 35 to 75 years ) , there was no difference in acs subtypes or in the proportion of males between those interviewed and those excluded .
the median age of the patients interviewed was 62 years , and 68% were males ( table1 ) . just over half of the patients ( 56% ) were born in australia ( 67% born in an english - speaking country ) , with the majority ( 80% ) currently residing in the city of melbourne .
most of those interviewed had received a high school level or higher education ( 70% ) , and almost half had previously undergone either first - aid ( 44% ) or nursing ( 2% ) training .
patients presented with nstemi ( 44% ) , ua ( 35% ) , and stemi ( 21% ) .
overall , two - thirds of patients ( n=127 ) recalled seeing or hearing the campaign advertising either unprompted or after viewing screen shots of the advertising ( figure1 ) . over half of those aware of the campaign estimated
they had seen the campaign during the period of paid advertising ( may to august 2013 ) : unprompted=55% and prompted=66% .
those aware of the campaign were more likely to be : younger ( median age 60 years versus 65 years , p=0.04 ) ; born in australia ( 63% versus 43% , p=0.007 ) or an english - speaking country ( 75% versus 54% , p=0.003 ) ; and first aid trained ( 50% versus 35% , p=0.04 ) ( table1 ) .
no other demographics or cardiac risk factors were associated with campaign awareness , including a history of previous acs events or being a melbourne resident . the proportion of acs patients who recalled seeing commercials or advertising about heart attacks unprompted or after viewing screen shots of the advertising ( n=199 ) .
the majority of patients ( 91% ) stated they knew at least 1 symptom or sign of heart attack ( symptom knowledge ) prior to this admission to hospital .
sources of this knowledge included the media ( 47% ) , personal experience ( 29% ) , first - aid training ( 19% ) , family members ( 21% ) , friends ( 11% ) , and local physicians ( 9% ) .
campaign awareness was not associated with increased symptom knowledge ( 91% versus 90% , p=0.80 ) .
however , the majority of those aware stated that the campaign increased their awareness of what is a heart attack ( 63% ) and the signs and symptoms of a heart attack ( 68% ) ( figure2 ) .
patients who stated the campaign influenced their knowledge of what is a heart attack were more likely to attribute their symptoms for this hospitalization to the heart ( 58% versus 41% , p=0.016 ) .
the influence of the campaign material on ( 1 ) awareness of what is a heart attack , ( 2 ) awareness of signs and symptoms , and ( 3 ) actions taken in response to symptoms ( restricted to those aware of the campaign ( n=127 ) .
almost half ( 43% ) of the patients who were aware of the campaign said it influenced their actions in response to symptoms ( figure2 ) .
this was highest among patients who were admitted for a stemi ( 62% ) , compared with nstemi ( 45% ) and ua ( 30% ) .
those who stated the campaign influenced their behavior were more likely to be younger than 65 years ( 44% versus 24% , p=0.02 ) .
the median patient delay time was 90 minutes ( iqr=20 to 661 ) , with almost half of patients ( 47% ) deciding to seek medical attention within 1 hour of symptom onset .
factors significantly associated with a patient delay time of 1 hour at the univariate level are given in table2 .
after adjustment for other important predictors and the propensity score , awareness of the campaign was significantly associated with patient delay time of 1 hour ( adjusted odds ratio [ aor]=2.25 , 95% ci:1.03 to 4.91 , p=0.04 ) ( table3 ) .
models testing the association of campaign awareness and patient delay of 1 hour and prehospital delay of 2 hours or indicates odds ratio ; stemi , st - elevation myocardial infarction . adjusted for propensity score .
the median prehospital delay was 210 minutes ( iqr=100 to 1035 ) , with 34% presenting to hospital within 2 hours of symptom onset .
table2 provides the factors significantly associated with a prehospital delay time of 2 hours . after adjustment , awareness of the campaign
was significantly associated with a prehospital delay time 2 hours ( aor=3.10 , 95% ci : 1.36 to 7.09 , p=0.007 ) ( table3 ) .
overall , 18% of patients visited their general practitioner ( gp ) before presenting to hospital .
patients admitted with unstable angina ( 26% ) or nstemi ( 20% ) were more likely to visit their gp than were patients experiencing stemi ( 2% , p=0.007 ) .
campaign awareness was not associated with lower rates of visiting a gp ( 20% versus 14% , p=0.24 ) , although those aware of the campaign who stated the campaign influenced their behavior were less likely to visit a gp ( 7% versus 22% , p=0.017 ) .
the ems system was used by 53% of patients , although 8% of these patients first attempted transport in a car or drove to a gp who called ems .
ems use was highest among patients admitted with stemi ( 69% ) , compared with nstemi ( 52% ) and ua ( 47% ) .
no difference was seen in ems use by campaign awareness ( 52% versus 57% , p=0.50 ) or in those who stated the campaign influenced their behavior ( 56% versus 53% , p=0.78 ) .
this study found acs patients who were aware of the heart foundation campaign were more than twice as likely to decide to seek medical attention and present to hospital in the first hours after symptom onset .
our study also confirmed previous published findings of factors related to prehospital delay and identified the characteristics of those with lower awareness of the campaign .
given the campaign was aimed at the general public and competing with other health care messages , awareness of the campaign at 64% among acs patients is reasonably high .
this is particularly so when considered in the context of a recent evaluation of a stroke awareness campaign , in which only 12% of patients or bystanders recalled exposure to campaign advertising.26 another australian report evaluating a heart attack campaign in late 1980s found similar campaign reach ( 72% ) among acs patients in a multicenter study.27 in our sample , campaign awareness was lower in older acs patients and those born in non english - speaking countries .
these groups could be targeted in future campaigns , along with the other factors associated with prehospital delay .
the factors associated with prehospital delay in our study are similar to those of previous australian and international reports.18,20,21,28 our study also confirms that a broad range of factors influence delay times,8,9 including sociodemographic ( eg , level of education ) , clinical ( eg , intermittent symptoms ) , perceptual ( eg , recognizing symptoms as heart related ) , behavioral ( eg , wanting to see a local doctor first ) , and emotional / psychological ( eg , not wanting to alarm or trouble others ) . however , our multivariable models were unable to test the association of all factors at an individual level , as this would have resulted in overfit in both models .
these factors were instead represented in the model in the propensity score , and therefore there may be additional factors independently associated with delays times to those identified by our study . of particular interest is the prehospital experience of stemi patients , who benefit the most from reduced delay times .
similar to previous studies,20,24 stemi was strongly associated with deciding to seek medical attention within 1 hour of symptom onset and present to hospital within 2 hours of onset .
stemi patients were also more likely to state the campaign influenced their actions in response to symptoms and to use ems as transport ( 69% versus 50% , p=0.02 ) .
overall our study found only half of patients presented to hospital by ems , 57% in those experiencing an acute myocardial infarction .
these percentages are unchanged from earlier australian reports.21,28,29 most studies to date have found no impact of mass media campaigns on ems use in patients admitted with acs.30,31 only 1 study using a multifaceted and community - based approach ( eg , targeted mass media , community engagement , and education for professionals , the public , and patients ) has been effective.32 ems use has also been shown to increase following campaigns for other acute conditions , including stroke33particularly when ems use was highlighted in all campaign materials and included in the main message of the campaign .
barriers to calling ems for acs symptoms include thinking self - transport would be faster , having a lower perceived seriousness of symptoms , embarrassment , and thinking it is not necessary to use ambulance.25,34 the warning signs advertisements sought to overcome some of these barriers by highlighting the consequences of waiting ( i did nt realize my heart muscle had already started to die ,
the longer you wait , the more your heart muscle dies ) and to call an ambulance even if unsure ( the [ emergency ] operator will work out if you need an ambulance ) .
however , our study suggests this approach did not increase ems use in acs patients .
a qualitative study describes the decision to call ems for symptoms as multifactorial and identified the immediate access to care and the safety of transport as important in making this decision.35 further research is needed to understand this decision - making process for acs symptoms and to identify what campaign messages can highlight the need and important benefits of ems use to increase ambulance use .
the prehospital delay times in our study ( median 210 minutes ) are comparable to those of recent international reports for all acs patients and in acs subgroups.19 however , when we compared our times with those in earlier australian reports of patient27,36 and total prehospital28,36 delay , we saw no reduction over the last 2 decades .
this may be explained by differences in the definitions of the timeframes and starting points between studies.37 we did note an upward trend in australian data for the percentage of patients who ( 1 ) decided to seek medical attention within 1 hour ( 38% in 1988,27 42% in 1989 precampaign,27 45% in 1989 postcampaign,27 to 47% in our 2013 - 2014 study ) and ( 2 ) who presented to hospital within 2 hours of symptom onset ( 28% in 199521 to 34% in our study ) .
other large interventional and observational studies have shown a mixed influence of campaigns on prehospital delay times.14 the largest randomized control trial , the rapid early action for coronary treatment ( react ) study conducted across 20 cities in the united states , found no impact of a community - based intervention on prehospital delay , although an increased use in ems was seen among acs patients.32 most other studies conducted in the united states have also failed to show any impact,31,32 whereas those in europe have been more favorable.30,38,39 this difference could be attributed to differences in the duration of data collection , healthcare systems , media environments , and insurance coverage.14,32,39 bett et al evaluated earlier australian campaigns and found no change in median prehospital delay in the weeks following 3 separate campaigns.27,36 in those evaluations , which were conducted across a large number of australian hospitals , there was no adjustment for important differences between samples or for other factors that are now known to influence prehospital delay.8 that study , as well as the international evaluations , also did not evaluate the awareness of the campaign among patients studied a major strength of our study .
another potential explanation for the success of the current campaign was that it was the first australian campaign to go beyond awareness of symptoms and action , which has also been the focus of most international campaigns that have been evaluated,14 and specifically address known barriers .
the first of the recent australian television advertisements ( it s ok to call ) shows a cardiologist discussing the need to call an ambulance and that false alarms are ok .
the second advertisement ( i wish i could have my heart attack again ) uses a white , male actor as a deceased heart attack patient reliving what he would do differently if he could have his heart attack again .
this is the first australian campaign to use patient experience to illustrate lesser known symptoms and highlight the risk of ignoring symptoms , the need for early action , and to use ems .
the use of a white male in the advertising and having it only available in english may explain the lower campaign awareness seen in non english - speaking australians .
future and international campaigns may need to consider using a broader ethnic demographic in advertising and using subtitles .
international campaigns may also need to address the barriers to seeking treatment specific to their own acs population , which are known to vary by region and cultural norms.11 our findings must be considered in light of the following limitations .
however , the hospital where recruitment occurred received patients from both metropolitan ( 80% ) and rural regions ( 20% ) during the study period ; thus , our findings are not restricted to metropolitan cases .
further , the demographics , risk factor profile , and proportion of acs subtypes of our sample were similar to those reported in the 2365 confirmed acs patients captured in the acs snapshot audit of 286 australian hospitals.40 nonetheless , our findings will require confirmation in a larger multicenter evaluation .
the campaign and our evaluation deliberately targeted a younger audience , so the findings may not apply to older acs patients .
the other limitations of the study relate to the collection of data on a retrospective event ; thus , the findings are subject to recall bias and prehospital delay may be related to other factors not measured . in summary ,
our study found awareness of the heart foundation s warning signs mass media campaign was reasonably high in a cohort of australian acs patients , and this was associated with shorter patient and prehospital delay times but not ems use .
future campaigns could target groups in whom awareness was low ( older patients and those not born in australia ) and highlight the need and important benefits of ems use .
data for our study were collected at only 1 site . however , the hospital where recruitment occurred received patients from both metropolitan ( 80% ) and rural regions ( 20% ) during the study period ; thus , our findings are not restricted to metropolitan cases .
further , the demographics , risk factor profile , and proportion of acs subtypes of our sample were similar to those reported in the 2365 confirmed acs patients captured in the acs snapshot audit of 286 australian hospitals.40 nonetheless , our findings will require confirmation in a larger multicenter evaluation . the campaign and our evaluation deliberately targeted a younger audience , so the findings may not apply to older acs patients .
the other limitations of the study relate to the collection of data on a retrospective event ; thus , the findings are subject to recall bias and prehospital delay may be related to other factors not measured . in summary ,
our study found awareness of the heart foundation s warning signs mass media campaign was reasonably high in a cohort of australian acs patients , and this was associated with shorter patient and prehospital delay times but not ems use .
future campaigns could target groups in whom awareness was low ( older patients and those not born in australia ) and highlight the need and important benefits of ems use .
drs bray , cartledge , and finn receive salary support from the national health and medical research council ( nhmrc ) centre for research excellence : australian resuscitation outcomes consortium ( no . 1029983 ) .
drs bray and stub are supported by co - funded nhmrc / heart foundation research fellowships ( no . 1090302/100516 and no . 1069985/100136 ) .
drs stewart , keech , and patsamanis were employed by the heart foundation at the time of this study . | backgroundthe aim of this study was to examine the awareness of a recent mass media campaign , and its influence on knowledge and prehospital times , in a cohort of acute coronary syndrome ( acs ) patients admitted to an australian hospital.methods and resultswe conducted 199 semistructured interviews with consecutive acs patients who were aged 35 to 75 years , competent to provide consent , and english speaking .
questions addressed the factors known to predict prehospital delay , awareness of the campaign , and whether it increased knowledge and influenced actions .
multivariable logistic regression was used to examine the association between campaign awareness and a 1-hour delay in deciding to seek medical attention ( patient delay ) and a 2-hour delay in presenting to hospital ( prehospital delay ) .
the median age was 62 years ( iqr=53 to 68 years ) , and 68% ( n=136 ) were male .
awareness of the campaign was reported by 127 ( 64% ) patients , with most of these patients stating the campaign ( 1 ) increased their understanding of what is a heart attack ( 63% ) , ( 2 ) increased their awareness of the signs and symptoms of heart attack ( 68% ) , and ( 3 ) influenced their actions in response to symptoms ( 43% ) .
after adjustment for other predictors , awareness of the campaign was significantly associated with patient delay time of 1 hour ( adjusted odds ratio [ aor]=2.25 , 95% ci : 1.03 to 4.91 , p=0.04 ) and prehospital delay time 2 hours ( aor=3.11 , 95% ci : 1.36 to 7.08 , p=0.007).conclusionsour study showed reasonably high awareness of the warning signs campaign , which was significantly associated with shorter prehospital decision - making and faster presentation to hospital . |
congenital talipes equinovarus is recognized as the down and in foot , often resembling a club - like appearance .
it is the most important cause of physical disability among all congenital musculoskeletal defects occurring in an otherwise normal child.1 for many years , the kite 's method was used for the correction of clubfoot , but it took enormous amount of time for correction and produced excellent results only in the hands of kite.2 in recent years , the ponseti method has become the gold standard for correction of clubfoot.12 however , most of the studies on ponseti method are done in younger children , before the walking age .
neglected clubfoot is the primary problem in developing nations , where social stigma , lack of education , poverty and lack of proper health services hinder the early presentation and treatment of a child with clubfoot .
the deformity becomes worse by walking as the weight bearing takes place on the side or dorsum of the foot , causing further contracture of the medial soft tissues and plastic deformation of bones.3 most of the literature on treatment of neglected clubfoot focuses on soft tissue release surgery , osteotomy or fixators .
the problem in developing country is the large number of patients and scarcity of skilled surgeons and ot time .
further , the good short term results have shown to deteriorate over longer followup resulting in rigid painful feet.45 the scars are cosmetically unacceptable .
this calls for a simpler method that can be easily applied to larger population with better results .
inspired by the success of ponseti method in idiopathic clubfeet , the method was tried in neglected feet as well in past , but in small series.6789 we here present one of the largest prospective studies published till date . we wanted to evaluate the effect of ponseti method of casting on neglected feet , to assess its failure and recurrence rate .
we also wanted to evaluate if there is any improvement in radiological parameters following serial casting by ponseti method .
further , we wanted to find if any correlation exists between total number of casts and initial pirani and dimeglio scores , and age of initial presentation of the patients .
41 feet of 30 patients with mean age of 3.02 years ( range 1 - 10.3 years ) were included in this prospective study children with virgin idiopathic clubfoot , with no previous treatment , in the age group of more than 1 year at the time of presentation were included in the study .
consent was taken prior to enrolment in the study after explaining the mode of treatment .
quantification of various components of clubfoot deformity was done using the dimeglio score10 and pirani score11 prior to application of every cast and at each followup .
anteroposterior ( ap ) and lateral standing radiographs of the feet were taken prior to treatment , immediately after correction and at the time of the latest followup .
child was placed on a specially designed step stool to take standing lateral x rays , with the lateral border of foot touching the cassette and rays perpendicular to the foot [ figure 1 ] . on the ap view
, the talo - calcaneal and the talar - first metatarsal angles were recorded . on the lateral view
the beatson - pearson index12 ( sum of the talo - calcaneal angle in the ap and lateral views ) was used for overall radiological assessment .
the casts followed standard ponseti protocol.13 initially , cast was applied in full supination for the correction of cavus .
percutaneous tenotomy of the tendo - achilles was done once the forefoot deformity was corrected with a fully reducible talar head which was assessed using pirani scoring ( lateral head talar sign in pirani scoring was 0).11 tenotomy was performed either under general or local anesthesia .
the technique was always percutaneous and in no case we had to resort to open lengthening [ figure 2a and b ] .
tenotomy of the tendo - achilles was done in all patients , even up to the age of 10.3 years .
a repeat tenotomy of tendo - achilles was done in children where adequate dorsiflexion , i.e. , at least ten degree of dorsiflexion was not achieved by the first tenotomy .
posttenotomy cast was removed at 3 weeks in patients aged <3 years and 4 weeks in patients aged more than 3 years . despite our initial apprehensions regarding tenotomy in older children
plantar flexion was checked at 3 months and 6 months using clinical and radiological studies .
clinically the strength was considered adequate when child could stand on toes unassisted and radiologically we used ultrasound to assess adequacy of the tendon . in no case was tendo - achilles found inadequate with most children found to stand on toes in 34 months unassisted .
no strict protocol is present in the available literature regarding the splinting in children of older age group .
it was applied for 23 h a day for the first 3 months in all the patients and advised for sleep time use up to the age of 4 years or 1 year in children older than 4 years .
clinical photographs showing ( a and b ) anteroposterior and lateral technique of taking x - rays clinical photographs showing ( a ) pretenotomy foot with equinus and ( b ) posttenotomy foot with correction of equinus during our study , we assessed the age at the time of presentation , severity of deformity by the pirani and dimeglio score at each visit , the number of casts required for the correction , amount of ankle dorsiflexion after tenotomy , need for any other surgery , change in the pirani and dimeglio score , and residual deformities .
the quantitative variables were expressed as mean standard deviation and compared between preoperative and postoperative followup using the paired t - test . also , the relation between the pirani and dimenglio score and age at presentation with the number of casts required was evaluated using pearson 's correlation coefficient .
statistical analysis was performed using spss version 15.0 software(ibm software , united states ) and a p < 0.05 was considered statistically significant .
the mean number of casts applied , to achieve final correction , were 12.8 casts ( range 8 - 18 casts ) .
the average dorsiflexion achieved was 12.26 ( range 320 ) at the end of treatment .
the average dorsiflexion achieved in age group 13 years was 15.48 , between 3 and 5 years was 8.54 and > 5 years was 7 [ figure 3 ] .
the older children had lesser dorsiflexion , yet the children and parents were satisfied with the final results as they were able to wear normal footwear and carry on most of the functions . in seven cases , where the dorsiflexion was less than ten degree after the tenotomy of tendo - achilles , we did a repeat tenotomy , after 1 month , to further improve the dorsiflexion .
scatter diagram showing that final dorsiflexion achieved depends on the age at which treatment is started .
less dorsiflexion with increasing age clinical photograph of 5-year - old child showing ( a ) neglected bilateral clubfeet at presentation ( b ) neglected bilateral clubfeet after 8 casts in tenotomy clinical photograph of 4-year - old child showing ( a ) neglected unilateral clubfoot before treatment ( b ) neglected unilateral clubfoot after 7 casts and tenotomy cavus was seen as one of the most severe deformities and in 5 children cavus remained even after complete correction of other deformities .
a percutaneous plantar fasciotomy was needed in these 5 feet for the correction of residual cavus .
three patients developed casting related complications of erythema and superficial sore , which were managed by generous padding in the subsequent casts .
one patient with bilateral clubfeet developed flat top talus probably as a result of more number of casts ( n = 18 ) and the dorsiflexion achieved was less , ( 3 ) even after tenotomy .
we calculated the correlation between the number of casts required for correction versus the initial age , pirani scores , and dimeglio scores .
there was a significant positive correlation between age and number of casts required ( r = 0.528 , p < 0.001 ) .
this has also been shown in the scatter diagram where an increasing trend is seen in the number of casts required for maximal correction with increasing age [ figure 6 ] .
also , age was negatively correlated with initial pirani score ( r = 0.510 , p = 0.001 ) and positively correlated with initial dimeglio score ( r = 0.439 , p = 0.004 ) .
figure 7 shows a scatter diagram showing a relationship between pirani score and age of patient . on the other hand , number of casts
required had no significant correlation with initial pirani score ( r = 0.234 , p = 0.141 ) while it was positively correlated with initial dimeglio score ( r = 0.413 , p = 0.007 ) .
scatter diagram where an increasing trend is seen in the number of casts required for maximal correction with increasing age scatter diagram , where the initial pirani scores show a decreasing trend with increasing age , despite the stubborn foot deformity the range of ap talo - calcaneal angle , precorrection , was from 0 to 14 , with a mean of 5.04. the postcorrection ap talo - calcaneal angles ranged from 15 to 35 , with a mean of 26.14. the range of lateral talo - calcaneal angle , pre correction , was from 0 to 16 , with a mean of 5.29. the range of postcorrection lateral talo - calcaneal angle was 1540 with a mean of 25.24. [ figures 8 and 9 ] show ap and lateral x - rays of our clinical case at pre- and post - treatment time .
the mean beatson - pearson index before treatment was 10.34. after correction our mean beatson - pearson index was 51.39.8 anteroposterior x - ray of foot before treatment showing ( a ) parallel talo - calcaneal relation ( b ) radiological improvement with talo - calcaneal angle nearing 30 in both planes lateral x - ray of foot before treatment showing ( a ) parallel talo - calcaneal relation ( b ) radiological improvement with talo - calcaneal angle nearing 30 in both planes 7 patients developed recurrence of the deformity .
the time to wear brace in these cases was less than 8 hours a day .
it was mainly a recurrence of equinus in 3 feet whereas adduction cavus and equinus in four .
these were treated by repeat ponseti casting and tenotomy in 4 feet and 3 patients required a tibialis anterior tendon transfer .
the children with neglected clubfoot deformity are unable to mix with their peers because of stigmatization , and as a result very few go to school .
most of the literature on treatment of neglected clubfoot has focused on extensive soft tissue release surgery , osteotomies , and various types of fixators or arthrodesis.451314 long term studies of surgical procedures have shown poor results with complications like painful feet , arthritis , stiffness of ankle and subtalar joint , and residual deformity.4514 the few studies , evaluating the use of ponseti method in children with neglected clubfeet have shown promising results.6789 loureno and morcuende treated 17 patients ( 24 feet ) with neglected clubfeet , with mean age of 3.9 years and found good results in 16 feet ( 66.6% ) without further surgery.6 verma et al . evaluated ponseti method in toddlers and found it to be very useful.7 khan and kumar treated neglected clubfoot in 21 children ( 25 feet ) over 7 years and found good results in 18 feet ( 85.7%).8 the comparison of these studies is described in table 1 .
comparison of various published studies the mean number of casts in our study before tenotomy was 12.8 weekly casts with an average of 3 months of immobilization .
loureno and morcuende6 treated with 9 biweekly casts which meant an immobilization of about 4.5 months that was slightly more than we observed .
however , khan and kumar8 and mehtani7 had treated patients in 12.1 and 10 casts , respectively , which was comparable to our study . also , the amount of final dorsiflexion achieved in our study was slightly more than other published series [ table 1 ] .
spiegel et al.9 in their study of 260 feet in the age group of 16 years observed mean dorsiflexion of 12.5 up to 2 years and 910 in 35 years of age , which is comparable to our result . in our study , all the 41 feet treated by the ponseti method of cast application achieved painless , supple , plantigrade , and cosmetically acceptable feet within a mean period of 3.6 months .
the recurrence rate of 17.07% in our series is also is comparable to that of most of the studies done in infants .
however , there were certain striking differences between the results of ponseti method in infants and older children .
the average dorsiflexion achieved in our study was 12.26 , which is more than that achieved by loureno and morcuende6 ( 5 ) but less than that achieved in younger children .
the feet could not be abducted to 70 as advised in younger children and passive abduction was possible up to 40 with a mean of 20.87. even loureno and morcuende in their study mentioned that abduction was attempted to approximately 3040 instead of 70 as recommended in infants . despite these limitations ,
we also compared the initial pirani and dimeglio scores for correlation with the number of casts required for correction and found that while dimeglio score had a moderate positive correlation ( 0.439 ) with the number of casts required , the pirani score had a low negative correlation ( 0.278 ) .
hence , pirani scoring is not a sensitive enough tool for assessing the deformity in children of older age group and gives fallacious low values , due to low scores for empty heel and posterior crease and medial crease , despite resistant , rigid deformities .
this is also shown by the scatter diagram , where the initial pirani scores show a decreasing trend with increasing age , despite the stubborn foot deformity [ figure 9 ] .
the number of casts required for correction also showed an increasing trend with the age . despite the encouraging results seen in our study
second , we found that older children had difficulty accepting the foot abduction brace and no definite protocol is available regarding bracing in older children .
we advocated at least 1 year of night time bracing even in older children but 5 patients who did nt tolerate the foot abduction brace had to be given a custom made ankle foot orthosis ( afo ) .
recurrence was seen in seven cases , but feet were still supple and responded to repeat ponseti casting unlike surgical recurrence .
an early tibialis anterior transfer to the third cuneiform , which would act as internal bracing might be helpful in preventing the relapses .
the larger patient samples and followup period would be required to establish fully the efficacy of ponseti method for treating neglected clubfoot but our results our encouraging . also , the bracing protocol is difficult particularly in older children .
we feel that ponseti method either fully takes care of this stubborn deformity or reduces the extent of residual surgical release and it should be a good alternative for treatment of neglected clubfoot .
| background : gentle passive manipulation and casting by the ponseti method have become the preferred method of treatment of clubfoot presenting at an early age . however , very few studies are available in literature on the use of ponseti method in older children .
we conducted this study to find the efficacy of ponseti method in treating neglected clubfoot , which is a major disabler of children in developing countries.materials and methods:41 clubfeet in 30 patients , presenting after the walking age were evaluated to determine whether the ponseti method is effective in treating neglected clubfoot .
this is a prospective study .
pirani and dimeglio scoring were done for all the feet before each casting to monitor the correction of deformity .
quantitative variables were expressed as mean standard deviation and compared between preoperative and postoperative followup using the paired t - test . also , the relation between the pirani and dimeglio score , and age at presentation with the number of casts required was evaluated using pearson 's correlation coefficient .
no improvement in dimeglio or ponseti score after 3 successive cast was regarded as failure of conservative management in our study.results:the mean age at presentation was 3.02 years ( range 1.1 - 10.3 years ) .
the mean followup was 2.6 years ( range 23.9 years ) .
the mean number of casts applied to achieve final correction were 12.8 casts ( range 8 - 18 casts ) .
the mean time of immobilization in cast was 3.6 months .
the mean dimeglio score before treatment was 15.9 and after treatment were 2.07 .
the mean pirani score was 5.41 before treatment and 0.12 after treatment .
all feet ( 100% ) achieved painless plantigrade feet without any extensive soft tissue surgery .
7 feet ( 17% ) recurred in our average followup of 2.6 years.conclusions:painless , supple , plantigrade , and cosmetically acceptable feet were achieved in neglected clubfeet without any extensive surgery .
a fair trial of conservative ponseti method should be tried before resorting to extensive soft tissue procedure . |
this is because it can provide site - specific information about protein motions over a large range of time scales .
over the past decade , n nmr relaxation experiments employing model - free analysis ( 1,2 ) have become the de facto standard used to characterize protein motions on a picosecond to nanosecond time scale .
however , as with any scientific method , this approach has certain limitations ( 3,4 ) .
perhaps the most obvious difficulty lies in the fact that n relaxation measurements are inherently time - consuming and tedious , often requiring many hours of data collection , processing and spectral analysis .
a second problem is that relaxation measurements are often seriously compromised by peak overlap , poor signal intensity or peak broadening .
a third problem is that the precision and accuracy of relaxation - derived measurements tends to deteriorate rapidly as the frequency of internal nanosecond motions approach that of the protein 's overall tumbling rate ( 5,6 ) .
this is because n relaxation rates become insensitive to internal fluctuations that are much slower than overall tumbling .
since calculations of relaxation rates are based on measuring peak intensities , their accuracy can be severely compromised by low signal - to - noise ratios .
this is especially true when dealing with larger ( > 150 residues ) proteins or proteins undergoing s ms conformational exchange .
another complication to the model - free formalism lies in the fact that its proper application often requires information about anisotropy of protein overall diffusion and , as a result , the method can not be used when the 3d structure is not known or when it is largely disordered .
these limitations with traditional relaxation measurements prompted us to develop a new , chemical - shift - based technique to characterize protein mobility from nmr data .
specifically , we wanted to develop an easy - to - use , robust approach that would not be affected by protein tumbling rates , uncertainties in peak intensities or lack of knowledge about the protein 's 3d structure .
this method is called the random coil index or rci ( 7 ) . as described in our previous publications ( 7,19 ) , the rci method exploits the fact that there is a remarkable amount of dynamic information intrinsic to nmr chemical shifts .
the connection between chemical shifts , especially random coil chemical shifts , and protein flexibility has been known for quite some time ( 811 ) .
random coil chemical shifts can be defined as shifts that result from a fast exchange among energy - weighted populations of all theoretically possible conformations of an unfolded polypeptide chain ( 12,13 ) .
the difference between an observed chemical shift for a given amino acid in a given protein , and its corresponding random coil value is called the secondary chemical shift .
secondary chemical shifts have been used for many years to qualitatively estimate the level of protein structural disorder ( 1418 ) . however , until recently , no quantitative relationships between secondary chemical shifts and protein dynamic parameters had been derived .
the rci is able to combine the chemical shift data from six different nuclei ( c , c , co , n , hn and hor any combinations thereof ) into a single parameter that closely correlates with amplitudes of backbone protein motions such as order parameters ( s ) and root mean square fluctuations ( rmsfs ) of structural ensembles .
previous descriptions of the rci method focused on explaining the algorithm , rationalizing its utility and assessing its accuracy . as a result , only a modest effort went into making the rci - based software user - friendly and flexible .
in an attempt to make the rci approach more accessible to the nmr community and much more robust , we decided to create an rci web server and to optimize the rci protocol for nmr assignments with different degrees of completeness and mis - referencing .
the rci server is a unique server , designed to support rapid ( 510 s ) , residue - specific determination of protein flexibility and protein mobility using only chemical shift assignment data as input .
it accepts almost any combination of nmr chemical shift assignments as its input and it outputs the expected values of the rmfs of md and nmr ensembles as well as model - free order parameters ( 1,2 ) . to improve the accuracy and reproducibility of the calculations ,
assessments of the rci server performance show an agreement between the values it calculates and experimentally obtained amplitudes of motions range from r = 0.770.82 depending on the type of experimental method .
the rci server is composed of two parts , a front - end web - interface ( written in python and html ) and a back - end consisting of several programs including rci ( 7 ) , csi ( 11 ) , refcor [ based on ( 19 ) ] , as well as several parsing and conversion utilities for handling different input files .
the csi program is written in ansi standard c , while rci , refcor , the input parsing and the conversion utilities are written in python .
the rci server accepts protein chemical shift assignments in standard bmrb nmr - star ( 20 ) and shifty ( 21 ) formats as input .
users can either upload an input file into the web server ( via a browse button ) or paste the data in a standard text box ( figure 1 ) .
users are also offered several options to adjust program operations to suit their specific needs .
to begin : ( 1 ) experimental chemical shifts are first uploaded by the user . ( 2 ) the input chemical shifts are then re - referenced ( if necessary ) by refcor .
( 3 ) the protein sequence is extracted from the nmr assignments and used to ( 4 ) determine the appropriate random coil chemical shifts ( 22 ) and ( 5 ) determine the neighboring residue correction factors for the i 1 and i 2 residues ( 23 ) .
the correction factors are applied to the random coil chemical shifts to obtain reference chemical shifts .
( 6 ) reference chemical shifts are then subtracted from the corrected experimental chemical shifts to obtain the secondary chemical shifts for the c , c , co , n , hn and h nuclei .
( 7 ) optionally , gaps in the chemical shift assignments , if any , are filled in by averaging the chemical shifts of neighboring residues .
( 9 ) secondary chemical shifts of are scaled to account for differences in their resonance frequencies , and , if below a certain floor limit ( currently 0.5 p.p.m . ) , replaced with this floor value . ( 10 ) initial rci values are calculated using the following expression .
1
where |c| , |co| , |c| , |n| , |nh| and |h| are the absolute values of the secondary chemical shifts ( in p.p.m . ) of c , co , c ,
a , b , c , d , e and f are weighting coefficients ( table 1 in the rci online help ) . left angle and right angle brackets ( < > ) indicate that the average is being calculated .
( 12 ) if the rci values are above a certain ceiling limit ( currently 0.6 ) , they are replaced with this ceiling value .
( 13 ) the final rci values are obtained after a second smoothing by three - point averaging .
( 14 ) finally , in the last step the expected values of model - free order parameters ( s ) , rmfs of md and nmr ensembles are calculated using the following empirical expressions .
2
3
4
a more detailed description of the aforementioned steps has been published elsewhere ( 19 ) .
figure 2.a flow chart describing the rci protocol . a flow chart describing the rci protocol . by default ,
the rci web server uses random coil reference chemical shifts and neighboring residue correction values originally published by schwarzinger and co - authors ( 22,23 ) .
this is the only set of random coil values and neighboring residue correction for residues i 1 and i 2 that were obtained under similar experimental conditions and , therefore , are expected to be quite consistent with each other .
however , users can also select different sets of random coil chemical shifts , including those values published by wang and jardetzky ( 24 ) , wishart et al .
users are also offered an option to select i 1 neighboring residue correction values published by wang and jardetzky ( 24 ) instead of the default values .
while these options do affect rci - based flexibility profiles , the differences in mean correlation coefficients for different combinations of random coil values and neighboring residue corrections ( table 2 of the rci online help ) are relatively small and may be considered statistically insignificant . by default ,
however , if unassigned residues are located in the middle of a well - defined secondary structure region ( e.g. -helix , -sheet ) , it is not unreasonable to expect that their flexibility will be similar to that of neighboring assigned resides . in these cases
, the rci web server allows users to predict the flexibility of unassigned residues based dynamic properties of adjacent regions .
the rci web server also has a somewhat similar option to fill small gaps in the per - residue distributions of secondary chemical shifts .
such gaps happen more often than a lack of assignments for all the six nuclei and should be dealt with separately .
the use of incomplete assignments generally reduces the quality of rci - based flexibility predictions ( see table 1 of the rci online help ) . by default ,
the rci web server fills small gaps in per - residue distributions of secondary chemical shifts by averaging secondary chemical shifts of residues i + 1 and i 1 , and , if not available , residues i + 2 and i 2 .
end effects. these are poorly understood phenomena that alter the chemical shifts of the n- and c - termini of proteins in unpredictable ways .
the rci web server offers an option to apply an end - effect correction to the rci values .
this end - effect correction is turned on by default . a detailed protocol describing how the end - effect correction is handled was published elsewhere ( 27 ) .
users should be aware that the improvement of rci predictions due to the end - effect correction is not related to the dynamic averaging of chemical shifts .
rather , it originates from the indirect correlation between the severity of the end effects and protein flexibility due to their shared dependence on the proximity to a terminal residue .
therefore , if the correction is applied , the rci profile at the terminal regions should be interpreted as a commonly observed trend of protein flexibility at termini of proteins .
the rci server offers users the option to disable the end effect correction or to exclude the first and the last three residues from predictions .
a particularly useful and important feature of the rci web server is its capacity to correct mis - referenced chemical shifts .
this automated reference correction is done using a local implementation of the refcor program . about 20% of newly deposited assignments in the bmrb database
were found mis - referenced by a recent survey ( 28 ) . given the relatively high level of mis - referencing in biomolecular nmr and given the fact that mis - referenced shifts can substantially reduce the performance of chemical - shift - based methods such as rci ( 7 )
, we believed that implementing this reference correction protocol was absolutely vital to maintaining the server 's performance .
the refcor reference correction protocol is based on the secondary structure predictions from the chemical shift index ( csi ) ( 29 ) and was published elsewhere ( 27 ) .
briefly , refcor uses csi - based predictions of secondary structure along with typical chemical shift values observed in different secondary structures are used to calculate the necessary reference correction for each nucleus .
since csi predictions are also affected by shift mis - referencing , the protocol is repeated several times using newly generated re - referenced shifts until the csi predictions become stable .
the reference correction option is always turned on by default although it can be switched off , if necessary .
an average rci run takes between 5 cpu s ( without chemical shift reference correction ) and 20 s ( with reference correction ) .
as might be expected , the rci web server displays the name of the input file , the options selected and a plot of the per - residue rci distribution .
users may download per - residue distributions of the rci , predicted model - free order parameters , predicted rmsfs of md and nmr ensembles as both text files and graphical images .
these files may also be obtained individually or as a single linked file containing all the results .
re - referenced chemical shifts and csi - based predictions of secondary structure are also available for download or direct viewing on the website . in addition to its extensive data output
, the rci web server also offers a comprehensive list of help pages to assist users in preparing their input files , in understanding the rci method and in interpreting the web server output .
this information is provided to make the rci protocol as transparent as possible and to facilitate protocol troubleshooting if required .
the rci web server was optimized and evaluated on a set of 18 proteins ranging in length from 56 to 283 residues ( 1585 residues in total ) , for which complete or nearly complete h , c and n chemical shifts were known ( table 3 of the rci online help ) .
a subset of 14 proteins was used to generate molecular dynamic ( md ) ensembles and optimize weighting coefficients in the rci expression [ equation ( 1 ) ] .
this was done by using a simple grid search to maximize rci correlation with rmsf values determined by molecular dynamics .
the weighting coefficients were obtained for all 63 possible assignment scenarios ( table 1 of the rci online help ) . to evaluate the performance of the algorithm for the training set
, we used a leave - one - out procedure by removing the query protein from rci training set prior to running the program .
the average correlation coefficients for the full training set and for all leave - one - out runs were identical ( r = 0.82 ) . to ensure that the rci algorithm had not been over - trained , four additional proteins , which were not previously included in the training set , were tested .
the average correlation coefficient between the rci determined md rmsf and the calculated md rmsf for these proteins was identical ( r = 0.82 ) to that of the training set ( see table 3 of the rci online help for information about the tested proteins ) . to validate
the relationship between rci predictions and the amplitudes of protein motions , the correlation of rci with model - free order parameters [ observed and predicted from the structure ( 30 ) ] and per - residue rmsf values of nmr ensembles were calculated .
the average correlation coefficients for the eighteen sets of order parameters and sixteen sets of nmr rmsfs were 0.77 and 0.81 , respectively .
the performance of the conversion expressions shown in equations ( 24 ) were assessed by calculating the average error between rci - predicted motional amplitudes and the corresponding experimentally and theoretically obtained parameters .
the average errors for the order parameters ( 18 proteins ) , the nmr rmsfs ( 16 proteins ) and the md rmsfs ( 18 proteins ) were 0.05 , 0.44 and 0.50 , respectively .
figure 1 in the rci online help shows examples of the good correlations obtained between rci and other measures of protein motional amplitudes ( e.g. nmr rmsf , md rmsf , s ) for such pharmaceutically important proteins as interleukin-4 and the hiv-1 gag protein . additional information about optimizing and testing the rci method ( i.e. details of md simulations , pdb ids and bmrb codes of proteins , etc . ) can be found in the original papers on the rci method ( 7 ) as well as on the rci online help pages .
in summary , we have described a web server that is capable of rapidly and accurately predicting protein flexibility using only chemical shift assignments as the input .
comparisons suggest that these predictions correlate well with other measures of protein mobility , such as model - free order parameters and root - mean square fluctuations ( rmsfs ) of nmr and md ensembles .
the approach is generally applicable to proteins of any size for which h , c and n shift assignments are available .
the web server is unique in its ability to extract dynamic information from nmr data without the prior knowledge of tertiary structure , without the need for favorable rates of rotational diffusion and without the need for exceptionally good spectral sensitivity .
we have already used the rci approach to monitor a number of interesting dynamic processes in proteins , such as the ph - induced conversion of the prion protein ( prp ) into its scrapie form ( prp ) and the dynamic response of picornaviral protease active sites to inhibitor binding ( manuscripts in preparation ) .
we believe the rci method may lead to important changes in the ways backbone protein flexibility is measured and reported by the scientific community . | protein motions play important roles in numerous biological processes such as enzyme catalysis , muscle contractions , antigen antibody interactions , gene regulation and virus assembly .
knowledge of protein flexibility is also important in rational drug design , protein docking and protein engineering .
however , the experimental measurement of protein motions is often difficult , requiring sophisticated experiments , complex data analysis and detailed information about the protein 's tertiary structure . as a result
, there is a considerable interest in developing simpler , more effective ways of quantifying protein flexibility .
recently , we described a method , called the random coil index ( rci ) , which is able to quantitatively estimate backbone root mean square fluctuations ( rmsfs ) of structural ensembles and order parameters using only chemical shifts .
the rci method is very fast ( < 5 s ) and exceedingly robust .
it also offers an excellent alternative to traditional methods of measuring protein flexibility .
we have recently extended the rci concept and implemented it as a web server .
this server allows facile , accurate and fully automated predictions of md rmsf values , nmr rmsf values and model - free order parameters ( s2 ) directly from chemical shift assignments .
it also performs automatic chemical shift re - referencing to ensure consistency and reproducibility . on average ,
the correlation between rci predictions and experimentally obtained motional amplitudes is within the range from 0.77 to 0.82 .
the server is available at http://wishart.biology.ualberta.ca/rci . |
parkinson s disease ( pd ) was first known as shaking palsy , a term coined in 1817 by james
parkinson .
it is a progressive neurologic disturbance in which the incidence rate increases
with age1 .
when approximately 6080% of the dopamine produced by the
substantia nigra dopaminergic neurons has been lost , the extrapyramidal system can no longer
effectively promote movements , and the symptoms of pd appear .
the clinical symptoms include
hypokinesia , bradykinesia , postural instability , rigidity , and tremor3 .
the gait of patients with pd is
characterized by reduced gait speed and short steps , improper trunk rotation or arm swings ,
difficulties in changes of direction , shuffling gait pattern , or acceleration gait
pattern4 .
many studies have been conducted on the gait of patients with pd . to improve the gait of
patients with pd , research into signals began in 19425 , and gait analysis was conducted in relation to external cues in
19676 . until recently , research into the
effects of methods using external cues has been conducted for various methods of improving
gait in pd patients1 , 7,8,9 . in their reviews of the gaits of pd patients with application of
external cues , rubinstein et al.10 and
darmon et
al.11 advised that external
cues could significantly improve the gaits of patients with pd or their gait - related
activities .
as external information in the form of visual or auditory cues is known to
improve motor performance and motor learning12 , 13 , most studies on motor learning have
generally used feedback such as training with visual or auditory cues .
recently , studies have been conducted on the use of feedback such as vibration stimuli .
haridas and zehr14 reported that the
sensory feedback during gait provided by stimuli through the skin caused reflex activities
during upper and lower extremity movements and affected the activity of the central pattern
generator ( cpg ) . when applied to the muscles , vibration acts as powerful proprioceptive
stimulus , strongly affecting the motion perception of not only healthy people but also
patients with various neurological disorders15 . in a spatiotemporal exploratory study of three patients with left
unilateral neglect , when work was performed
while vibration stimuli were applied to the
muscles , the subjects detected remarkably more objects16 .
sorensen et al.17 found that vibration stimuli applied to the ankles played an
important role in controlling posture and balance during gait because the stimuli caused the
lower extremity to provide information about the movement of the center of gravity to the
central nervous system . by reviewing previous studies related to improvement of the gait of patients with pd
, it
can be seen that studies on motion perception or vibration stimuli are insufficient ,
although many studies have been conducted on the dependency on external cues in many
approaches for improving motor control or gait ability in patients with pd .
therefore , the
purpose of this study was to compare temporal and spatial gait parameters while applying
vibration in patients with pd .
the subjects had an average age of 61.825.57 ,
an average height of 155.363.71 cm , and an average weight of 60.154.38 kg .
approval for
the study was obtained from the institutional review board of the national evidence - based
healthcare collaborating agency , and written informed consent was obtained from each patient
before starting the study .
eight vibrators ( 10 g , diameter of 1 cm ) were made especially for this study .
they were
attached to the muscle bellies ( tibialis anterior , gastrocnemius , biceps femoris , and rectus
femoris ) on both sides of the lower extremities of the subjects .
the subjects wore short
pants to minimize external stimuli , and were barefoot . to identify the effects of vibration
stimuli during walking
, subjects were tested with respect to spatiotemporal gait parameters
with and without vibration in both lower extremities using a motion analysis system
. a hawk
digital system ( 60 hz , motion analysis , santa rosa , ca , usa ) was used to measure the
spatiotemporal parameters during walking .
the amplified motion analysis signals were sampled
online with the evart 5.0 software and were analyzed using the cortex 64 and orthotrak 6.6.4
software .
the wilcoxon signed - rank test was performed to identify the differences in
spatiotemporal parameters between with and without vibration .
comparison between the spatiotemporal gait parameters with and without vibration in pd
patients revealed that stride length and walking speed with vibration were significantly
increased compared with those without vibration ( p<0.05 ) .
although the other parameters
were also improved , there were no statistically significant differences ( table 1table 1 .
comparison of spatiotemporal parameters between with and without vibration in
patients with pdvariablewithout vibrationwith vibrationcadence ( steps / min)110.2 ( 9.6)111.6 ( 10.4)stride length ( cm)110.0 ( 15.1)114.0 ( 14.5 ) * step length ( cm)55.7 ( 7.3)58.1 ( 6.9)step width ( cm)11.5 ( 1.8)10.9 ( 1.6)walking speed ( cm / sec)101.7 ( 15.7)107.1 ( 17.3 ) * single support time ( % cycle)61.6 ( 1.8)60.5 ( 1.8)double support time ( % cycle)11.7 ( 3.0)11.1 ( 1.4)*significant difference between with and without vibration ( p<0.05 ) ) . * significant difference between with and without vibration ( p<0.05 )
pd is a progressive degenerative neuronal disease occurring because of a deficiency in the
dopaminergic neurons in the basal ganglia .
patients with pd show movement impairment
symptoms such as bradykinesia , tremor , rigidity , and impaired balance18 , and these symptoms cause changes in their gaits .
many
studies on pd patients spatiotemporal gait variables have reported that their gait patterns
showed a short stride length and low walking speed3 ,
19 , 20 .
vibration stimuli act as powerful proprioceptive stimuli that strongly affect motion
perception in patients with neurological disorders15 and , in particular , can cause changes in the motor control patterns
of patients with damage to their basal ganglia21 . because the basal ganglia serve as a motor control function in
making sequential movements , if they are not properly controlled , movements will become
slower and muscle activities will decrease22 .
muscle stimuli overcome the suppression of the thalamus , which
plays an important role as a gateway and acts as an integration between the cerebral cortex
and subcortical signals , although only for a short time , and induce thalamocortical
activation , thereby improving visual perception and processing speed23 . in this study ,
when vibration stimuli were applied to improve the gaits of patients with
pd , their stride lengths and walking speeds significantly increased .
given this , application
of the vibration stimuli to patients with pd having damaged basal ganglia seemed to cause
the patients motor control patterns to improve , thus improving the gait patterns of the
patients who had shown short stride lengths and low walking speeds .
previous studies also reported that when feedback was not properly provided , movements
decreased gradually24 . as feedback ,
either external information in the form of visual or auditory cues known to improve motor
performance and motor learning or vibration stimuli that stimulate proprioception to improve
motor perception are used .
rubinstein et al.10 noted that visual cues were one of most effective methods for
stride length adjustment , and lim et al.1
stated that the application of auditory cues was effective for improving walking speed .
vibration stimuli that brought about increases in pd patients stride lengths and walking
speeds are an effective method , like visual or auditory cues , which are known to be an
effective method for improving gait in patients with pd .
therefore , vibration stimuli can be
said to be a type of feedback suitable for gait training or research intended to improve the
gait of patients with pd .
because this study was conducted in patients with pd at levels 1.52.5 of hoehn and yahr s
scale , the results of this study can not be generalized to all patients with pd ; however ,
they do suggest that application of vibration stimuli to the lower extremities can help
functional ability during gait in patients with parkinson s disease . | [ purpose ] the purpose of this study was to investigate the effect of muscle vibration in
the lower extremities in patients with parkinson s disease ( pd ) during walking .
[ subjects ]
nine patients with pd participated in this study and were tested with and without
vibration ( vibration at 60 hz ) .
[ methods ] eight oscillators of vibration were attached to
the muscle bellies ( tibialis anterior , gastrocnemius , biceps femoris , and rectus femoris )
on both sides of the lower extremities with adhesive tape in this study .
spatiotemporal
gait parameters were measured using a motion analysis system . [ results ] stride length and
walking speed with vibration
were significantly increased compared with those without
vibration in pd patients .
[ conclusion ] these results suggest that the application of
vibration to lower extremity muscles in patients with pd may improve the parkinsonian gait
pattern . |
radiation - induced sarcoma ( ris ) is a rare late event after radiation therapy ( rt ) , with a reported incidence of 0.03%- 0.2% in 5 years and overall up to 0.8% .
it is known to have a worse prognosis than sporadic soft tissue sarcoma ( sts ) , with a 5-year disease - specific survival ( dss ) rate of 32%-58% in recent reports [ 3 - 5 ] .
gladdy et al . reported that ris had a worse dss than sporadic sts with a hazard ratio of 1.7 .
dineen et al . also recently reported a lower dss of ris than of sporadic sts . because ris arises in the previously treated area
. emphasized the low possibility of curative resection with data showing 62% r0 resection . in the study by neuhaus et al .
, r0 - 1 resection was achieved in approximately 37% of the patients and was the only factor showing significant correlation with the dss .
in addition , adjuvant rt for ris should be administered with caution because of the higher risk of rt - related toxicities .
therefore , concerns about ris are emerging among cancer survivors because the survival rate and the survival time after cancer treatment are increasing as a result of advanced treatments including the use of targeted agents .
most studies of ris in asia are case reports , and significantly under - reported compared to western countries .
ris patients tend to be referred to a large tertiary center because of the surgical complexity , therefore we report herein the incidence and experience in management of ris at a single large tertiary referral center for 15 years .
after receiving institutional review board approval , we retrospectively reviewed our institutional sarcoma registry from january 2000 to april 2014 . out of the 3,674 patients in the registry , we identified 33 patients who met the following criteria for ris , which were modifications of cahan s criteria : ( 1 ) pathologic confirmation of sarcoma that was histologically unique from the primary cancer , ( 2 ) occurrence of the sarcoma in the field of radiation , and ( 3 ) radiation exposure at least 6 months before development of the sarcoma .
medical records were reviewed for the patients age , sex , primary cancer treatment , ris characteristics , ris treatment , and follow - up data .
the radiographic image of ris and the histopathology report were assessed for identification of the tumor location , size , and histology .
all available medical records from the follow - up were reviewed for evaluation of the disease status and toxicity .
the toxicities from re - irradiation were evaluated using the common terminology criteria for adverse events ( ctcae ) ver .
the overall survival rate was calculated from the time of the ris diagnosis until death .
actuarial survival curves were plotted from the ris diagnosis using the kaplan - meier method . a p - value less than 0.05 was considered to indicate a statistically significant difference .
the overall survival rate was calculated from the time of the ris diagnosis until death .
actuarial survival curves were plotted from the ris diagnosis using the kaplan - meier method . a p - value less than 0.05 was considered to indicate a statistically significant difference .
analyses were performed using pasw ver . 18.0 ( spss inc . , chicago , il ) .
the number of cases increased from four cases in the years 2000 - 2003 to 14 cases in the years 2012 - 2014 with corresponding proportion of ris among sarcoma patients of 0.5% to 1.6% ( fig .
the median age at the primary radiotherapy was 44 years ( range , 0 to 71 years ) .
three patients were treated before age 20 ( for bilateral retinoblastoma , wilms tumor , and nasopharyngeal cancer ) , and 20 patients were treated in their 40s and 50s .
the median age at the ris diagnosis was 55 years ( range , 22 to 85 years ) , and the median latency was 12.1 years ( range , 1.9 to 28.8 years ) .
the most common type of primary cancer was breast cancer ( n=9 ) , followed by uterine cervical cancer ( n=8 ) , and head and neck cancer ( n=7 ) .
all patients received a dose greater than 45 gy , with the exception of three patients with wilms tumor , retinoblastoma , and lymphoma treated with 10.8 gy , 36 gy , and 30 gy , respectively .
four patients underwent intracavitary brachytherapy in addition to external - beam rt for treatment of cervical cancer ; 58% of patients underwent chemotherapy as their primary cancer treatment .
ris was located in the trunk in 75.8% of the cases , and in the head and neck in 24.2% .
the most common histology was osteosarcoma ( 36.4% ) , followed by malignant fibrous histiocytoma ( 33.3% ) and angiosarcoma ( 6.1% ) .
the median tumor size was 4.8 cm ( range , 1.5 to 12.5 cm ) , and tumors were located deep in the muscle fascia in 27 cases ( 81.8% ) .
the median follow - up period was 23.1 months ( range , 2.2 to 174.8 months ) .
nineteen patients underwent surgery with curative intent , while 14 patients were treated conservatively . a list of the details of the treatment of 19 patients with curative surgery is shown in table 2 .
the median overall survival period of all patients was 2.9 years , and their 5-year survival rate was 44.7% . in univariate and multivariate analyses ,
the age at diagnosis and the treatment aim showed association with the overall survival ( table 3 ) .
the median duration of the overall survival period and the 5-year survival rate of patients treated with curative surgery were 9.6 years and 65% , respectively , and that of the conservatively treated patients , 0.7 years and 0% ( p=0.001 ) ( fig .
local recurrence occurred in nine patients ( 47% ) , and distant metastasis , in seven patients ( 37% ) .
four patients were misdiagnosed with bone metastasis from their primary tumor and underwent palliative rt before biopsy .
three patients underwent palliative radiotherapy , one patient was treated with gamma knife surgery of the recurred mass , and one patient underwent adjuvant rt after curative surgery .
five patients , including the familial retinoblastoma patient who developed grade 4 skin necrosis after re - irradiation , showed radiation toxicity after re - irradiation ( table 4 ) .
the number of cases increased from four cases in the years 2000 - 2003 to 14 cases in the years 2012 - 2014 with corresponding proportion of ris among sarcoma patients of 0.5% to 1.6% ( fig .
the median age at the primary radiotherapy was 44 years ( range , 0 to 71 years ) .
three patients were treated before age 20 ( for bilateral retinoblastoma , wilms tumor , and nasopharyngeal cancer ) , and 20 patients were treated in their 40s and 50s .
the median age at the ris diagnosis was 55 years ( range , 22 to 85 years ) , and the median latency was 12.1 years ( range , 1.9 to 28.8 years ) .
the most common type of primary cancer was breast cancer ( n=9 ) , followed by uterine cervical cancer ( n=8 ) , and head and neck cancer ( n=7 ) .
all patients received a dose greater than 45 gy , with the exception of three patients with wilms tumor , retinoblastoma , and lymphoma treated with 10.8 gy , 36 gy , and 30 gy , respectively .
four patients underwent intracavitary brachytherapy in addition to external - beam rt for treatment of cervical cancer ; 58% of patients underwent chemotherapy as their primary cancer treatment .
ris was located in the trunk in 75.8% of the cases , and in the head and neck in 24.2% .
the most common histology was osteosarcoma ( 36.4% ) , followed by malignant fibrous histiocytoma ( 33.3% ) and angiosarcoma ( 6.1% ) .
the median tumor size was 4.8 cm ( range , 1.5 to 12.5 cm ) , and tumors were located deep in the muscle fascia in 27 cases ( 81.8% ) .
the median follow - up period was 23.1 months ( range , 2.2 to 174.8 months ) .
a list of the details of the treatment of 19 patients with curative surgery is shown in table 2 .
the median overall survival period of all patients was 2.9 years , and their 5-year survival rate was 44.7% .
in univariate and multivariate analyses , the age at diagnosis and the treatment aim showed association with the overall survival ( table 3 ) .
the median duration of the overall survival period and the 5-year survival rate of patients treated with curative surgery were 9.6 years and 65% , respectively , and that of the conservatively treated patients , 0.7 years and 0% ( p=0.001 ) ( fig .
local recurrence occurred in nine patients ( 47% ) , and distant metastasis , in seven patients ( 37% ) .
four patients were misdiagnosed with bone metastasis from their primary tumor and underwent palliative rt before biopsy .
three patients underwent palliative radiotherapy , one patient was treated with gamma knife surgery of the recurred mass , and one patient underwent adjuvant rt after curative surgery .
five patients , including the familial retinoblastoma patient who developed grade 4 skin necrosis after re - irradiation , showed radiation toxicity after re - irradiation ( table 4 ) .
to the best of our knowledge , this is the first study reporting on the incidence and survival of ris in asia in a significant number of patients .
our data showed that , although rare , the number of patients diagnosed with ris in our institution is consistently increasing with time .
this may be due to the significant increase of rt usage in cancer therapy in south korea since the first cobalt-60 teletherapy installation in 1963 .
the increased survival time of cancer patients could also be attributed to the increasing incidence of ris .
investigated the question of whether or not a previous rt history is a worse prognosticator . in their analysis ,
a previous rt history was not a prognostic factor , but unlike sporadic sts , ris tended to occur in a central location and to be associated with incomplete surgery , which are known poor prognosticators in sarcoma .
. stated , previous rt might obscure the anatomic and tumor planes , which might make radical resection challenging . consistently in our data , only 57.6% of the patients could be treated with curative intent , and the survival outcome differed significantly according to the treatment aim .
in addition , the adjuvant rt for ris is limited by the concern for possible complications regarding re - irradiation , which can hinder optimal ris management .
an adjuvant rt of 42 - 63 gy is known to reduce local recurrence of sts after conservative surgery . however , as shown in this study , rt for ris results in a high rate of rt - related complications , which is inevitable in high - dose regions . due to the rarity and the histologic diversity within ris
, it is difficult to analyze the efficacy of secondary adjuvant rt after curative resection of ris . extrapolating from sporadic sts , adjuvant rt
should also be performed for ris , but care is required in the process of consultation with the patient , rt planning , and prescription of the rt dose .
also , high - precision rt techniques with increased conformality , such as intensity - modulated radiation therapy ( imrt ) , should be used in such re - irradiation cases to minimize the toxicity .
the question of whether or not the use of imrt , stereotactic body radiation therapy , or proton therapy would reduce ris incidence is yet to be determined . from the follow - up observation data on atomic bomb survivors who showed no excess of sarcoma after exposure to radiation of a few
gy , we acknowledge that ris is less likely to arise in very low - dose regions , and more likely to arise in intermediate- to high - dose regions of above 5 gy , with some evidence of a dose - response relationship .
however , as studies regarding ris , including this study , lack data on the initial dose prescribed for the ris lesion due to the loss or incompleteness of the topographic primary rt planning information , accurate dose - incidence evaluation is limited .
increased conformality with high - precision rt techniques would reduce the high - dose region , but integral doses in irradiated regions can be increased . in this
context , hall and wuu insisted that imrt would double the incidence of secondary malignancy . on the contrary , in a risk estimation study , imrt or proton therapy did not increase the risk of secondary cancer , but it was assumed that proton therapy reduced the incidence .
these findings should be demonstrated by clinical data in future studies . in this study , four ris patients misdiagnosed with bone metastasis underwent palliative rt before the biopsy , which is not an optimal treatment for ris .
although rare , the possibility of ris must be considered when malignancy is suspected in the previously irradiated region , and pathologic confirmation must be considered before the treatment . in this respect , identifying predisposing factors for development of ris could be of clinical concern . in the us surveillance , epidemiology and end results analysis by berrington de gonzalez et al . , a higher standardized incidence ratio for subsequent primary sarcoma was observed for patients treated at a younger age than those treated at older ages .
in addition , several studies reported multiplicative effects of radiotherapy and chemotherapy for the subsequent risk of sarcoma [ 18 - 21 ] . however , there is a limitation in explaining the occurrence of ris with these clinical factors giving way to the idea of possible genetic predisposition of a patient for development of ris .
pointed out that in the early 2000s , there was no molecular or pathological marker that was helpful in accurately distinguishing ris from spontaneous sarcoma , and little has changed since then . a study by kacker et al .
found that myc gene amplification was prevalent in ris . on the other hand , the study by kadouri et al .
, investigating the role of p53 and brca mutation in ris development , found a higher frequency of brca mutation in ris patients , but was insignificant , thus they concluded that brca mutation should not be considered when making the treatment decision .
an in vivo study by kansara et al . reported evidence that rb1 is required for development of radiation - induced osteosarcoma .
comparing the transcriptome of sporadic sarcoma with that of ris showed 135 gene signatures which could differentiate ris from sporadic sarcoma , which indicated mitochondrial dysfunction with a hallmark of chronic oxidative stress .
however , studies on genetic factors related to ris are still in their early stage .
future studies on the biological background of ris should be conducted in two major parts : that on the genetic susceptibility to ris to distinguish high - risk patients before rt , and that on the molecular radiation signature that can distinguish ris from sporadic sts beyond cahan s criteria .
in this study , we found that 0.9% of the sarcoma patients in the registry of a large tertiary center met the ris criteria .
although prognosis of ris is poorer than that of sporadic sts , a surgical approach with curative intent is needed if possible , from which we can expect a favorable survival outcome , as seen in this study . considering the aforementioned difficulties in ris treatment , future study should focus on selection of ris patient candidates using molecular markers before rt and should compare the risks and benefits of administering rt before the treatment .
in addition , enhancing the precision of rt would contribute to the reduction of ris or related complications in the future , but warrants further investigation . | purposethe purpose of this study is to report on the incidence and the experience in management of radiation - induced sarcoma ( ris ) at a large single center in korea for 15 years.materials and methodswe retrospectively reviewed the sarcoma registry of a large institution from january 2000 to april 2014.resultsout of the 3,674 patients listed in the registry , 33 patients ( 0.9% ) diagnosed with ris were identified .
the median latency of ris was 12.1 years .
the number of cases of ris increased from four cases in the years 2000 - 2003 to 14 cases in the years 2012 - 2014 .
the most common histology was osteosarcoma ( 36.4% ) .
the median follow - up period was 23.1 months , the median overall survival ( os ) of all patients was 2.9 years , and their 5-year survival rate was 44.7% .
univariate and multivariate analyses showed association of the age at diagnosis ( p=0.01 ) and the treatment aim ( p=0.001 ) with the os . the median os and the 5-year survival rate of patients treated with curative surgery ( n=19 ) were 9.6 years and 65% , respectively , and of the conservatively treated patients , 0.7 years and 0% ( n=14 ) .
re - irradiation was delivered to nine patients , and radiation toxicity was observed in five patients.conclusionin this study , ris accounted for 0.9% of the cases of sarcoma , with increasing incidence . despite the association of curative resection with increased survival , it could be applied to only 58% of the patients . considering the limited treatment options for ris , conduct of a genetic study to identify the underlying mechanism of ris
is needed . |
procalcitonin ( pct ) is elevated in serum of patients with severe infections of bacterial or parasitic origin . increased levels
it has been recently demonstrated that pct levels in falciparum malaria correlate with parasitemia and disease severity , and that markedly elevated values ( > 25 ng / ml ) are associated with a high risk of fatal outcome . in this study , pct levels were investigated in patients with tertian malaria , which is generally mild without severe organ complications and which is usually non - fatal .
patients with malaria who are seen at the site of the study follow a standard protocol for prospective assessment of relevant parameters .
thirty seven unselected , consecutive patients with tertian malaria due to plasmodium vivax or plasmodium ovale seen during 2000 and 2001 and in whom all necessary data were available , were analysed .
concomitant infections with a possible impact on pct levels were excluded by history , clinical examination , and laboratory tests .
patients who were raised in an area endemic for tertian malaria were defined as semi - immune ( n = 12 ) .
blood samples were drawn immediately before treatment ( day 0 ) and on follow - up until day 7 .
pct serum levels were determined using an immuno - luminometric assay ( lumitest pct , brahms diagnostica gmbh , berlin , germany ) according to the manufacturer 's specifications .
routine laboratory investigations included differential blood count , haemoglobin , platelets , standard coagulation tests , creatinine , neopterin , c - reactive protein , lactate dehydrogenase and liver function tests .
patients were monitored by physical examination , ecg , abdominal ultrasound , and chest radiography .
two patients , in whom the plasmodium species could not be differentiated by the first blood sample , received mefloquine .
results were compared with data obtained at the same site in a cohort of plasmodium falciparum patients and published earlier .
all patients had a low or modest parasitemia ( < 1% ) , haemoglobin concentrations > 12 g / dl ( except two patients with 8.4 and 10.7 g / dl ) , normal renal and liver function , and no signs of concomitant infections .
the disease progression was always mild with a rapid parasite clearance in response to anti - malarial treatment .
initial pct concentrations varied between < 0.1 ng / ml and 47 ng / ml and , in all patients , dropped with declining parasite counts and clinical recovery ( figure 1 ) .
pct levels before treatment did not correlate with parasite burden , laboratory values , reported duration of illness , body temperature or fever attacks , or the presence of semi - immunity . before treatment ,
two thirds of the patients showed normal ( < 0.5 ng / ml ) or slightly elevated pct levels ( geometric mean 0.44 ng / ml , range < 0.1 2.1 ) . in a subset of patients ( 12/37 ) , however , pct was markedly elevated ( geometric mean 14.4 ng / ml ; range 4.8 47 ) .
pct in these patients was in the range of patients with severe and complicated falciparum malaria ( > 2.5 ng / ml , figure 2 ) .
the two groups with low or high pct were undistinguishable by means of history , clinical signs , or laboratory parameters .
serum procalcitonin ( pct ) concentrations in 37 patients with tertian malaria immediately before treatment ( day 0 ) , and on follow - up .
box : median , 25 , and 75percentile ; whiskers : 5and 95percentile ; star : 1and 99percentile ; open squares : mean ; filled circles : geometric mean .
pct levels before treatment in patients with tertian malaria ( triangles ) compared to falciparum malaria ( open squares : survivors ; filled squares : patients that died ) .
cluster a : semi - immune patients ; cluster b : non - immune patients with mild disease ; cluster c : non - immune patients with severe and complicated disease .
the horizontal line at 2.5 ng / ml is the lower cut - off for complicated falciparum malaria and arbitrarily discriminates a high level from a low level group in tertian malaria .
in contrast to falciparum malaria and other infectious diseases in which pct is induced , markedly elevated pct levels could be observed in this cohort in one third of the patients without any correlation to disease severity .
this could not be attributed to concomitant infections or complications . while the study was not statistically powered to significantly detect weak correlations ,
although the pathophysiology of pct has not been determined yet , the question remains , what is so different in tertian malaria .
in contrast to bacterial or other infectious diseases , tertian malaria is characterized by surges in parasitic density and fever every 48 hours , leaving time in - between for the defence system to recover .
. it would be interesting to find out whether such host factors indicated an individual risk of developing severe falciparum malaria .
the unique feature of pct induction in tertian malaria raises interesting questions about the clinical and pathophysiological role of pct in infectious diseases and in malaria in particular .
ss assessed the clinical parameters and participated in the design of the study and the statistical analysis . | backgroundprocalcitonin ( pct ) is closely correlated with parasite burden and clinical outcome in falciparum malaria .
the role of pct in tertian malaria has not previously been investigated.patients and methodspct serum levels in 37 patients with tertian malaria were analysed .
clinical and laboratory parameters were assessed and statistically correlated both to the initial pct levels and during the course of the disease.resultspct levels rose for one day after commencing treatment and declined thereafter .
however , there was no significant correlation with parasite burden , clinical parameters , laboratory values , or the presence of semi - immunity . before treatment
, the majority of patients showed normal or slightly elevated pct levels ( < 2.5 ng / ml ) , but pct was markedly elevated ( 4.8 47 ng / ml ) in one third of the population .
the two groups did not differ by any other of the assessed parameters .
thus , while the post - treatment course of pct resembles falciparum malaria , the lack of correlation between disease severity and even high pct levels in a large proportion of patients is intriguing.conclusionsthere is a fundamental difference in the relationship of pct with tertian malaria not seen in other infectious diseases in which elevated pct levels have been observed .
this suggests distinct pathophysiological pathways in malaria . |
kohn sham ( ks )
density functional theory ( dft ) provides
a powerful theoretical framework for performing calculations on a
subsystem exactly embedded in its full , quantum - mechanical environment . the quantity that mediates the interaction between subsystems is
the electronic density , which is partitioned into two terms the ks energy consists of terms relating
to subsystems a and b
and an interaction term containing all nonadditive parts of the energy : this last term includes an explicitly known
coulomb contributionand , depending on the details of the subsystem
partitioning , simple electrostatic interaction of electrons in a with
nuclei in b and vice versa .
exchange - correlation
effects between subsystems are included throughwhich of course must be approximated in practical
calculations .
if the subsystem densities a and b are constructed from mutually orthogonal
orbitals , the kinetic
energy of the whole system is simply given by the sumin general , however , there will also be a
nonadditive termwhich must be approximated or ,
in more recent versions of the theory , computed exactly using optimized
effective potential ( oep ) methods .
the current
authors have demonstrated that oep methods can be used
in an iterative scheme to obtain the kohn
sham ground state
density for molecular systems , including those that exhibit strongly
overlapping subsystem densities .
reiher and co - workers
used oep methods to analyze and compare accurate embedding potentials
in approximate embedding schemes , and
oep methods were used to solve for a unique partitioning of a system
by allowing the subsystems to share a common embedding potential .
finally , several groups have utilized these dft embedding methods
to describe the interface between high - level and low - level subsystems
in wave - function - in - dft ( wf - in - dft ) approaches . in what follows
, we avoid the complications
of oep through three
simple , robust innovations : ( 1 ) we replace the iterated dft - in - dft
with a single conventional ks calculation .
( 2 ) we completely avoid
the issue of kinetic energy nonadditivity through the use of a level
shifting projection operator to keep the orbitals of one subsystem
orthogonal to those of another . and , ( 3 ) we develop a simple but effective
perturbation theory to eliminate practically all dependence on the
level shift parameter .
embedding methods that maintain orthogonality
between subsystem
orbitals have been in use for decades .
what has not been recognized
is that these can be used to formulate a formally exact dft embedding
scheme , equivalent to but much simpler than the recently developed
oep - based methods . the philips
kleinman pseudopotential approach
starts by level shifting the core orbitals to produce valence orbitals implicitly orthogonalized to the core .
even frozen core approximations , which have been in use in quantum
chemistry at least since the 1950s , amount
to a sort of hartree fock ( hf)-based embedding scheme in which
the core and valence subsystems are described by mutually orthogonal
orbitals .
similar ideas operate in the incremental scheme introduced
by stoll and co - workers , in the region
method of mata and et al . , and in henderson s
embedding scheme .
further details of
the large amount of literature in this area can be found in a recent
and comprehensive review .
the pauli
principle is enforced through a level - shift projector
in the ab initio model potential ( aimp ) method , which in turn derives from earlier work by huzinaga and cantu .
a similar strategy was employed by rajchel et
al . for the description of intermolecular interactions , but there
they iterated equations for each subsystem .
projection is sometimes employed in the embedding used in the fragment
molecular orbital method ( see for example the recent article ref ( 23 ) and references therein ) .
the novelty of the current work resides in the important simplification
that arises by performing a conventional kohn sham calculation
plus localization to determine fragment orbitals , the recognition
that this amounts to a formally exact dft - in - dft embedding scheme ,
and the elimination of practically all dependence on the level - shift
parameter through perturbation theory ( see below ) .
self - consistently
iterated embedded dft calculations are exactly
equivalent to full ks theory on the whole system .
if the only aim
is to perform dft - in - dft embedding , it is currently more practical
to perform ks theory on the whole system .
we now demonstrate that
this remains true even if the initial mean - field calculation is merely
a prelude for wf - in - dft embedding .
the sum of the atomic - orbital
( ao ) density matrices from the embedded
one - electron equations is equal to the total ks density matrix : + = .
the occupied ks orbitals { i } can
be rotated among each other , and one rotation produces the subsystem
orbitals { ia } { ib}. let us assume that
the whole system and both subsystems a and b are closed - shell and
that the total number of electrons is partitioned as na + nb = n ; the extension of the following arguments to open - shell subsystems
is straightforward .
to produce the correct total electron density ,
a ks calculation
on subsystem a requires an embedding potential that correctly describes
the electrostatic and exchange - correlation interactions with the electrons
in subsystem b , while also properly enforcing pauli exclusion between
the subsystem densities .
this is in fact the role of the nonadditive
kinetic potential vnad , but an alternative scheme can be reached by simply level
shifting the orbitals of b to very high energy for the calculation
on a. then , a correctly embedded calculation on the na electrons in subsystem a has the energy expression1where h is the core hamiltonian
( kinetic plus external potential ) in the ao basis .
we propose
a fock operator for electrons in subsystem a that includes
the appropriate derivative of the energy , plus a level - shift term
to preserve the orthogonality relation ia|jb = 0:2wherethe term p elevates the energy of the ith orbital in subsystem
b to ib + , and provided that is sufficiently
large and positive , the eigenstates of f are then mutually orthogonal to the orbitals in subsystem
b. evaluation of the gradient in eq 2 produces
the fock operator for subsystem a that we employ in the calculations
below : in hf theory or in ks theory with hybrid functionals ,
the additional ( possibly scaled ) exchange term k[ + ] appears in the usual
way . as in other dft - in - dft embedding schemes , this approach
provides
the flexibility to perform a completely different type of electronic
structure calculation for subsystem a , using a core hamiltonian that
contains the additional embedding terms from above:3the total energy for a normalized wave function
in the a subsystem is then calculated aswhere is the hamiltonian built with the embedding potential added to the
core hamiltonian , the central terms correct the exchange - correlation
energy contribution , and the final term is the energy of the electrons
in the b subsystem .
the code is implemented in the development
version of the molpro
software package .
the current version of the code ,
and all calculations presented here , employ the supermolecular basis
set .
future versions will employ subsystem calculations in a spatially
local set of atomic orbitals ; we see no technical barriers to this
extension .
we begin by demonstrating the efficacy of the projection technique
described above , using the example of embedding the 10 electrons of
the oh moiety of ethanol in the environment produced by the
ethyl subsystem .
ks orbitals from a pbe calculation in the 6 - 31 g * basis are localized
using the pipek
mezey localization scheme , and the five orbitals clearly associated with the oh
group are identified . note that this step is performed automatically by finding those orbitals with a mulliken population on any active
the embedded core hamiltonian matrix h is then constructed according
to eq 3 , with = 10eh , and a 10-electron kohn sham calculation
is performed .
if the projection technique has functioned perfectly ,
the resulting orbitals will be orthogonal to those associated with
subsystem b , the energy expression in eq 1 will
yield precisely the ks energy of the whole system , and tr
p will exactly vanish .
the value of tr p is approximately 20 eh , and the energy is somewhat too low . in the limit of infinite ,
the agreement with kohn
sham theory will be exact by construction ,
so the error arises from the finite value of this parameter . upon
increasing the value of , we do indeed find that the error
in the energy decreases ( figure 1 , uncorrected ) ,
although very large values result in numerical noise associated with
the double - precision numerics of the program .
error in the uncorrected
( e[; ] ) and
corrected ( e[; ] + tr p ) energy
expressions relative to full ks on ethanol
using pbe/6 - 31 g * , demonstrating that the perturbation theory correction
yields essentially exact embedding energies over a wide range of . a more stable approach is to compute the
limit using perturbation theory .
we can introduce a perturbed operator f that satisfies the requirements f0 = f + p andby adopting the form(here , for clarity , we have dropped the a
and b superscripts . ) expanding the operator f as a power series in and making the usual
perturbative expansion of energy and wave function , we obtainwhich yields at first - order the contribution e1 = 0|p|0. in the ao basis e1 =
tr p , and as shown in
table 1 , this correction brings the embedded
energy into near - perfect agreement with the ks energy .
higher orders
in this perturbation expansion could be used to correct the embedded
orbitals , or to further correct the energy , although nothing in our
calculations so far suggests that this will be necessary . in
fact
, it turns out that the energy expression e[; ] + tr p agrees with the full ks energy
to 7 peh , which is essentially exact in
double precision .
this excellent agreement is very insensitive to
the choice of , as shown in figure 1 . for values
< 10eh , the electronic structure is qualitatively incorrect , and for
> 10eh numerical noise
begins
to become an issue .
but for all values of in between , spanning
5 orders of magnitude , the discrepancy from full ks theory is below
20 neh .
similar results are found
for other molecules , and the projection
approach can also be employed for hf embedding .
for example , a hf - in - hf
embedded calculation on pyradine with subsystem a composed of the
five pipek
mezey orbitals associated with the nitrogen atom
and with = 10eh reproduces
the total hf energy to within 0.1 neh .
the method is also very stable for wf - in - dft calculations , which we
will demonstrate through ccsd(t)-in - pbe calculations on ethanol , using
the same subsystem partitioning as before .
the calculations are performed
( i ) without the perturbative correction to the total energy , ( ii )
with the pertubative correction obtained using the hf subsystem density
for the active subsystem , and ( iii ) with the perturbative correction
obtained using the unrelaxed ccsd density for the active subsystem ;
the differences between the total energies with = 10 and = 10 are then 470 neh , 50 neh , and 0.74 neh , respectively .
a key motivation
for dft embedding schemes is the possibility of embedding accurate
wave function calculations in a dft environment . here ,
we demonstrate
the simplicity and robustness of the current scheme with a few , sample
calculations .
first , we consider the deprotonation energy of
gas - phase ethanol , an example for which the pbe / aug - cc - pvdz level
of theory is in fairly good agreement with ccsd(t)/aug - cc - pvdz .
two
embedded calculations are performed , using either oh ( 10 electrons )
or ch2oh ( 18 electrons ) as the active subsystem .
the results are shown in table 2 , where it
can be seen that embedded ccsd(t)-in - pbe with the 10-electron active
subsystem slightly overcorrects the pbe value , by approximately 6
meh .
the calculation with the 18-electron
active subsystem produces a result in much better agreement , differing
from the total ccsd(t ) energy by a little over 1 meh .
these results are not entirely unexpected , since in
the first calculation the system is partitioned across a bond which
changes polarization during the deprotonation process .
the reactant and transition
state structure were optimized at the b3lyp/6 - 311g*++ level of theory using gaussian .
three coupled - cluster ,
ks , and embedded activation energies are shown in table 3 .
activation energies calculated at the ccsd(t ) , dft , and
ccsd(t)-in - dft levels of theory are shown in table 3 ; each of these calculations is performed using the cc - pvtz basis set with aug - cc - pv(t+d)z on chlorine .
dft calculations with approximate functionals
often underestimate barriers , but in the present case , the pbe and
blyp barriers are actually negative .
the embedded results are consistently much closer to the reference
coupled - cluster results , with the accuracy improving as more atoms
are included in subsystem a. the basis set is cc - pvtz with
aug - cc - pv(t+d)z on chlorine .
recent research in the group of one of us has focused on the use
of embedded many - body expansions to compute accurate structure and
energetics of molecular crystals .
the
idea builds on earlier attempts to capture many - body effects through
embedding , and it offers an important step toward systematically
improving accuracy by improving the embedding scheme , rather than
the number of terms in the many - body expansion .
we illustrate this
approach for the case of a symmetric model water trimer . for
simplicity ,
the trimer is optimized in a planar geometry , in which
each of the three monomers have equivalent geometries and environment
( see structure in figure 2 ) .
the geometry is
optimized with these contraints using mp2/6 - 31g*. denoting the monomer ,
dimer , and trimer ccsd(t ) energies as e1cc , e2cc , and e3cc , we can immediately express the coupled - cluster binding energy ( relative
to water molecules in their cluster geometry ) in the form ebindcc = e3cc 3e1cc .
binding curve relative to monomers for a planar
water trimer as
a function of the distance from the center of mass to the oxygen atoms .
shown
are the binding curves from hf theory , ccsd(t ) many - body expansion
truncated at the two - body level ( mbe2 ) , embedded two - body expansion
( embe2 ) , and from full trimer ccsd(t ) .
in the lower panel , the error for
embe2 is compared to that of the simple additive scheme hf-12 , in which the hf binding curve is corrected using the two - body
ccsd(t ) correlation energies .
several important approximations can be defined
without embedding ,
and two of these are shown in figure 2 .
truncating
the many - body expansion at the two - body level gives the approximation4where e2cc = e2cc
the hf approximation to the binding energy is defined by ebindhf = e3hf 3e1hf .
both of these approximations result
in errors of around 5 meh in the total
binding energy of around 20 meh .
since
our hf - in - hf embedding calculations require hf on the full trimer ,
we focus our attention on the correlation contribution to binding ,
and we use the notation e e
the one - body correlation correction without
embedding vanishes ( by analogy to eq 4 ) , but
with embedding , the contribution added to the trimer energy is different
from that subtracted as part of the monomer energy .
thus , one obtains
the approximationwhere 1corr is the correlation
energy from a cc - in - hf calculation on a single monomer .
a further
correction can be made by considering the two - body embedded correlation
contribution , givingwhere 2corr
this embe2 approximation can be
seen in figure 2 to be in essentially perfect
agreement with ccsd(t ) across
all geometries .
also shown in the figure is the error in this scheme
( compared to ebindcc ) , which can be seen to be on the order of
10 eh in the binding region .
the
results are strikingly better than those obtained using the simple
unembedded hf-12 approach defined asebindhf-12 = ebindhf + 3e2corr , especially at short range .
the ideas presented here
derive from a long history of ensuring
that the pauli principle is obeyed through level shifting a subset
of the orbitals .
but the interesting and novel point is that the dft
embedding techniques recently developed by us and
by others can be cast exactly into the very simple and robust framework described
here .
it should be noted that this projector embedding method
is not
without drawbacks .
it is limited to applications for which the electronic
structure can be reasonably described using ks theory , because the
projector can only be formed if ks orbitals are available .
this limitation
will become problematic in cases for which the dft description is
fundamentally broken , or if it is impractical to perform a full ks
calculation on the entire system .
furthermore , the new method describes
embedding in the environment by a projection operator , rather than
a simple , local potential . in its favor
, however , the current
approach relies on existing ,
stable , well - developed technologies .
it requires nothing more than
a ks code , an orbital localization scheme , some elementary matrix
operations in the atomic orbital basis , and any wave function method
that can accept an arbitrary core hamiltonian .
since these ingredients
are available in practically all molecular electronic structure codes ,
we anticipate no barriers to widespread adoption of the approach . | density functional theory ( dft ) provides a formally exact
framework
for quantum embedding .
the appearance of nonadditive kinetic energy
contributions in this context poses significant challenges , but using
optimized effective potential ( oep ) methods , various groups have devised
dft - in - dft methods that are equivalent to kohn sham ( ks ) theory
on the whole system .
this being the case , we note that a very considerable
simplification arises from doing ks theory instead .
we then describe
embedding schemes that enforce pauli exclusion via a projection technique ,
completely avoiding numerically demanding oep calculations .
illustrative
applications are presented using dft - in - dft , wave - function - in - dft ,
and wave - function - in - hartree fock embedding , and using an embedded
many - body expansion . |
nanoparticles are diverse class of small - scale ( < 100 nm ) substances with novel properties like small size , large surface area , specific shape , and surface activity .
toxicity of nanomaterials refers to the interaction of nanomaterials with the biological systems and induction of toxic responses .
once inside the body , they get distributed to various organs or may remain in the same site and can be structurally modified or metabolized .
when inhaled , they can translocate out of the respiratory tract via different pathways and mechanisms . when ingested
when in blood circulation , they can be distributed throughout the organism and will be taken up by liver , spleen , bone marrow , heart , and other major organs [ 2 , 3 ] .
depending on the duration of exposure , these materials can translocate from circulation to internal organs .
hence , the potential for significant biological response at each of these sites requires investigation .
magnetic nanoparticles are widely used in biomedical applications such as contrast agents in magnetic resonance imaging ( mri ) and in tissue repairing , detoxification of biological fluids , hyperthermia , drug delivery , cell separation , drug targeting , and so forth .
ferrite particles coated with biocompatible phases like hydroxyapatite are employed for hyperthermia treatment of cancer . as bare metal and metal oxide nanoparticles
are toxic to biological systems , coating with biocompatible phases is often done to increase the biomimetic nature of the particles suiting for various biomedical applications .
dextran ( c6h10o5 ) , a branched polysaccharide , obtained by microbiological synthesis is used to coat iron nanoparticles .
dextran being nontoxic , biodegradable , and hydrophilic may facilitate the intracellular uptake of dextran coated magnetic iron particles [ 3 , 4 ] .
the major toxicological issue associated with the manufactured nanomaterials is that some of them are redox active ; they can transport across cell membranes and interact with subcellular organelles . as a consequence of all these properties , nanoparticles can have direct interaction with individual target cells , either with the external membrane or inside the cell at the site of action .
advances in nanotechnology led to the exposure of humans to engineered nanomaterials and hence it became necessary to evaluate the potential human health effects before these materials are fully exploited [ 1 , 2 ] .
experimental evidences have shown that metal and metal oxide nanoparticles induced dna damage and apoptosis through ros generation and oxidative stress [ 10 , 11 ] .
it has been reported that magnetic nanoparticles administered intraperitoneally can cross blood - brain barrier where they have the potential to affect cerebral functions . due to the high reactivity of ros , most cellular components are likely to be the targets of oxidative damage : lipid peroxidation , protein oxidation , gsh depletion , and dna single strand breaks .
all of these events ultimately lead to cellular dysfunction and injury . for this reason ,
as long as there exists a balance between oxidative stress and antioxidant defense system , human body is maintained in an optimal health state .
antioxidant defense system includes both low - molecular - weight free radical scavengers , such as the tripeptide glutathione ( gsh ) , as well as antioxidant enzymes such as superoxide dismutase ( sod ) , glutathione reductase ( gr ) , and glutathione peroxidase ( gpx ) . the immunotoxicology of engineered nanoparticles is a growing concern in the present era .
the potential immunotoxicity and mechanisms of action of these particles have not received sufficient attention .
nanoparticles can interact with immune system in several ways and can enhance or suppress immune function depending on their physicochemical characteristics .
it was reported that the surface modification of carbon nanotubes recognizes scavenger receptor and alleviates nf-b activation and reduces its immunotoxicity .
similarly , shell - cross - linked knedel - like nanoparticles induced lower immunotoxicity than their non - cross - linked analogs as was demonstrated by elsabahy et al . . here ,
dfnps intended to be used for targeted drug delivery applications were in - house synthesized and characterized .
the primary focus of the present study was to examine the immunotoxic potential and cellular response of dfnps .
thiobarbituric acid ( tba ) , reduced glutathione ( gsh ) , oxidized glutathione ( gssg ) , and dithiobis-2-nitrobenzoic acid ( dtnb ) were purchased from sigma chemical co. , st .
louis , mo , usa . pyrogallol ( pg ) , diethylene triamine pentaacetic acid ( dtpa ) , and trichloroacetic acid ( tca ) were purchased from merck , germany .
rnase , ethanol , bromophenol blue , ethidium bromide , 100 bp dna ladder , and taq polymerase were purchased from fermentas , usa ; mouse oligonucleotide primers for interleukin 1 ( il 1 ) , interleukin 10 ( il 10 ) , tumour necrosis factor beta ( tnf ) , and beta actin ( actin ) were procured from eurogentec , belgium ; rpmi-1640 from himedia , mumbai , india ; rt sybr green rox q pcr master mix , rneasy lipid tissue mini kit , qiazol lysis reagent , and rt first strands kit were from qiagen , hilden , germany , genelute mammalian genomic dna miniprep kit was from sigma / aldrich , usa . mouse t and b cell selection kit was from stem cell technologies inc . , canada .
all the other chemicals used were of analytical grade and were purchased from qualified local vendors .
the equipment used was as follows : spectrophotometer ( shimadzu , japan ) , laminar air flow ( mark air particulars , india ) , incubator shaker ( new brunswick scientific , usa ) , biophotometer ( eppendorf , germany ) , and steam sterilizer ( nat steel , india ) .
all animals were handled humanely , without making pain or distress and with due care for their welfare .
the care and management of the animals will comply with the regulations of the committee for the purpose of control and supervision of experimental animals ( cpcsea ) , government of india .
all the animal experiments were carried out after prior approval from institutional animal ethics committee and in accordance with approved institutional protocol .
the body weight ranges between 17 and 23 g and was maintained in a 12 h light / dark cycle at a constant temperature of 22 3c with free access to standard pellet diet and water .
in addition to this , each animal cage was identified by labels having details such as experiment number , name , animal number(s ) , and date of experiment .
all the animals were acclimatized for a period of 5 days before initiation of experiment .
briefly , the stoichiometric mixtures of fecl3 and fecl24h2o ( fe / fe : 2 : 1 ) were heated at 70c in n2 atmosphere .
ferrite nanoparticles were precipitated by the addition of 3 m naoh dropwise for 1 h followed by heating with stirring for about another 1 hr .
the precipitate was then washed with deionized water three times to get uniformly dispersed spherical magnetite particles .
all reactions were carried out in n2 atmosphere to prevent oxidation of magnetite to magnemite .
surface coating of ferrite nanoparticles with dextran was done by overnight stirring ( at 37c ) of ferrite nanoparticles in a solution of dextran of appropriate concentration .
the precipitate was then washed and lyophilized to get dextran coated ferrite nanoparticles ( dfnps ) .
the size determination of synthesized dfnps was studied by transmission electron microscopy ( tem ) analysis .
the spleen cells were seeded at a density of 20,000 cells / well in a 96-well plate at 37c in 5% co2 atmosphere .
after 24 h of culture , 200 , 400 , 600 , 800 , and 1000 g / ml of dfnps ( in triplicate , cells alone as negative control ) were added onto the suspension culture of spleen cells .
the cells were incubated at 37 1c for 24 1 h and examined microscopically for morphological changes and quantitated by mtt assay .
20 l of mtt dye solution ( 5 mg / ml in phosphate buffer ph 7.4 ) was added to each well .
after 4 h of incubation , the mtt was removed and formazan crystals formed were solubilized with 200 l of dmso .
the absorbance of each well was read on a microplate reader ( elx 808iu ultramicroplate reader , bio - tek instruments , usa ) at 540 nm .
the generation of ros was monitored by employing 2,7,dichlorodihydrofluorescein diacetate ( h2dcfda ) [ invitrogen ] which is nonfluorescent unless oxidized by the intracellular ros .
a dose dependent measurement of the ros generation was done by preincubating 20,00040,000 spleen cells with h2dcfda at a concentration of 100 m .
then , the cells were exposed to 200 , 400 , 600 , 800 , and 1000 g / ml of dfnps ( in triplicate ) for 2 h , at 37c .
cells were then washed in serum - free medium and resulting fluorescence intensity was read in a fluorescence microplate reader using an excitation wavelength of 488 nm and emission wavelength of 535 nm .
immunotoxicity is to assess the potential adverse effects on the immune system , which is an important component of the overall evaluation of drug / chemical / nanomaterial toxicity . in this study ,
100 mg of dfnps was injected intraperitoneally to 18 albino mice for immunological stimulations .
six animals each were sacrificed at 0 days ( animals not exposed to nanoparticles ) and at the end of 7 , 14 , and 21 days [ total 24 albino mice ] after exposure . at the end of each observation period ,
gross necropsy was carried out , and spleen and liver were collected for the following studies . spleen was excised from experimental animals and splenocytes were isolated to study the cell proliferation by tritiated ( h ) thymidine incorporation assay .
single cell splenocytes suspension was used to separate t and b lymphocytes using kit ( easysep b cell enrichment kit , t cell enrichment kit ) according to the protocol described in the kit using an automated cell separator ( robosep ) .
both the t and b lymphocytes ( 2,00,000 cells / well ) were cultured in a 96-well plate in rpmi-1640 medium supplemented with 10% fbs , streptomycin ( 100 g / ml ) , and penicillin ( 100 units / ml ) for 24 h at 37c in a co2 incubator . after 48 h of incubation , h - thymidine at a concentration of 1
ci / ml was added to each well and incubated further for 24 h at 37c .
cells were harvested after 72 h and radioactivity in terms of counts per minute ( cpm ) was measured by liquid scintillation counter [ 21 , 22 ] .
10% liver tissue homogenate was prepared in phosphate buffer ( 0.1 m , ph 7.4 ) using an ice - chilled glass homogenizing vessel in a rotor stator homogenizer at 900 rpm .
the resultant supernatants were maintained in an ice bath until being used for the estimation of total protein , lipid peroxidation , reduced glutathione , and antioxidant enzymes ( glutathione reductase , glutathione peroxidase , and superoxide dismutase ) using standard protocols with slight modifications .
total proteins in the liver homogenate of mice exposed to dfnps were estimated by the method of , using bovine serum albumin as standard .
the extent of lipid peroxidation ( lpo ) in the liver homogenate of mice exposed to dfnps was determined as the concentration of malondialdehyde ( mda ) generated by the thiobarbituric acid reactive substances ( tbars ) , as described by .
reduced glutathione ( gsh ) level in the liver homogenate of mice exposed to dfnps was determined by the method of , with slight modifications in which ellman 's reagent or dtnb ( 5,5-dithiobis-(2-nitrobenzoic acid ) ) reacts with gsh to form a spectrophotometrically detectable product at 412 nm . the change in absorbance at 412
nm is a linear function of the gsh concentration in the reaction mixture and is based on the reaction of gsh with dtnb to give a compound 5-thionitrobenzoic acid ( tnb ) that is absorbed at 412 nm .
the glutathione reductase ( gr ) activity in liver homogenate of mice exposed to dfnps was determined by measuring the reduction of gssg in the presence of nadph as described by .
thus , one gr unit is defined as the reduction of one m of gssg per minute at 25c and ph 7.6 .
activity of glutathione peroxidase ( gpx ) in the liver homogenate of mice exposed to dfnps was assayed by the method described by .
the remaining gsh after the enzyme catalyzed reaction was complexed with dtnb , which absorbs at maximum wavelength of 412 nm . enzyme activity was expressed as g of gsh consumed / min / mg protein .
the superoxide dismutase ( sod ) in liver homogenate of mice exposed to dfnps was done using modified pyrogallol autoxidation method spectrophotometrically measured at 420 nm . all measurements were carried out using uv spectrophotometer-1601 , shimadzu , japan .
total m - rna was isolated from splenocytes exposed to dfnps ( 200 , 600 , and 800 g / ml ) and bare ferrite nanoparticles ( 600 , 800 g / ml ) , following the manufacturer 's protocol , using trizol reagent ( sigma , usa ) . 150 ng of m - rna was used for c - dna synthesis of il-10 , il-1 , tnf , and actin in a reaction volume of 20 l using rt first strand kit ( qiagen , germany ) and the synthesis was carried out on the eppendorf master cycler , germany .
the mouse oligonucleotide forward and reverse primer sequences used to determine specific m - rna gene expressions are depicted in table 1 .
the real time pcr reaction was carried out with rt sybr green rox q pcr master mix of total reaction volume of 25 l , and real time pcr amplifications were done using a chromo 4 system , bio - rad ( mj research , ca ) for 40 cycles as per manufacturer 's protocol .
the level of gene expression is reported as the ratio between the mrna level of the target gene and the actin , a reference gene using the comparative 2 method .
ndna was isolated from liver tissues of mice used for immunotoxicity studies , as per the manufacturer 's protocol ( sigma , usa ) .
the quantity and quality of the isolated ndna were estimated ( nanodrop , eppendorf , germany ) .
ndna isolated from in vivo experimental groups was amplified using mouse actin specific forward primer ( f - gcgtggggacagccgcatctt ) and reverse primer ( r - atcggcagaaggggcggaga ) ( eurogentec , belgium , accession number hq675031.1 ) at a concentration of 100 ng/l per reaction mixture .
pcr of ndna was carried out as per standard conditions in eppendorf master cycler ; germany .
purity and integrity of the amplified products were checked by purity factor ( 260/280 nm ) and agarose gel electrophoresis . the amplified pcr product ( actin ) from the above immunotoxicity studies was purified with a qiaquick pcr purification kit ( qiagen , hilden , germany ) according to the manufacturer 's instructions .
actin ndna products were sequenced using mouse actin forward and reverse primers in ab1 prism 3730 genetic analyzer dna sequencer with a big dye terminator cycle sequencing ready reaction kit ( applied biosystems japan co. , ltd . , tokyo , japan ) .
abi sequence scanner was used for sequencing and alignment of sequence was done using bioedit software . after sequence analysis and alignment ,
using sequence navigator program version 2.1 , all sequences were submitted to ncbi website ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) and blast sequence similarity search was conducted .
statistical significance between the control and experimental values was compared by student 's t - test .
ferrite nanoparticles were prepared by the standard coprecipitation method and coated with dextran to yield dfnps .
the transmission electron microscopic ( tem ) images indicate that a very uniform size distribution of dfnps particles and was found to be less than 25 nm ( figure 1 ) .
cytotoxicity of dfnps was evaluated in cultured splenocytes ; the results are depicted in figure 2(a ) .
no marked decrease in cell viability was observed after 24 hrs of exposure for all the concentration tested .
it is well known that most of the manufactured nanoparticles cause cell injury by increasing reactive oxygen species formation .
the amount of ros generated after 24 hrs of exposure to dfnps is illustrated in figure 2(b ) .
the results of the study suggest that the ros generation in all the samples was well comparable with control values and was not statistically significant .
the animals exposed to dfnps were monitored throughout the experimental period for any unusual changes .
the increase in body weight and feed intake was normal and none of the animals showed any abnormality or behavioral changes during the experimental period .
gross examination of carcasses of control and dfnps exposed mice did not reveal any abnormality in the organs examined .
lymphocyte proliferation is one of the markers to predict the immune response induced upon nanoparticle exposure .
the proliferation assay was evaluated in both t and b lymphocytes isolated from spleen of animals exposed to dfnps for different time periods .
it was observed that , at 7 days after exposure , a slight increase in thymidine incorporation was observed when compared to control although the effect was statistically insignificant .
decrease in lymphocyte proliferation was observed for t cells after 21 days of exposure and after 14 and 21 days for b cells .
figure 4(a ) shows the malondialdehyde ( mda ) production in the liver homogenate of mice after exposure to dfnps .
the results indicate that the level of lpo production in the control and 21 days postexposure group was 4.51 1.7 and 5.83 0.5 nmoles / mg proteins , respectively .
it was observed that the level of lpo was not significantly increased at any time periods ( 7 , 14 , and 21 days after exposure ) when compared to control .
the level of reduced glutathione ( gsh ) in the liver homogenate of mice exposed to dfnps is illustrated in figure 4(b ) .
the result suggests that a time dependent decrease in the level of gsh was observed in the liver homogenates of mice with the significant decrease in 21 days postexposure group ( 1.12 0.2 ) when compared to control ( 1.49 0.1 ) . the level of glutathione reductase ( gr )
, glutathione peroxidase ( gpx ) , and superoxide dismutase ( sod ) was illustrated in figure 5 .
the result of the study indicates that the production of gr was significantly increased at the end of 7 days and decreased at the end of 21 days after exposure of dfnps ( 0.34 0.02 ) in mice when compared to control ( 0.29 0.02 ) .
similarly , there was significant increase in the production of sod in the liver homogenate of mice 21 days after exposure of dfnps ( 0.15 0.00 ) when compared to control ( 0.12 0.01 ) .
in addition , there was no increase in the level of gpx observed at any time points which was well comparable to control values .
as evident from figure 6(a ) , il-10 mrna expression was increased by 3-fold in 600 g / ml dfnps cells and 2-fold in 800 g dfnps exposed cells on comparison of cells treated with similar concentration of bare ferrite particles .
no marked difference was noticed in il-1 mrna expression for both dfnps and bare ferrite nanoparticles treated cells ( figure 6(b ) ) . eventually , there was a dramatic decrease in m - rna expression of tnf in dfnps particles in a concentration dependent manner when compared with bare ferrite particles .
differences were observed in both concentrations ( 600 g and 800 g / ml ) of dfnps when compared to bare ferrite nanoparticles ( figure 6(c ) ) .
the values are expressed in mean se . from figure 7(a ) ( ( b ) , ( c ) , and ( d ) ) ( treated groups , forward actin primer ) when compared to figure 7(a ) ( a ) ( control group , forward actin primer ) it was evident that bases 10 to 20 of the sequence were altered in 7 , 14 , and 21 days ' treated mice liver , respectively .
likewise , it is apparent from figure 7(b ) ( ( b ) , ( c ) , and ( d ) ) ( treated groups , reverse actin primer ) when compared with figure 7(b ) ( a ) ( control group , reverse actin primer ) bases 10 to 20 showed irregular base mismatches .
figure 7(b ) ( a ) ( control group , reverse actin primer ) had a poly a tail whereas in figure 7(b ) ( b ) ( 7 days ' treated group ) such tail was tainted in the sequence .
bioengineered nanoparticles are being considered for a wide range of biomedical applications , from magnetic resonance imaging to drug delivery systems .
the development of novel nanomaterials for biomedical applications must be accompanied by careful scrutiny of their biocompatibility .
attention should be paid on the possible interactions between nanoparticles and cells of the immune system . in the present study ,
dfnps intended to be used for targeted drug delivery applications were in - house synthesized , characterized , and examined for the immunotoxic potential and cellular response .
the detailed characterization of dfnps was carried out using dynamic light scattering for hydrodynamic size profiling , transmission electron microscope for particle size analysis , x - ray diffraction technique for phase purity analysis , thermo gravimetric analysis for quantifying the dextran in dfnps , and fourier transform infrared spectral analysis for coating efficiency .
the result of the study indicated that the synthesized dfnps were an authentic particle and have a size less than 25 nm [ 17 , 18 ] . on entry into the body , nanoparticles can move ahead and get absorbed in liver , spleen , and bone marrow through blood and lymph .
it was also known that nanoparticles exposure may result in ros production and splenocytes apoptosis , change cytokine production , and decrease immune response .
thus , it is extremely crucial to evaluate the altered immune responses induced upon nanoparticle entry . in this study ,
systemic immune response induced by dfnps was evaluated under both in vitro and in vivo conditions .
spleen is the largest lymphoid organ which plays a critical role in both innate and adaptive immune responses ; splenocytes were opted for initial cytotoxicity screening .
it was found that no obvious cytotoxicity was noted in spleen cells after dfnps exposure even though a marginal increase in splenocytes activity was observed .
the oxidative stress experienced by the cell was assessed by the measurement of intracellular reactive oxygen species ( ros ) level .
increases in intracellular ros ( oxidative stress ) are potentially toxic to the cells which , if not neutralized by antioxidant defenses ( e.g. , glutathione and antioxidant enzymes ) , could lead to membrane dysfunction , protein degradation and dna damage , and finally cell death . for measuring the ros , the cell permeable probe ( i.e. , 2,7-dichlorodihydro - fluorescein diacetate , h2dcfda ) was used for the present study .
from the results , it was very clear that the ros generation was well comparable with control values that predict a nontoxic response at the cellular level .
the systemic administration of dfnps ( 7 , 14 , and 21 days ) for immunotoxicity evaluation in albino mice showed that the general physical conditions of the experimental animals were normal during the experimental period .
all the animals were sacrificed and there were no abnormalities observed during the gross necropsies of animal at the end of each observation period .
the results of the proliferation assay indicated that there was a significant reduction in the h - thymidine incorporation in both the t and b cells after 14 and 21 days after exposure of dfnps which needs further investigation .
the concentration of mda in biological materials has been widely utilized as an indicator of oxidative damage to unsaturated lipid .
measurement of mda , the byproduct of lpo , provides an exact and well - established index of oxidative damage since it is very reactive and takes part in cross - linking with biomolecules .
it is well known that lipid peroxidation occurs naturally in small amounts in the body , mainly by the effect of several ros or by the action of several phagocytes [ 33 , 34 ] . in the present study , no changes in the level of lipid peroxidation in liver
but there was a significant reduction of gsh observed after 21 days of exposure and this may be the result of direct reaction of dfnps with gsh .
reported that after administration of nanoparticles ; gsh can act as a conjugating agent in their metabolism .
when these nanoparticles induce oxidative stress by generating h2o2 or hydroperoxides , gsh can also be oxidized in a reaction catalyzed by gsh - px .
the depletion of gsh in tissues leads to impairment of the cellular defense against ros , and may result in peroxidative injury [ 35 , 36 ] . regarding the antioxidant enzymes ,
it was observed that the level of gr and sod was significantly altered 21 days after exposure period .
the cells replenish the lost gsh by converting the peroxides formed from free radicals into gsh .
thus , in the present study , the increase in gr activity observed after 7 days of exposure may result in the conversion of peroxides formed by ros into gsh .
after 21 days of exposure , although a slight decrease in gr activity was observed , it was significantly higher compared to control .
gr and gpx are the two most important enzymes in the gsh - gssg cycle and may be activated by increased hydrogen and/or lipid peroxide production .
antioxidant activity of gsh - px involves neutralization of h2o2 , reduction of lipid hydroperoxidases , and maintenance of normal membrane permeability .
it was also observed that there was no increase in the gpx production , when dfnps were exposed to mice .
the finding of the cytokines analysis of the present study are in line with the literature suggesting that tumor necrosis factor - beta ( tnf ) is a cytokine that is inhibited by interleukin 10 .
it was observed that the increase in the concentration of dfnps increases the expression of il-10 , an anti - inflammatory cytokine , and decreases the expression of tnf which mediates a large variety of inflammatory , immunostimulatory , and antiviral responses .
earlier studies revealed no macrophage or dendritic cell secretion of proinflammatory cytokines when silicone - coated iron oxide nanoparticles were administered .
similarly , the present study revealed that no increase in proinflammatory cytokines occurs upon administration of dfnps .
sequence analysis of cytoskeletal actin revealed regions from 20 to 50 bases were conserved in treated mice .
earlier studies of cytoskeletal actin mrna sequencing of mouse provided information about the conserved sequences as well as the posttranscriptional regions in the sequence . the conserved sequence of actin in mice for mir-644a target site was provided in 2012 .
the 5 end of the mouse actin gene contains sequence elements which mediate the stimulatory effects of serum growth factors and are responsive to both positive and negative regulators of gene expression of several genes .
base mismatches in the nonconserved region observed may not significantly lead to mutations and altered protein synthesis but may result in the short frame shift mutations .
animals appeared to be normal during the course of the experimental period after exposure to dfnps .
the dfnps ( < 25 nm ) are found to be noncytotoxic in spleen cells .
it was also evident that the dfnps do not influence cellular proliferation , lpo , gsh , and antioxidant enzymes .
hence , it can be concluded that the dfnps were nontoxic at cellular level and nonimmunotoxic when exposed to albino mice , under laboratory conditions simulation .
base alterations in the nonconserved region may not significantly lead to mutations and distorted protein synthesis of the cytoskeletal actin gene but might result in the diminutive frame shift mutations . | in this study , dextran coated ferrite nanoparticles ( dfnps ) of size < 25 nm were synthesized , characterized , and evaluated for cytotoxicity , immunotoxicity , and oxidative stress by in vitro and in vivo methods .
cytotoxicity was performed in vitro using splenocytes with different concentrations of dfnps .
gene expression of selected cytokines ( il-1 , il-10 , and tnf ) secretion by splenocytes was evaluated .
also , 100 mg of dfnps was injected intraperitoneally to 18 albino mice for immunological stimulations .
six animals each were sacrificed at the end of 7 , 14 , and 21 days .
spleen was subjected to immunotoxic response and liver was analyzed for antioxidant parameters ( lipid peroxidation , reduced glutathione , glutathione peroxidase , superoxide dismutase , and glutathione reductase ) .
the results indicated that dfnps failed to induce any immunological reactions and no significant alternation in antioxidant defense mechanism .
also , mrna expression of the cytokines revealed an increase in il-10 expression and subsequent decreased expression of il-1 and tnf . eventually , dna sequencing of liver actin gene revealed base alteration in nonconserved regions ( 1020 bases ) of all the treated groups when compared to control samples .
hence , it can be concluded that the dfnps were nontoxic at the cellular level and nonimmunotoxic when exposed intraperitoneally to mice . |
after tooth extraction , the healing process initiates however , alveolar socket never reaches its previous dimensions either buccolingually or apicocoronally .
additionally , the remaining bone structure after healing undergoes a progressive resorption ( catabolic remodeling ) .
resorption in the first 6 months after extraction is very fast ; however , it slows down and mildly continues for the rest of the person s life .
successful replacement of lost teeth through conventional prosthodontic treatments or implant placement is challenging in an atrophic alveolar ridge .
[ 1 - 2]bone loss compromises the support , retention , stability , and esthetics of dental prosthetics along with the health of the remaining tissues .
bone mass is regulated through the process of remodeling which includes two phases including bone formation by osteoblasts and bone resorption by osteoclasts .
besides
recent investigations have demonstrated that central
and peripheral nervous systems play a significant role in regulation of bone mass .
leptin stimulates the sympathetic
nervous system [ 6 - 7 ] and this system intrinsically results in increased expression of rankl
( receptor activator of nuclear factor kappa - b ligand ) receptor on their surface by stimulating the 2-adrenergic receptors on the surface of osteoblasts . the interaction between
rankl and rank ( receptor activator of nuclear factor kappa - b ) on the surface of osteoclasts precursors , transforms them into osteoclasts .
considering the above mentioned findings , various researchers have suggested the use of 2-adrenergic receptor antagonists in prevention or treatment of bone resorption .
[ 7 - 18 ] several reports discuss the decreased risk of fracture in those using propranolol which is a
non - specific 2-adrenergic receptor antagonist .
[ 7 - 18 ] takeda et al . and bonnet et al . demonstrated that a non - specific 2-adrenergic receptor antagonist like propranolol can enhance bone formation in female rats that had undergone ovariectomy .
if beta - blockers are truly capable of preventing bone resorption in humans , the above mentioned findings can be successfully implemented in oral cavity for extraction socket healing or prevention of progressive alveolar bone resorption following teeth loss .
therefore , the present study was conducted to evaluate the effect of 2-adrenergic receptor antagonists on number of osteoblasts and osteoclasts in alveolar socket following tooth extraction in animal models .
a total of 40 , eight - week old wistar rats with a mean weight of 27050 g were selected from an inbred colony . during the study period , the rats were divided into 10 groups of 4 each and kept in an standard cage in a room under controlled conditions with a temperature of 212c and 50%7% moisture in 12 hours dark and 12 hours light cycle and free access to standard food and water .
the rats were brought to the lab one week before the initiation of study to make them acquainted to the environment .
the samples were then randomly divided into 2 equal groups of test and control . color coding system was used for identification of animals to blind the study .
it should be mentioned that the present study was conducted regarding the ethical guidelines of animal model studies designed by the research deputy of the ministry of health and was approved by the ethics committee of animal studies , tabriz university of medical sciences .
maxillary right first molars of rats in both test and control groups were extracted with an excavator ( no.55 , smic , china ) under general anesthesia with i m ketamine hydrochloric acid 44 mg / kg ( rotexmedica , germany ) .
hemostasis occurred approximately 5 minutes after tooth extraction and antibiotics or other medications were not administered .
first , injectable propranolol ( tolidaroo , iran ) , a non - specific beta - adrenergic receptor antagonist , was dissolved in sterile saline solution right before injection .
it was then injected using an insulin syringe at a dosage of 0.1 mg / kg / day intraperitoneally during the whole study period and at a specific time ( 11.00 a.m ) for all samples in the test group ( n=20 ) .
the mentioned dosage has been proposed by bonnet et al . as the lowest dosage capable of preventing bone loss without any cardiovascular effect .
the same amount of saline solution
was injected to the control group ( n=20 ) with the same method .
injections continued in both groups until their sacrifice ( figure 1 ) . the best protocol between control groups ( group c ) and test groups ( group t ) . at days 7 , 14 , 21 and 28 post - extraction , 5 samples of any of the case and control groups were randomly selected and sacrificed using diethyl ether ( pars chemie , iran ) .
. then it was preserved in ethylene diamine tetra acetic ( edta ) 10% at 4c and ph=7.2 for 21 days in order
to become decalcified and embedded in paraffin .
tissue blocks were cut serially into 5-m sections parallel to the sagittal plane using a microtome .
we selected sections which contained tooth socket
and its surrounding bone structure and were stained with hematoxylin - eosin .
osteoclasts and osteoblasts were counted twice by one blinded researcher at 4 random areas in the alveolar socket wall .
collected data were analyzed using spss version 13 software and included descriptive statistics ( mean and standard deviation ) , unpaired t - test and one - way anova using fisher s protected
least significant difference method .
maxillary right first molars of rats in both test and control groups were extracted with an excavator ( no.55 , smic , china ) under general anesthesia with i m ketamine hydrochloric acid 44 mg / kg ( rotexmedica , germany ) .
hemostasis occurred approximately 5 minutes after tooth extraction and antibiotics or other medications were not administered .
first , injectable propranolol ( tolidaroo , iran ) , a non - specific beta - adrenergic receptor antagonist , was dissolved in sterile saline solution right before injection .
it was then injected using an insulin syringe at a dosage of 0.1 mg / kg / day intraperitoneally during the whole study period and at a specific time ( 11.00 a.m ) for all samples in the test group ( n=20 ) .
the mentioned dosage has been proposed by bonnet et al . as the lowest dosage capable of preventing bone loss without any cardiovascular effect .
the same amount of saline solution
was injected to the control group ( n=20 ) with the same method .
injections continued in both groups until their sacrifice ( figure 1 ) . the best protocol between control groups ( group c ) and test groups ( group t ) .
at days 7 , 14 , 21 and 28 post - extraction , 5 samples of any of the case and control groups were randomly selected and sacrificed using diethyl ether ( pars chemie , iran ) .
. then it was preserved in ethylene diamine tetra acetic ( edta ) 10% at 4c and ph=7.2 for 21 days in order
to become decalcified and embedded in paraffin .
tissue blocks were cut serially into 5-m sections parallel to the sagittal plane using a microtome .
we selected sections which contained tooth socket
and its surrounding bone structure and were stained with hematoxylin - eosin .
osteoclasts and osteoblasts were counted twice by one blinded researcher at 4 random areas in the alveolar socket wall .
collected data were analyzed using spss version 13 software and included descriptive statistics ( mean and standard deviation ) , unpaired t - test and one - way anova using fisher s protected
least significant difference method .
in the first week following tooth extraction , number of osteoclasts in the control group was significantly higher than the test group receiving propranolol
( table 1 and figure 2a and 2b ) .
mean and standard deviation ( sd ) of osteoblasts ( ob ) and osteoclasts ( oc ) in the control ( c ) and test ( t ) groups at weeks 1 to 4 post - extraction
in the control group , accumulation of osteoclasts was observed not only in the interdental septum but also on the socket floor ( figure 2a and 2b ) .
also , a significant increase in number of osteoclasts in both groups was observed in the first week compared to the following weeks ( p < 0.05 ) ( figure 3a and 3b and figure 4a and 4b ) .
a : accumulation of osteoclasts on the socket wall in the control group .
b : socket wall in the test group at week one following tooth extraction ( 10x magnification ) .
a : comparison of the number of osteoclasts in the test group ( t - oc ) during the 4 weeks .
b : comparison of the number of osteoclasts in the control group ( c - oc ) during the 4 weeks .
b : accumulation of osteoblasts in the socket in the test group at week 3 post - extraction ( 40x magnification ) .
the number of osteoblasts in the control group reached its maximum rate in the second week and remained constant in the 3 and 4
weeks .
the number of osteoblasts in the test group receiving propranolol increased similar to the control group but reached its maximum rate at week 3 and demonstrated a significant increase compared to the control group ( p < 0.05 )
( figure 4a and 4b and figure 5a and 5b ) .
however , a significant reduction in osteoblast number was observed in week 4 ( figure 5a and 5b ) .
figure 6
demonstrates the difference in trabecular thickness and number and also the width of bone marrow space in the bone covering alveolar socket floor in test and control rats .
a : comparison of the number of osteoblasts in the test group ( t - ob ) during the 4 weeks .
b : comparison of the number of osteoblasts in the control group ( c - ob ) during the 4 weeks .
a : new bone formation in tooth socket in the control group .
b : new bone formation in tooth socket in the test group at week 4 following the tooth extraction ( 10x magnification ) .
in the present experimental study , we found that 2 adrenergic receptor antagonists could decrease the number of osteoclast and increase the number of osteoblast during extraction socket healing .
our study results revealed that in the first week following tooth extraction , when bone resorption is the dominant process , a significant increase was detected in the number of osteoclasts in both groups compared to
the following weeks and the number of osteoclasts in the control group was significantly higher than the test group receiving propranolol ( p < 0.05 ) .
the mechanism of action of beta - blockers in regulating bone mass is through the stimulation of osteogenesis and decreasing bone resorption by preventing the differentiation
of osteoclasts through rank - rankl system.[7 - 19 ] since beta - blockers are widely used for treatment of cardiovascular diseases ,
epidemiologic studies in this respect are feasible .
studies have demonstrated that use of beta - blockers has been associated with lower risk of fractures .
considering the association of sympathetic nervous system with bone metabolism , medical researchers have seriously focused on the application of beta - blockers for prevention and treatment of osteoporosis.[9 - 18 ] in dentistry , the relationship between beta - blockers and bone loss has been the focus of attention for some time now .
kim et al .
induced alveolar bone loss by superior cervical ganglionectomy and oral challenge with p. gingivalis .
we found that the number of osteoclasts in the control group was significantly higher than the test group receiving propranalol which our results are in agreement of okada et al .
they assessed the effects of a beta - blocker on alveolar bone loss induced by p. gingivalis
infection in rats to examine the involvement of sympathetic nervous system in periodontal breakdown and indicated that beta - blockers inhibited osteoclast differentiation
and prevented alveolar bone loss by controlling the alveolar bone mass metabolism .
shimizu et al .
also demonstrated that sympathetic nervous activity can affect bone loss induced by occlusal hypo - function in rats .
decreased number of osteoclasts following beta - blocker administration that has been reported by other researchers as well [ 20 - 21 ]
is indicative of the role of sympathetic nervous system in regulating bone mass and confirms the hypothesis of beta - blocker use for prevention of alveolar bone resorption following tooth extraction .
the number of osteoblasts reached its maximum rate in week 2 in the control and in week 3 in the pro- pranolol group and showed a significant increase in the test group compared to the control group .
the above mentioned findings are in accord with animal model studies on extraction socket healing in rats indicating that osteogenesis initiated in the middle of the 2
week post - extraction and slowed down at the end of the 3 week when the tooth socket is mostly filled with newly formed bone .
[ 23 - 24 ]
however , in rats receiving propranolol although the number of osteoblasts at week 2 increased similar to the control group , in contrast to the controls , this increase continued until
week 3 and number of osteoblasts revealed a significant difference with that of the controls ( figure 4 ) .
in fact ,
duration of osteogenesis phase in the propranolol group was longer than that in the control group which is in agreement with other studies reporting increased osteogenesis in rats receiving propranolol .
[ 7 - 19 ]
our results clearly demonstrate the difference in trabecular number and thickness and also the width of bone marrow space in the bone covering alveolar socket floor in test and control rats .
however , considering our obtained results , a hypothesis can be proposed regarding the prevention of alveolar bone resorption following tooth extraction by using beta - blockers .
in a conclusion , 2 adrenergic receptor antagonists decrease the number of osteoclasts and increase the number of osteoblasts during extraction socket healing . | statement of the problem : various researchers have suggested the use of 2-adrenergic receptor antagonists in prevention or treatment of bone resorption .
purpose : this study aimed to evaluate the effect of 2-adrenergic receptor antagonists on number of osteoclasts and osteoblasts involved in the healing of extraction socket of maxillary first molar in rats .
materials and
method : maxillary first molars of 40 rats were extracted and divided into two groups .
the test group received 0.1 mg / kg propranolol intraperitoneally daily .
the controls received normal saline . at days 7 , 14 , 21 and 28 post - extraction
, 5 rats were euthanized from each group .
maxillary bone was resected and the mean number of osteoblasts and osteoclasts in tooth socket was measured .
results : after 1 week , the number of osteoclasts in the controls was significantly higher than the test group .
a significant increase in the number of osteoclasts in both groups at week 1 was observed compared
to the following weeks ( p < 0.05 ) .
the number of osteoblasts in the controls at second week reached its maximum rate but stayed constant in the 3rd and 4th weeks .
osteoblasts in the test group increased
similar to the controls but reached its maximum at 3rd week and showed a significant increase compared to the controls ( p < 0.05 ) .
conclusion:2 adrenergic receptor antagonists decrease the number of osteoclasts and increase the number of osteoblasts during extraction socket healing . |
esthetics can influence the selection of the prosthesis . in screw - retained implant restorations ,
the screw- access hole is unesthetic ; because the gray color of screw hole can not be eliminated by opaque composite materials .
all prostheses have inherent advantages and disadvantages ; clinicians should assess them and choose the appropriate one .
the porcelain veneer in porcelain fused to metal ( pfm ) restorations is susceptible to chipping due to different factors such as inappropriate porcelain -metal bonding , incompatible thermal expansion coefficients of the porcelain and the metal substructure , use of a metal alloy with low modulus of elasticity and trauma from occlusion .
four different modes of fracture have been reported in the surfaces : ( 1 ) adhesive failure between the metal and the oxide layer ; ( 2 ) cohesive failure within the oxide layer ; ( 3 ) adhesive failure between the oxide and ceramic layers ; ( 4 ) and cohesive failure within the ceramic layer .
faulty design of the metal substructure , excessive porcelain thickness with inadequate metal support and technical flaws in the porcelain application are among the suggested causes of failure .
tooth - supported restorations have shown lower five - year risk of ceramic fracture in comparison with implant supported fixed partial dentures ( fpd ) ( 2.9% versus 8.8%)[4 , 5 ] .
possible somatic causes that may account for the differences in ceramic failure rates between restorations include the lack of neurologic feedback and the periodontal reflex mechanism that are not present as a protective mechanism resulting in generation of high masticatory loads .
two main techniques have been recommended to repair a fractured porcelain : the direct method and the indirect method . for screw - retained implant restorations ,
the repair procedure can be performed extra - orally ( indirect method ) . in direct methods ,
the disadvantages of this technique include poor wear qualities , low strength and poor esthetics due to the lack of color stability and shade - matching to the remaining porcelain .
fabrication of a pin - retained casting with a fused porcelain veneer or fabrication of an overlay
the purpose of this study is to describe the use of a separate over casting crown design to cover the screw access hole of implant screw - retained prosthesis in the esthetic zone .
a 51 year - old woman was referred to the prosthodontics department of shahid beheshti university , school of dentistry to assess teeth 12 and 13 ( fdi two digit tooth numbering system ) screw - retained fixed implant restorations ( figure 1 ) .
four years ago , she received dental implants to restore edentulous spaces in both jaws with implant -supported fixed prosthesis ( figure 2 ) ; 6 months after delivery of the prosthesis , porcelain chipping occurred in teeth 12 and 13 , which was repaired with composite resin .
but , the composite detached from the porcelain once every 5 6 months and she was forced to go to dental office for repair .
clinical examination revealed that the anterior segment of the maxilla had a six - unit screw - retained fpd ( figure 1 ) .
four implants in the maxillary right and left lateral and canine areas supported this fpd ( figure 2 ) .
the extreme buccal angulation of the implant replacing teeth 12 and 13 resulted in a buccally- located screw access hole , which compromised esthetics and potentially weakened the porcelain around the screw holes in the six - unit screw - retained fpd . in lateral excursions canine
it was decided to overcome this esthetic and structural obstacle by using a separate over casting crown design to cover the metal substructure of the screw - retained prosthesis .
after choosing a suitable stock tray , alginate ( tropicalgin , zhermack , italy ) impression was made from the upper jaw .
then , two implant analogs ( ( biomet 3i implants , uk ) were connected to the abutment connections of both laterals . after using a separator ( gingifast separator , zhermack , italy ) , gingival mask ( gingifast , zhermack , italy )
also , both canine abutment connections and tissue surface of pontics were covered with gingival mask to protect and facilitate the separation of fpd from the cast and then the impression was poured with type 4 dental stone ( vel - mix , ernst hinrichs gmbh , germany ) .
all the porcelain on the labial and palatal one - third of the lateral and canine teeth was removed with green stone and then metal was removed in the same places to gain space for metal and porcelain of the over casting crowns ( figures 4 - 1 and 4 - 2 ) . finally deep chamfer finish line was designed in the palatal surface with chamfer diamond bur . in the labial surface because of the limitation in metal volume , light chamfer finish line was designed .
die spacer ( euro classic die spacer , kerr , orange , ca ) was applied to the prepared frame but not in the screw access holes . for adequate marginal adaptation ,
a band of about 1 mm , not painted , adjacent to the preparation margin immediately .
vaseline was applied to the prepared frame to lubricate it and then full contour resin pattern ( gc resin pattern , tokyo , japan ) was fabricated on it .
this pattern penetrated the screw access holes until 1 mm from the head of screws remained .
once the final contour of the resin pattern was completed , the pattern was cut back over a uniform thickness of about 1 mm to provide room for the porcelain fused to the cast metal substructure .
finally , thickness of metal framework was 0.2 mm in the cervical area to 0.5 mm in the incisal area . investing and
casting procedures were done and porcelain was applied to the metal framework ( figures 5 - 1 and 5 - 2 ) .
six - unit screw - retained fpd was placed into the patient s mouth and was secured by tightening the screws ( figure 6 ) .
splinted over - casting crown was tried in the mouth and color coordination was done by applying stain ( figure 7 ) .
then , a small amount of gutta - percha was placed over the head of screws .
the splinted over casting crown was glazed and cemented with temporary cement ( temp bond ne , kerr , orange , ca , usa ) ( figure 8) .
after choosing a suitable stock tray , alginate ( tropicalgin , zhermack , italy ) impression was made from the upper jaw .
then , two implant analogs ( ( biomet 3i implants , uk ) were connected to the abutment connections of both laterals . after using a separator ( gingifast separator , zhermack , italy ) , gingival mask ( gingifast , zhermack , italy )
also , both canine abutment connections and tissue surface of pontics were covered with gingival mask to protect and facilitate the separation of fpd from the cast and then the impression was poured with type 4 dental stone ( vel - mix , ernst hinrichs gmbh , germany ) .
all the porcelain on the labial and palatal one - third of the lateral and canine teeth was removed with green stone and then metal was removed in the same places to gain space for metal and porcelain of the over casting crowns ( figures 4 - 1 and 4 - 2 ) . finally deep chamfer finish line was designed in the palatal surface with chamfer diamond bur . in the labial surface because of the limitation in metal volume , light chamfer finish line was designed .
die spacer ( euro classic die spacer , kerr , orange , ca ) was applied to the prepared frame but not in the screw access holes . for adequate marginal adaptation ,
a band of about 1 mm , not painted , adjacent to the preparation margin immediately .
vaseline was applied to the prepared frame to lubricate it and then full contour resin pattern ( gc resin pattern , tokyo , japan ) was fabricated on it .
this pattern penetrated the screw access holes until 1 mm from the head of screws remained .
once the final contour of the resin pattern was completed , the pattern was cut back over a uniform thickness of about 1 mm to provide room for the porcelain fused to the cast metal substructure .
finally , thickness of metal framework was 0.2 mm in the cervical area to 0.5 mm in the incisal area .
investing and casting procedures were done and porcelain was applied to the metal framework ( figures 5 - 1 and 5 - 2 ) .
six - unit screw - retained fpd was placed into the patient s mouth and was secured by tightening the screws ( figure 6 ) .
splinted over - casting crown was tried in the mouth and color coordination was done by applying stain ( figure 7 ) .
then , a small amount of gutta - percha was placed over the head of screws .
the splinted over casting crown was glazed and cemented with temporary cement ( temp bond ne , kerr , orange , ca , usa ) ( figure 8) .
it is very common to use a resin - based composite for intraoral repair of the fractured porcelain of pfm crowns or fpd .
micromechanical retention is an important factor for resin - metal bonding and this method fails in many cases .
using composite materials for repairing screw access holes is very difficult since implementing color to match with the surrounding porcelain in cases with large screw access holes is hard and problematic .
also , by increasing the forces on the prosthesis in lateral excursions , composite separation is likely as in this case .
another problem which arises is high wear of composite material . a novel overlay casting
restoration [ 1216 ] explained here is useful when a huge part of the porcelain is missing and metal is exposed or where metal support for composite is missing .
the reliability of this technique is higher in comparison to resin used in the direct method .
support and reinforcement of porcelain are provided by the metal substructure that uses a separate over casting crown design . moreover
, oven heat evaporates the water , which has been absorbed by the old porcelain in the oral environment during its service and increases the likelihood of its cracking .
one reason which causes increase in generation of masticatory force in the implant supported fpd is lack of periodontal ligament .
therefore , porcelain not supported by a metal substructure renders it prone to chipping and removing the previous porcelain and rebaking is unreasonable .
our method has many advantages namely predictable results , lower cost , easiness and time saving at chair side [ 12 , 13 ] .
the simplicity of our method makes it suitable for repair of fractured porcelain surrounding screw access holes of metal - ceramic restorations in screw retained implant restorations .
screw - retained fpds generally have screw access holes , which can compromise esthetics and weaken the porcelain around the holes .
this study described the use of a separate over - casting crown design to cover the screw access hole of implant screw - retained prosthesis in the esthetic zone with notable advantages . | screw - retained implant restorations have an advantage of predictable retention as well as retrievability , and obviate the risk of excessive sub - gingival cement commonly associated with cement retained implant restorations .
screw - retained restorations generally have screw access holes , which can compromise esthetics and weaken the porcelain around the holes .
the purpose of this study is to describe the use of a separate overcasting crown design to cover the screw access hole of implant screw - retained prosthesis for improved esthetics . |
for a successful bacterial colonization of the oral cavity , adherence mechanisms are essential , otherwise the bacteria get washed away and swallowed by the salivary flow .
oral bacteria have evolved several mechanisms to withstand the salivary flow and succeed in adhesion to and subsequently form biofilm on surfaces of the oral cavity .
streptococcus gordonii has been considered to play an important role in cariogenesis because it readily colonizes the clean tooth surfaces and is capable of forming biofilm . among the oral bacteria
, s. gordonii appears to have highest affinity to hard surfaces of the oral cavity .
s. gordonii biofilm forms an important component of human dental plaque by virtue of its ability to adhere to tooth surfaces .
formation of dental plaque precedes cariogenesis ; thus , interfering with s. gordonii adhesion and biofilm formation of hard tissue is likely to improve the oral health .
dietary agents that interfere with adhesion of and biofilm formation by bacteria has been the focus of intensive research because such natural agents are likely to be nontoxic to the host .
furthermore , the identified active components can be used as supplement to oral health hygiene product negating the necessity to adhere to a particular diet .
perhaps most important advantage of searching dietary agents is that approval of clinical trials would be easier to obtain , as toxicity is not an issue . in this respect , cranberry juice and isolated fractions / constituents which inhibit adhesion of bacteria to various surfaces
phenolic compounds of cranberry were shown to prevent adherence of uropathogen to animal cells [ 4 , 5 ] .
the cranberry components were also shown to reduce the risk of cardiovascular disease , periodontal disease , and inhibit host inflammatory response .
earlier studies demonstrated a high - molecular - weight mass , nondialyzable material ( ndm ) prepared from cranberries to contain polyphenolic compounds that inhibited the secretion of proteolytic enzymes by periodontopathogens , adhesion of a number of bacterial species [ 10 , 11 ] , and were also shown to interfere with coaggregation of oral bacterial species and biofilm formation by streptococcus mutans [ 3 , 12 ] .
the polyphenol fraction of cranberry was reported to decrease the hydrophobicity of streptococcal species [ 12 , 13 ] . in the present study
, we sought to determine the ability of the high - molecular - weight component from cranberry ( ndm ) to interfere with biofilm formation by s. gordonii in general and in particular on dental composites and titanium discs .
we hypothesize that the cranberry ndm will have a beneficiary role by interfering with streptococcal biofilm formation on dental materials .
prevention or reduction of oral bacterial load on the surfaces of the oral cavity will have a beneficial role in improving the oral health .
ndm was obtained as described previously from concentrated cranberry juice made from the american cranberry , vaccinium macrocarpon , and provided by ocean spray cranberries , inc .
briefly , ndm was obtained after lyophilization of the material retained in a dialysis bag ( 12,000 molecular weight cutoff ) following extensive dialysis .
the retentate of the bag designated ndm , is soluble in water up to 4 mg / ml , devoid of proteins , carbohydrates , and fatty acids , and was found to exhibit tannin - like properties suggests that it is rich in phenolic compounds ( e.g. , proanthocyanidins ) .
further analysis performed by ocean spray inc . revealed that this fraction is devoid of sugars , acids , and nitrogen and contains 0.35% anthocyanins ( 0.055% cyanidin-3-galactoside , 0.003% cyanidin-3-glucoside , 0.069% cyanidin-3-arabinoside , 0.116% peonidin-3-galactoside , 0.016% peonidin-3-glucoside , and 0.086% peonidin-3-arabinoside ) and 65.1% proanthocyanidins [ 9 , 12 ] .
streptococcus gordonii challis ( atcc , rockville , md ) was grown in trypticase soy broth ( tsb ; difco labs ) for 48 hours at 37c .
cells were washed in pbs and resuspended to contain 5 10 cells / ml . following the treatment of bacteria with ndm
, s. gordonii cells were incubated with 50 l of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-{(phenylamino)carbonyl}-2h - tetrazolium hydroxide ( xtt ) for 4 hours at 37c . in this assay ,
the tetrazolium salt xtt is cleaved to an orange - colored formazan product by mitochondrial dehydrogenase in viable cells . at the end of incubation period ,
the absorbance of the resulting supernatant was measured at 490 nm using an elisa reader ( bio rad laboratories ) .
prior to the measurement of number of cells in the biofilm , a standard curve was prepared with known numbers of bacteria . following the treatment of bacteria with ndm
, s. gordonii cells were incubated with 50 l of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-{(phenylamino)carbonyl}-2h - tetrazolium hydroxide ( xtt ) for 4 hours at 37c . in this assay ,
the tetrazolium salt xtt is cleaved to an orange - colored formazan product by mitochondrial dehydrogenase in viable cells . at the end of incubation period ,
the absorbance of the resulting supernatant was measured at 490 nm using an elisa reader ( bio rad laboratories ) .
prior to the measurement of number of cells in the biofilm , a standard curve was prepared with known numbers of bacteria .
we also used the crystal violet staining method to assess the effect of ndm on the bacterial biofilm formation in a 96-well microtiter plate .
adherent bacteria in the wells were fixed with methanol for 15 min , extensively washed with distilled water , and then stained with 0.4% crystal violet ( 100 l ) for 15 min .
wells were rinsed with distilled water and dried at 37c for 2 h. after adding 100 l of 95% ( v / v ) ethanol to each well , the plate was shaken to release the stain .
all assays were run in triplicate , and the means sd of three independent experiments were calculated .
freshly cultured bacterial suspension containing 5 10 cells / ml was prepared , from which 0.1 ml suspension was placed in each of a 96-well microtiter plate .
an equal volume of serially diluted ndm ( 5.0 to 400 g / ml ) in phosphate - buffered saline ( pbs ; 0.1 m na2hpo4 , 0.1 m kh2po4 , 0.15 m nacl , ph 7.4 ) was added to each well containing the bacteria and incubated for 24 hours at 37c . after the incubation , unattached bacteria were washed off by rinsing the plate with pbs .
the microtiter plate was replenished with fresh tsb media ( 0.2 ml ) supplemented with 0.2% sucrose and incubated for an additional 24 hours . at the end of incubation
, nonadherent cells were removed and the metabolic activity of the bacteria in the biofilm was assessed by xtt assay and by staining with crystal violet , as described above . for control purposes , 2.0 ml of standard bacterial suspension
was immersed in a beaker of boiling water for 5 minutes and then treated with xtt reagent as the experimental cells .
espe composite material was molded into a 6 mm disc and cured for 10 seconds as recommended by the manufacturer . in a similar manner , amalgam discs ( 6 mm )
an aliquot ( 0.5 ml ) of standard bacterial suspension was incubated with cranberry ndm ( 5 to 200 g / ml ) for 60 min at 37c .
bacteria were then added to the discs placed in a 24-well culture dish and further incubated for 24 hours at 37c .
after gentle washing to remove the nonadherent bacteria , discs were incubated with 0.5 ml of tsb supplemented with 0.2% sucrose for an additional 48 hours to facilitate biofilm formation .
at the end of incubation , discs were rinsed once and then incubated with xtt reagent to assay for their metabolic activity .
bacteria were stained using the live / dead baclight kit ( molecular probes - invitrogen , carlsbad , ca ) .
this stain distinguishes live cells from dead bacteria based on membrane integrity and two nucleic acid stains .
the green fluorochrome ( syto 9 ) can penetrate intact membranes , while the larger red fluorochrome ( propidium iodide ) penetrates only compromised membranes of dead bacteria .
cells treated with ndm ( 25 g / ml for 60 minutes ) or not ( control ) were stained with the dye for 15 minutes in dark .
bacterial cells ( 5 10 cells ) were pelleted , and the pellet was resuspended in 1.0 ml of ndm ( 25 g / ml ) and incubated for 60 min .
the cells were stained with live / dead kit ( molecular probes - invitrogen , carlsbad , ca ) fluorescent dyes , calcein am , and ethidium homodimer diluted according to the manufacturer 's recommendation for 15 minutes in dark at room temperature .
similar numbers of bacteria were incubated with 70% isopropanol for 45 minutes to generate dead cells , which were also stained similarly ( dead cell control ) .
the effect of ndm on bacterial cells was analyzed by facscan flow cytometry ( becton dickinson ) using 520 20 nm excitation for measuring calcein green fluorescence emission and ethidium homodimer red fluorescence emission using 615 30 nm .
the data was processed with flowmax software ( partec ) , and electronic gating with the software was used to separate positive signals from noise . between 7,500 and 50,000 events
were acquired using linear amplification for forward and side scatter and logarithmic amplification for fluorescence .
samples were measured in triplicate , and selected samples were controlled with epifluorescence microscopy to confirm the bacterial nature of stain .
bacterial samples were stained with the live / dead baclight bacterial viability kit ( invitrogen l-13152 ) , a rapid epifluorescence staining method as specified by the manufacturer .
the bacteria were incubated for 15 minutes in dark and then examined for the difference in live and dead cells between ndm - treated and control s. gordonii by confocal microscope .
images were analyzed by using comos software ( bio rad ) , and green and red images were merged and formatted on confocal assistant software ( bio rad ) .
data are expressed as the means standard deviations of three independent experiments with triplicate samples in each experiment .
to investigate the effect of ndm on metabolic activity of s. gordonii in biofilm , bacteria were treated with serially diluted ndm and then measured their metabolic activity by xtt metabolic assay .
cranberry ndm inhibited s. gordonii metabolic activity in a dose - dependent manner ( table 1 ) . a 50% reduction in bacterial metabolic activity
was seen when treated with 7.5 g / ml of ndm , while 50 g / ml of ndm inhibited by 96% . total inhibition in metabolic activity
colony forming units of the ndm treatment of bacteria revealed that viability was not affected at any of the concentrations tested suggesting that the inhibition of metabolic activity was not related to ndm toxicity to bacteria ( data not shown ) .
the results ( figure 1 ) show that the time of treatment and concentration of ndm affected the s. gordonii metabolic activity .
pretreatment of bacteria with 2.5 g / ml of ndm for 15 minutes reduced bacterial metabolic activity as estimated by xtt assay by 20% of the bacteria when compared to the untreated bacteria ( control ) .
bacteria treated with 25 g / ml ndm showed metabolic reduction of 66% ( figure 1 ) .
higher concentrations of ndm ( 100 g / ml ) completely abolished the metabolic activity ( data not shown ) .
contrary to the reduction in metabolic activity of s. gordonii by ndm treatment , there was no reduction in biofilm mass when stained with crystal violet ( figure 2 ) .
biofilm mass was reduced by only 8% of the control when bacteria were treated with 100 g / ml . there was no significant difference in biofilm mass between the groups treated with different concentrations of ndm .
the data suggests that the cranberry ndm at relatively low concentrations does not appear to inhibit biofilm formation as estimated by the total mass but the metabolic activity of the formed biofilm was affected by ndm . in figures 1 and 2 ,
one ml aliquots of standard bacterial suspension in triplicate was incubated with varying concentrations of ndm ( 5.0 to 200 g / ml ) for 15 , 30 , 45 , and 60 minutes . after the incubation
, bacteria were centrifuged and the pellet was washed with pbs once and suspended in 0.2 ml of tsb culture media supplemented with 0.2% glucose for an additional 48 hours and measured their metabolic activity in the biofilm by xtt .
another parallel set of bacterial cells treated in a similar way was stained with crystal violet , and the optical density was measured at 550 nm . for control purposes ,
1.0 ml of standard bacterial suspension was immersed in a beaker of boiling water for 5 minutes and then treated as above with xtt and crystal violet .
next , we investigated the effect of cranberry ndm on preformed s. gordonii biofilm and assayed metabolic activity by xtt and biofilm mass by crystal violet method .
the results ( table 2 ) demonstrate that the ndm failed to detach the bacteria from the preformed biofilm mass , even at a concentration of 3 mg / ml . on the other hand ,
it is of interest to note that the bacterial metabolic activity was reduced by ndm treatment ( table 2 ) .
ndm at a concentration of 100 g / ml for 60 minutes reduced the metabolic activity by 50% , but the same concentration had no effect on biofilm mass . increasing
the length of treatment of preformed biofilm up to 8 hours also showed similar results ( data not shown ) .
the results ( figure 3 ) demonstrate that the ndm treatment of s. gordonii showed a reduction in metabolic activity of bacteria adherent to both titanium and polystyrene surfaces .
greater than 50% reduction in metabolic activity was seen with 10 g / ml concentration of ndm .
higher concentration of ndm ( > 25 g / ml ) reduced the metabolic activity of the adherent bacteria by greater than 98% .
in contrast , ndm at the same concentrations reduced total bacterial mass by only 58 percent , when stained with crystal violet ( data not shown ) , consistent with the data shown in figure 2 .
we tested the ndm effect on metabolic activity of biofilm created on amalgam , composite , and hydroxyl apatite discs by xtt assay as described previously .
the results ( table 3 ) of the study showed that ndm treatment inhibited s. gordonii biofilm metabolic activity on the two dental restorative materials as well as on polystyrene surface .
25 g / ml of ndm inhibited approximately 50% of bacterial metabolic activity on all three discs .
no measurable inhibition of metabolic activity was seen when bacteria were treated with ndm concentrations greater than 100 g / ml .
in contrast , the total bacterial mass of the biofilm bacteria was reduced by only 58% as determined by crystal violet staining ( data not shown ) consistent with the data shown earlier .
the data obtained in this study so far suggested that the cranberry ndm inhibited the metabolic function of s. gordonii without being bactericidal . in order to confirm this observation , we stained the biofilm of s. gordonii created on polystyrene with live / dead baclight according to the recommended protocol by the manufacturer ( invitrogen ) .
stained bacteria were first viewed by a fluorescent microscope and then by confocal microscope .
figure 4 shows that ndm treatment did not appear to cause cell death , and both ndm treated ( figure 4(a ) ) and untreated bacteria ( figure 4(b ) ) appear to contain similar proportion of live and dead bacteria .
the results of flow cytometry ( figures 5(a ) and 5(b ) ) indicate that ndm treatment did not change in the ratio of live to dead bacteria consistent with our microscopic observation that the ndm has no cytotoxic effect on s. gordonii cells .
early colonization on the tooth surface and subsequent biofilm formation by s. gordonii and their ability to coaggregate with several oral microorganisms result in the formation of dental plaque .
the plaque and biofilm formation leads to caries and subsequently leads to periodontal disease if left untreated .
constituents of cranberry were demonstrated to decrease the hydrophobicity of streptococcal species [ 12 , 16 ] .
cranberry high - molecular - weight component was shown to inhibit secretion of glucosyl and fructosyltransferases by oral streptococci and coaggregation of bacteria .
the high - molecular - weight component of cranberry was shown to be highly soluble in water , lacking proteins , carbohydrates , and fatty acids [ 17 , 18 ] .
previous studies have shown that relatively high concentrations of ndm ( e.g. , 0.52 mg / ml ) were required to cause 80% or more reduction of biofilm formation by s. mutans on saliva - coated ha and by p. gingivalis on polystyrene surfaces . in the present study , we show that lower concentrations of ndm ( 0.050.1 mg / ml ) selectively inhibited the metabolic activity of oral bacteria , s. gordonii .
this conclusion is based on ( i ) the effect of ndm on metabolic activity of preformed biofilm that was more profound than on the total biofilm mass as measured by crystal violet stain and ( ii ) the confocal microscopy stain with live / dead stain showing no effect on the ratio of viable / dead s. sangius .
the effect of ndm on metabolic activity may indirectly interfere with the ability of the bacteria to adhere and form biofilm onto various dental surfaces as shown in the present study and in other studies [ 7 , 12 , 16 ] .
previous studies have shown that ndm affects various physicochemical properties of uropathogenic bacteria [ 4 , 9 ] .
the present study shows an effect on metabolic activity , and further studies are needed to see how these two effects are connected .
perhaps most important is to study how these effects of relatively low concentrations of ndm on metabolic activity of s. sangius biofilm influences cariogenicity of the bacteria such as acid formation . either way
, ndm seems to affect s. sangius adhesion and biofilm formation mainly by inhibiting metabolic activity of the cariogenic bacteria .
it is expected that supplementing oral health product such as mouth wash with ndm will affect not only s. mutans total counts as shown previously but also s. sangius cariogenic activity .
our study revealed the beneficial role of cranberry ndm in reducing the s. gordonii metabolic activity in the biofilm created on various dental surfaces such as titanium implant material , amalgam , and composite materials . the potential use of cranberry ndm in oral rinse merits further investigation , since it appears to benefit the health of the oral cavity , by reducing the metabolic activity of s. gordonii . | previous studies demonstrated that a cranberry high - molecular - mass , nondialyzable material ( ndm ) can inhibit adhesion of numerous species of bacteria and prevents bacterial coaggregation of bacterial pairs .
bacterial coaggregation leads to plaque formation leading to biofilm development on surfaces of oral cavity . in the present study
, we evaluated the effect of low concentrations of ndm on streptococcus gordonii metabolic activity and biofilm formation on restorative dental surfaces .
we found that the ndm selectively inhibited metabolic activity of s. gordonii , without affecting bacterial viability .
inhibiting the metabolic activity of bacteria in biofilm may benefit the health of the oral cavity . |
alternative medicine refer to a broad set of health care practices that are not part of that country s own tradition or conventional medicine and are not fully integrated into the dominant health - care system . according to who they are used interchangeably with traditional medicine in some countries .
homeopathic medicine , herbal medicine and acupuncture are the most common , albeit not the unique , cam forms in european countries .
homeopathy is a method of medical practice that aims to improve the level of health of an organism through the administration of medicinal products selected individually according to the principle of similarity ( see text ) . since homeopathy is strictly individualized and takes into account the physical , emotional , mental , constitutional , biographical and environmental state , it is a medicine of the whole person .
the term homeopathy comes from the greek ( omoios = similar , pathos = suffering ) .
integrative medicine is not simply the combination of conventional medicine with complementary and alternative medicine .
the consortium of academic health centers for integrative medicine defines it as the practice of medicine that reaffirms the importance of the relationship between practitioner and patient , focuses on the whole person , is informed by evidence , and makes use of all appropriate therapeutic approaches , healthcare professionals and disciplines to achieve optimal health and healing .
alternative medicine refer to a broad set of health care practices that are not part of that country s own tradition or conventional medicine and are not fully integrated into the dominant health - care system . according to who they are used interchangeably with traditional medicine in some countries .
homeopathic medicine , herbal medicine and acupuncture are the most common , albeit not the unique , cam forms in european countries .
homeopathy is a method of medical practice that aims to improve the level of health of an organism through the administration of medicinal products selected individually according to the principle of similarity ( see text ) . since homeopathy is strictly individualized and takes into account the physical , emotional , mental , constitutional , biographical and environmental state , it is a medicine of the whole person .
the term homeopathy comes from the greek ( omoios = similar , pathos = suffering ) .
integrative medicine is not simply the combination of conventional medicine with complementary and alternative medicine .
the consortium of academic health centers for integrative medicine defines it as the practice of medicine that reaffirms the importance of the relationship between practitioner and patient , focuses on the whole person , is informed by evidence , and makes use of all appropriate therapeutic approaches , healthcare professionals and disciplines to achieve optimal health and healing . | some physicians have incorporated some forms of complementary and alternative medicine ( cam ) or related medicinal products in their clinical practices , suggesting that an unconventional treatment approach might be seen as an integration rather than as an alternative to standard medical practice . among the various cams ,
homeopathy enjoys growing popularity with the lay population , but it is not acknowledged by academia or included in medical guidelines . the major problem is to establish the effectiveness of this clinical approach using the strict criteria of evidence - based medicine .
this issue of the journal of medicine and the person collects contributions from some of the most prestigious centers and research groups working in the field of homeopathy and integrative medicine .
these contributions are not specialized information but are of general interest , focusing on this discipline as one of the emerging fields of personalized medical treatment . |
samples : serum samples were taken from 337 pet rabbits presented to 34
veterinary facilities in different 20 prefectures in japan .
healthy rabbits were brought to the veterinary clinics for either general
health checks or other disease conditions . among the rabbits ,
the rest showed variable clinical
conditions , such as embraced tumors or disorders of the digestive , respiratory or urinary
systems .
the examined rabbits
ranged in age from 3 months to 10 years old , and the average age was 3.4 years .
the breeds
of the tested rabbits and their numbers were as following : 16 holland lop , 28 netherland
dwarf , 3 lion , 9 angora , 3 rex , 2 dutch , 1 american fuzzy lop , 1 jersey wooly , 272 others
and 2 unknown rabbits .
all rabbit serum samples referred to our laboratory had been tested
to assess the seroprevalences of t. gondii and n. caninum
antibodies .
parasites : t. gondii ( rh and me49
strains ) and n. caninum ( nc1 strain ) tachyzoites were maintained in human
foreskin fibroblast ( hff ) cells cultured in dulbecco s modified eagle s medium ( dmem , gibco ,
grand island , ne , u.s.a . ) supplemented with 7.5% heat - inactivated fetal bovine serum ( fbs ) .
for purification and lysis of tachyzoites , the infected cells were washed with cold
phosphate - buffered saline ( pbs ) .
cell pellets were resuspended in medium and passed through
a 27-gauge needle and then through a 5.0 m - pore filter ( millipore ,
bedford , ma , u.s.a . ) .
after centrifugation at 2,000 g for 5 min at 4c ,
the pellet was resuspended with ripa buffer ( 50 mm tris - hcl , ph 7.5 , 150 mm nacl , 1 mm edta ,
0.25 mm sodium deoxycholate , 0.1% triton x-100 and 1% nonidet p-40 ) . the tachyzoite lysates
were recovered after centrifugation at 2,000 g for 5 min .
recombinant protein production : recombinant proteins of t.
gondii surface antigen 2 ( tgsag2 ) and n.
caninum surface antigen 1 ( ncsag1 ) were generated according to
previous reports [ 20 , 21 ] .
generation of polyclonal anti - tgsag2 antisera : polyclonal
anti - tgsag2 antisera were produced in balb / c mice .
immunization of mice
was performed as described previously . in brief ,
mice were immunized subcutaneously at three different inoculation sites with 10
g of recombinant tgsag2 protein emulsified with an
equal volume of freund s complete adjuvant .
two weeks later , mice were immunized with the
same dose of antigen emulsified with freund s incomplete adjuvant .
another dose of the
antigen with the incomplete adjuvant was given on day 28 .
seven days later , mice were
sacrificed under isoflurane anesthesia , and blood was collected for serum extraction .
the experiments were
conducted according to the guidelines for the care and use of laboratory animals issued by
obihiro university of agriculture and veterinary medicine .
elisa : one hundred microliters of antigen ( either tachyzoites lysate or
recombinant protein ) in a coating buffer ( 50 mm carbonate , ph 9.6 ) at concentration of 2
g / ml were coated onto elisa plates ( nunc , roskilde ,
denmark ) and incubated overnight at 4c .
plates were washed 5 times with pbs containing
0.05% tween 20 ( pbs - t ) and blocked with pbs containing 1% skimmed milk ( pbs - sm ) for 1 hr at
room temperature ( rt ) .
after washing with pbs - t , 100 l of rabbit serum
diluted at 1:500 with pbs - sm were added to duplicate wells and incubated for 1 hr at rt .
after washing 5 times with pbs - t
, the plates were incubated with horseradish peroxidase
( hrp)-conjugated anti - rabbit igg ( ge healthcare uk limited , buckinghamshire , u.k . ) or igm
( southernbiotech , birmingham , al , u.s.a . ) diluted at 1:2,000 with pbs - sm at rt for 1 hr .
plates were further washed 5 times before the substrate solution [ 0.1 m citrate buffer , ph
4 , 0.003% h2o2 and 0.3 mg / ml 2 , 2-azino - bis
( 3-ethylbenzothiazoline-6-sulfonic acid ) ; sigma - aldrich ] was added to each well in 100
l aliquots .
the absorbance at 415 nm after 30 min of incubation at rt
was measured using an elisa reader ( corona microplate reader mtp-120 ; corona , tokyo , japan ) .
the cutoff point was determined as the average value of seronegative rabbit samples ( n=24 )
plus 3 sd .
latex agglutination test : the lat was performed according to the kit
manufacturer s instructions ( toxocheck - mt , eiken chemical , tokyo , japan ) .
samples were
considered positive when agglutination was observed at a dilution of 1:32 or greater .
western blot analysis : lysates of t. gondii ( rh strain )
and n. caninum were dissolved in sodium dodecyl sulfate polyacrylamide gel
electrophoresis ( sds - page ) sample buffer ( 62.5 mm tris - hcl , ph 6.8 , 2% sds , 140 mm
2-mercaptoethanol , 10% glycerol and 0.02% bromophenol blue ) heated at 95c for 5 min and
separated on a 12% polyacrylamide gel .
all separated proteins were electrically transferred
onto polyvinylidene fluoride ( pvdf ) membranes ( immobilon - p transfer membrane , millipore ,
billerica , ma , u.s.a . ) using a western blot apparatus ( horizeblot type ae-6677 , atto
bioscience & biotechnology , tokyo , japan ) . after blocking for 1 hr in pbs - sm
, membranes
were probed with the rabbit serum diluted at 1:500 with pbs - sm for 1 hr . the membranes were
washed 3 times for 10 min with pbs - t and then probed with hrp - conjugated anti - rabbit igg
diluted at 1:2,000 in pbs - sm for 1 hr at rt . after washing with pbs - t
, the membranes were
immersed in the detection solution ( 0.1 m tris , ph 8.5 , 1.25 mm luminol acid , 0.2 mm
coumaric acid and 0.075% h2o2 ) and exposed to x ray film . prestained
molecular mass standards ( genedirex , biospeed , las vegas , nv , u.s.a . ) were used .
immunoprecipitation : one hundred micrograms of t. gondii
lysate were mixed with 2 l of either seronegative or seropositive rabbit
serum and kept on ice for 1 hr . twenty microliters of protein g sepharose ( ge healthcare ,
uppsala , sweden ) were added to each sample and rotated at 4c for 1 hr . beads were washed 5
times with pbs , and then protein samples were separated on 12% polyacrylamide gel and
electrically transferred onto pvdf membranes .
, long beach , ca ,
u.s.a . ) or mouse polyclonal anti - sag2 antibodies were used to probe the membranes at a
dilution of 1:500 in pbs - sm . after washing 3 times with pbs - t
, membranes were probed with
hrp - conjugated anti - mouse igg diluted at 1:2,000 in pbs - sm incubated for 1 hr at rt .
indirect immunofluorescence assay ( ifa ) : the indirect immunofluorescence
was performed as described previously with some
modifications .
glass coverslips with confluent monolayers of hff cells were infected with
t. gondii ( me49 strain ) and incubated in a co2 incubator for
24 hr at 37c . after washing 3 times with pbs ,
cells were fixed and permeabilized with 4%
formaldehyde and 0.2% triton x-100 in pbs ( ph 7.2 ) for 15 min .
cells were then washed 3
times with pbs and blocked for 30 min in 3% bsa / pbs .
coverslips were incubated with rabbit
serum samples diluted at 1:100 , 1:500 and 1:1,000 in 3% bsa / pbs .
after washing 3 times with pbs , coverslips were incubated with alexa fluor 594-conjugated
goat anti - rabbit igg and alexa fluor-488 conjugated goat anti - mouse igg ( invitrogen ) diluted
at 1:1,000 in 3% bsa / pbs .
mowiol ( 4.8 g mowiol , 12 g glycerol in 50 mm tris hcl , ph 8.5 ) was used as a mounting
medium .
samples were examined on a leica tcs nt confocal laser scanning microscope ( leica
microsystems , wetzlar , germany ) using the appropriate settings .
green and red fluorescence
and differential interference contrast ( dic ) images were recorded using the leica powerscan
software .
statistical analysis : the results of the different serological tests were
estimated by the percentage of agreement , the sensitivity and specificity and the kappa
values using ( http://vassarstats.net/ ) .
detection of specific antibodies raised by natural infections of t. gondii
or n. caninum : to assess the presence of toxoplasmosis or neosporosis in
pet rabbits , igg and igm elisa tests were carried out to screen all rabbit sera ( 337 ) using
tachyzoites lysate of t. gondii or n. caninum and
tgsag2 or ncsag1 recombinant protein as antigens .
seropositive samples were further examined by lat , western blotting and ifa . for t.
gondii , igg - elisa identified 9 positive sera according to the calculated cutoff
value ( 0.6 ) , 6 samples scored an od value below 1 , 1 sample scored an od value between 1 and
2 , and the remaining 2 sera had od values of more than 2 .
only one sample was found to be
positive in the igm elisa ( table 1table 1.results of the serological tests performed on sera from pet rabbitstested parasiteno .
of rabbitselisa iggelisa igmlatwestern blotifaabs ( od)abs ( od)0.6112 20.65112 2 t . gondii337612001333n .
all rabbit sera that showed positive reactivity with lysate antigen were
also reactive with the recombinant protein .
sample that scored od values below 0.6 ( 328 )
were considered negative by elisa test .
d ) ifa : indirect immunofluorescence antibody assay . to include a wide range of samples , the latex agglutination test was applied to all samples
that showed an od value above 0.3 ( 35/337 ) using the recommended dilutions .
agglutination
was observed only in 3 samples ( od value above 1 ) at dilutions of 1:2,048 and 1:128 ( table 1 ) , while the remaining 32 samples did not
show any agglutination and were therefore considered negative . in addition , all samples that
showed od values above the elisa cutoff point ( 0.6 ) were analyzed by western blotting and
ifa .
both tests resulted in 3 positive sera that had shown positivity also in the lat ( fig .
a ) lanes 1 , 2 and 3 represent seropositive rabbit samples for
t. gondii .
the analysis
was performed by using an equal volume of purified t. gondii
tachyzoite lysate of the me49 strain as the antigen .
b ) specific bands of sag1 can be
observed at 30 kda ( left panel ) , and those of sag2 can be observed at 22 kda ( right
panel ) .
lane 2 shows mouse anti - sag1 monoclonal ( left panel ) and mouse
anti - sag2 polyclonal ( right panel ) antibodies , respectively .
2.immunoprecipitates of either seronegative ( lane 2 ) or seropositive ( lane 3 ) samples
reacted with either the mouse anti - sag1 monoclonal antibody ( left panel ) or mouse
anti - sag2 polyclonal antibody ( right panel ) .
the black arrow represents the specific band of sag1 ( left ) and sag2 ( right )
precipitated only by the positive sera .
arrowheads represent igg heavy ( upper ) and
light ( lower ) chains , respectively .
rows 1 , 2 and 3 represent t. gondii - seropositive
samples ( left panel , red ) .
the mouse
anti - t . gondii imc1 antibody ( green ) was used for
counterstaining .
we found that the only serum samples that scored elisa od values higher
than 1.0 were detectable by lat , western blot and ifa ( 3/337 ) .
a ) lanes 1 , 2 and 3 represent seropositive rabbit samples for
t. gondii .
the analysis
was performed by using an equal volume of purified t. gondii
tachyzoite lysate of the me49 strain as the antigen .
b ) specific bands of sag1 can be
observed at 30 kda ( left panel ) , and those of sag2 can be observed at 22 kda ( right
panel ) . lane 1 shows seropositive rabbit sample naturally infected with t.
gondii .
lane 2 shows mouse anti - sag1 monoclonal ( left panel ) and mouse
anti - sag2 polyclonal ( right panel ) antibodies , respectively .
immunoprecipitates of either seronegative ( lane 2 ) or seropositive ( lane 3 ) samples
reacted with either the mouse anti - sag1 monoclonal antibody ( left panel ) or mouse
anti - sag2 polyclonal antibody ( right panel ) .
the black arrow represents the specific band of sag1 ( left ) and sag2 ( right )
precipitated only by the positive sera .
arrowheads represent igg heavy ( upper ) and
light ( lower ) chains , respectively .
rows 1 , 2 and 3 represent t. gondii - seropositive
samples ( left panel , red ) .
gondii imc1 antibody ( green ) was used for
counterstaining . on the other hand , when rabbit samples tested for the presence of n.
caninum antibodies , although igg - elisa showed 10 cases over the cutoff value
( 0.6 ) , 9 of them had od values below 1 , and one case had an od value between 1 and 2 ( table 1 ) .
no specific band or reactive staining was
observed for n. caninum in both western blot and the ifa .
detection of specific antigens in sera of naturally infected rabbits with t.
gondii : in western blotting , positive sera generated multiple distinct bands
between 14 and 130 kda ( fig .
certain protein
bands were noticeably common in all positive sera at 30 and 22 kda , the same molecular
weights as the sag1 and sag2 antigens . to confirm this ,
western blot analyses were
performed to compare the sizes of identified bands in the sera of infected rabbits with the
mouse anti - sag1 monoclonal and anti - sag2 polyclonal antibodies .
further
confirmation was performed by immunoprecipitation using seropositive and seronegative
t. gondii rabbit serum samples that were tested previously by elisa and
lat .
the staining pattern in the ifa of positive
sera showed a strong signal on the surface of the parasites ( fig .
3 ) . the results of elisa , ifa and western blot were statistically calculated to obtain the
specificity and sensitivity and the kappa value to assess their ability to detect t.
gondii antibodies in rabbit sera in comparison with the lat as a reference test
( table 2table 2.specificity and sensitivity of the elisa , ifa and western blot in detecting
t. gondii - specific antibodies in rabbit sera compared with the
results in the reference lat testparametervalue of performed serological tests compared with
the latelisaifawestern blotsensitivity ( % ) 100 ( 9/3)100 ( 3/3)100 ( 3/3)specificity ( % ) 98 ( 328/334)100 ( 334/334)100 ( 334/334)concordance ( % ) 98.2100100kappa value0.4911 ) .
rabbits are especially popular as pets in japan ; dogs and cats may be the most frequent
household animals , but the popularity of rabbits is increasing . therefore , their health
status is also important to consider .
moreover , questions often arise concerning the hazards
in the surrounding environments for both pet rabbits and the people around them .
t.
gondii and n. caninum are protozoan parasites known to infect
humans and animals [ 12 , 37 ] .
human beings can be infected with t. gondii and
possibly with n. caninum [ 16 , 28 , 38 ] by
ingestion , the major route , of either oocysts in contaminated food and water or meat
containing the parasite cysts .
the ability to distinguish whether the original infection was
by tissue cyst or oocysts and discover whether the outcomes are different is a great concern
nowadays .
because cats and dogs are so abundant in the environment and indeed are
increasing , contamination of the human environment with oocysts will continue to increase ,
and exposure will be unavoidable . furthermore , recent studies reported that the majority of
congenital infections and postnatal acute infections in the united states originated from
oocysts , and this will increase the concern about
the severity and outcomes of infections primarily induced by oocysts . because the oocyst is
the only source of infection in rabbits , pet rabbits are considered good indicators for
assessment of the oocyst contamination in the human environment .
therefore , in view of the
worldwide importance of these parasites and their infective stages , the seroprevalences of
both t. gondii and n. caninum in pet rabbits were
estimated in this study .
serodiagnostic tools are important methods for detecting parasitic infections ; one of these
tools is the elisa using tachyzoite - derived antigens or generated recombinant proteins
[ 7 , 19 ] . in
this study ,
igg and igm elisas were used to analyze all rabbit sera by using the whole
tachyzoites - derived antigen of either t. gondii or n.
caninum .
the use of such antigens may produce false positive results due to
cross - reaction with other closely related parasites or unknown reasons . to validate our results , elisas based on the recombinant
proteins tgsag2 and ncsag1
similar
data were obtained , indicating that in this case , the antigenicity of the recombinant
proteins did not show any difference compared with the lysates .
therefore , it was necessary
to perform other tests , such as the lat , western blotting and ifa .
these three tests
identified only three positive sera that previously showed od values > 1 in the igg
elisa . also , we identified sag1 and sag2 as major antigens detected by the positive rabbit sera .
these findings were confirmed by western blotting and immunoprecipitation , as shown in the
results .
although the tgsag2 recombinant proteins have already been
established as specific antigens for diagnosis of t. gondii via elisa
, our results confirmed that sag1 and sag2 can
be used as useful target antigens for specific testing of rabbit sera to detect t.
gondii infection by other serological methods .
concerning n.
caninum antibodies in rabbit sera , although elisa detected 10/337 positive sera ,
western blot and the ifa did not reveal any positivity .
therefore , it could be plausibly accepted that toxoplasmosis was not common and that
neosporosis was not prevalent among pet rabbits in japan . additionally , in this study , we
compared the positivity of the results between the elisa and the other serological methods .
the rabbit sera tested by elisa showed 2.67% positivity for t. gondii and
2.96% for n. caninum .
however , the seropositivity was only 0.89% for
t. gondii and 0% for n. caninum using western blotting
or the ifa .
therefore , it is reasonable to accept that using more than one method in
serodiagnosis is recommended for accurate investigation .
since we used different tests here for serological detection , it was important to emphasize
the capability of each test .
therefore , the results of elisa , ifa and western blot were
statistically compared with those of the lat as a reference test for detection of t.
gondii antibodies ( table 2 ) . among
the tests used , the elisa showed a higher positive rate of detection of t.
gondii - specific igg antibodies than the ifa and western blot ( table 1 ) . although elisa achieved very high sensitivity in
detection ( 100% ) , the six negative samples that were detectable by elisa and not by lat
resulted in 98% specificity for elisa .
the kappa value of 0.49 could be interpreted as a
moderate level of agreement . while , the sensitivity and specificity of the ifa and western
blot were both 100% when the lat was used as a reference test associated with high agreement
indicated by maximum kappa value ( 1 ) as summarized in table 2 .
therefore , we could conclude from this analysis that the lat , ifa and
western blot had similar capabilities for detection of t. gondii antibodies
in rabbit sera .
however , the availability of these tests for rapid screening is not similar ,
because the ifa and western blotting are more sophisticated techniques that might not be
applicable to large number of samples or as a field test .
moreover , both tests were unable
to detect the antibody titer , while the lat is convenient , more applicable and provides the
advantage of both qualitative and quantitative assessment of the infection status .
concerning n. caninum , the results of elisa and western blot showed 97% and
100% specificity , respectively , when the ifa was used as a standard test , while the
sensitivity could not be calculated ( data not shown ) . in reference to the seroprevalence of t. gondii in rabbits in different
countries , tested domestic rabbits reared on farms and in backyards and pet shops in
northern mexico showed that antibodies to t. gondii were found in 16.3% of
429 rabbits .
in korea , a similar type of rabbits
was surveyed from breeding farms , and 10.6% of them showed t.
gondii - specific igg antibodies . in
egypt ,
high seroprevalences of t. gondii were also detected , 37.5% and
11.34% , in locally bred and commercial rabbits . in
the studies described above
, the high seroprevalences of t. gondii
infection could be correlated to factors , such as rearing outdoors or in a pet shop , feeding
of possibly contaminated vegetables and cohabitation with cats or dogs . additionally , considerably high prevalences
for example , in china , the seroprevalence of t.
gondii antibodies was 23.4% in free domestic rabbits from a rural area .
also , a study in spain reported 14.2% t. gondii positivity among 456 wild
rabbits captured or hunted from forests . on the other hand , a study on wild cottontail
rabbits in northern italy mentioned low prevalences of both t. gondii and
n. caninum infections , 2.08% and 2.78% , respectively . in the present study ,
the seroprevalence of t. gondii was 0.89% in pet
rabbits of japan . referring to the previously mentioned studies performed on domestic or
wild rabbits ,
results can not be directly compared , although the susceptibilities were quite
similar because the rabbits were from the same species .
it is important to consider the
surrounding environment and rearing conditions of the examined rabbits in this study . in
general ,
wild rabbits or even domestic rabbits that are reared or have access to the
outdoors might be more susceptible to such parasitic infections than pet rabbits confined
indoors , bearing in mind the risk of direct contact with the contaminated environment . in
japan ,
rabbits are considered pets and are mostly kept indoors or in cages as in a zoo .
although they are sometimes given time to range freely outside ,
this is different to some
extent from the case of domestic farm rabbits reared for meat purposes and to a great extent
from wild rabbits .
given the unavailability of data concerning recovery of oocysts of the
aforementioned parasites from the human environment , little is known about their potential
hazards . in conclusion , the present study indicates that there is probably a low level of oocyst
contamination in the human environment according to estimation of the prevalences of
toxoplasmosis and neosporosis in pet rabbits .
we concluded that t. gondii
was not common among healthy pet rabbits and those suffering from neurological disorders .
furthermore , as far as could be checked , there were no apparent n. caninum
infections in pet rabbits .
assessment of contamination of the surrounding environment with
oocysts of the parasites and its implications for humans and other animals requires further
attention and investigation . | abstractthe potential contamination of toxoplasma gondii and
neospora caninum oocysts in the human environment is a concern from the
public health viewpoint .
however , estimation of their seroprevalences in humans can not be
performed in a manner that distinguishes between oocysts and tissue cysts as a source of
infection .
rabbits are considered popular pet animals in japan that can acquire natural
infections by the aforementioned parasites only through the ingestion of oocysts .
therefore , this study was conducted to estimate the seroprevalences of t.
gondii and n. caninum in pet rabbits in japan as an indicator
of the possible oocyst contamination in the environment surrounding human beings .
serum
samples of 337 rabbits were examined by different serological methods .
enzyme - linked
immunosorbent assays were performed to measure the titer of igg and igm antibodies .
samples revealed to be seropositive by elisa were further analyzed by a latex
agglutination test , western blotting and an indirect immunofluorescence assay .
the rates
of seropositivity for t. gondii were 0.89% ( 3/337 ) and 0.29% ( 1/337 ) in
igg and igm elisa , respectively .
sag1 and sag2 were detected as major antigens by the
positive rabbit sera in western blotting associated with strong staining observed by ifa
in t. gondii tachyzoites .
regarding n. caninum , none of
the serum samples showed a specific reaction in both western blotting and the ifa .
the
results of this study indicate low seroprevalences of toxoplasmosis and neosporosis in pet
rabbits in japan , suggesting low oocyst contamination in the human environment . |
to investigate the role of the chinese herbal medicine xianhuayin on the reversal of 7,12-dimethylbenz[a]anthracene ( dmba)-induced premalignant mucosal lesions in the oral buccal pouch of golden hamsters .
the animals were randomly divided into a non - diseased control group ( n=5 ) and an experimental group including 50 animals in which the buccal mucosa had been painted with dmba ( 0.5% in acetone ) to generate an oral mucosa premalignant lesion .
animals in the experimental group were further divided into xianhuayin - treated group ( n=30 ) , untreated premalignant lesion group ( n=10 ) and normal saline ( ns)-treated group ( n=10 ) .
the cheek ( buccal ) pouch mucosa of the golden hamsters in each group was observed with light and electron microscopy eight weeks after intragastric administration with ns or xianhuayin .
in the non - diseased control group , the buccal mucosa was keratinized and stratified squamous epithelium under a light microscope . in the untreated premalignant lesion group ,
scanning electronic microscopic ( sem ) analysis showed that surface of the cells exhibited honeycomb structures in the hamster of untreated - group .
the cells were morphologically irregular , overlapped and loosened in the untreated premalignant lesion group .
transmission electronic microscopic ( tem ) analysis showed that buccal mucosal epithelial cells were morphologically regular in the non - diseased control group .
desmosomes and tonofibrils were reduced and the nucleus was morphologically irregular in the untreated premalignant lesion group . in the xianhuayin - treated group ,
the widening intercellular gap was gradually reduced , desmosomes and the cells becoming morphologically regular .
no significant difference was observed between the hamsters in ns - treated group and those in the untreated premalignant lesion group .
xianhuayin is effective in the reversal of dmba - induced premalignant lesions in the buccal pouch of golden hamsters . | aimto investigate the role of the chinese herbal medicine xianhuayin on the reversal of 7,12-dimethylbenz[a]anthracene ( dmba)-induced premalignant mucosal lesions in the oral buccal pouch of golden hamsters.methodologythe animals were randomly divided into a non - diseased control group ( n=5 ) and an experimental group including 50 animals in which the buccal mucosa had been painted with dmba ( 0.5% in acetone ) to generate an oral mucosa premalignant lesion .
animals in the experimental group were further divided into xianhuayin - treated group ( n=30 ) , untreated premalignant lesion group ( n=10 ) and normal saline ( ns)-treated group ( n=10 ) .
the cheek ( buccal ) pouch mucosa of the golden hamsters in each group was observed with light and electron microscopy eight weeks after intragastric administration with ns or xianhuayin.resultsin the non - diseased control group , the buccal mucosa was keratinized and stratified squamous epithelium under a light microscope . in the untreated premalignant lesion group ,
variable degrees of epithelial dysplasia was observed .
the irregular epithelial mucosa gradually became distinct in the xianhuayin - treated group .
scanning electronic microscopic ( sem ) analysis showed that surface of the cells exhibited honeycomb structures in the hamster of untreated - group .
the cells were morphologically irregular , overlapped and loosened in the untreated premalignant lesion group .
most of the cell surface exhibited honeycomb structure in the xianhuayin - treated group .
transmission electronic microscopic ( tem ) analysis showed that buccal mucosal epithelial cells were morphologically regular in the non - diseased control group .
desmosomes and tonofibrils were reduced and the nucleus was morphologically irregular in the untreated premalignant lesion group . in the xianhuayin - treated group ,
the widening intercellular gap was gradually reduced , desmosomes and the cells becoming morphologically regular .
no significant difference was observed between the hamsters in ns - treated group and those in the untreated premalignant lesion group .
significant therapeutic efficacy was observed in the group receiving xianhuayin.conclusionxianhuayin is effective in the reversal of dmba - induced premalignant lesions in the buccal pouch of golden hamsters . |
streptococcus pneumoniae ( spn ) may cause a wide spectrum of diseases ranging from otitis and pneumonia to invasive forms such as meningitis or sepsis . among pneumococcal diseases , pneumonia has the higher clinical burden in terms of morbidity , mortality and hospitalization rate , with heavy implications for worldwide health systems . in particular , higher incidence and mortality rates of pneumococcal community - acquired pneumonia ( cap ) cases , with related costs , are registered among elderly .
incidence values vary among different countries and surveys , but generally mortality rate increases with age and comorbidities . in the future
, this will lead to an inevitable increase of hospitalizations , with an economic burden that should not be underestimated .
therefore , pneumococcal vaccination is extensively recommended for subjects aged 65 y and for those aged 5064 y at high risk ( hr ) for specific health conditions , such as diabetes , cardiovascular diseases , nephropathies , hypertension and chronic obstructive pulmonary disease ( copd ) . in italy ,
the ministry of health recommends the use of conjugated 13-valent vaccine ( pcv13 ) for children aged < 2 y , while the immunization with 23-valent polysaccharide vaccine ( ppv23 ) is recommended for adults aged 65 y and for hr subjects aged 2 y. however ,
the levels of immunization coverage among adults are so far insufficient , and probably this depends on the doubts about polysaccharide vaccine efficacy .
in fact , clinical studies showed that this formulation is unable to induce an adequate and durable immune response , especially in hr individuals and against non - invasive pneumococcal diseases .
the 13-valent conjugate vaccine , recently indicated for the immunization of all the ages , has showed adequate safety and immunogenicity levels and seems to induce a durable protection against pneumococcal diseases , both invasive and not .
several studies suggest that immunological properties of pcv are higher than those of ppv23 , while safety and tolerability are comparable .
this study aimed to realize an economic evaluation about the immunization with pcv13 in the adult population in campania region , south italy . for this purpose we performed , considering a period of 5 y , a budget impact analysis ( bia ) and a cost - effectiveness analysis which considered 2 immunization targeted cohorts .
figure 1 shows the pneumococcal cap cases expected in the first group ( hr subjects aged 5079 ) with and without vaccination .
figure 1 . expected cases with and without vaccination in the first targeted group ( hr subjects aged 5079 ) .
costs needed for the vaccination of the first targeted cohort in 5 y of follow - up are reported in table 1 .
the total costs per year with or without immunization of the first cohort are reported in table 2 . during the first year , the implementation of the vaccination program requires more than 2-fold the resources needed for the treatment of pneumococcal cap cases expected without vaccination .
the bia for the base case scenario confirms the great initial expense and the following savings , up to 29 005 660 after 5 y ( table 3 ) .
table 4 considers savings and avoided cases achievable in 5 y with vaccination . for the first targeted group ,
total pneumococcal cap cases expected with a vaccination program were assumed to be 509 , while those expected without vaccination were estimated to be 4083 ( with a reduction of 3574 cases ) .
therefore , the final savings per pneumococcal cap case is equal to 8116 , what relationship between avoided costs and avoided cases .
the sensitivity analysis was performed by varying the effectiveness of the vaccine ( + /10% ) : estimated savings after 5 y could be 34 662 134 in the first case , and 23 402 745 in the worst scenario ( data not shown ) . as for the second targeted group ( hr subjects aged 5064 plus 65 y - old subjects ) ,
expected cases with and without vaccination in the second targeted group ( hr individuals aged 5064 + those aged 65 y ) .
table 5 shows vaccination costs in 5 y. the costs per year with or without immunization of the second cohort are reported in table 6 . in this case also , at the first year the costs with vaccination seem to be higher than those for the treatment of pneumococcal cap cases in the second scenario ; however , even from the second year it s possible to observe notable savings . *
bia showed at the end of the follow - up achievable savings equal to 10 006 017 ( table 7 ) .
the number of expected cases ( 2694 ) among the second targeted cohort without vaccination could be reduced by vaccination to 337 . with this strategy ,
sensitivity analysis showed savings equal to 10 879 772 when considering a 10% increase in the effectiveness of the vaccine and equal to 9 132 263 with a 10% decrease ( data not shown ) .
the new paradigm of evidence based medicine for decision - making has widely grown in recent years , making available to public health care policies and to professionals and managers as well tools to assess the clinical and welfare rationality of public choices .
the evidence - based health care approach currently support most of the control activities and decision - makers choices , thus becoming a real asset in all health care organization levels : national first , and then regional .
this study represents an example of how these indicators could be useful to manage and control diseases with a high burden .
although pneumococcal diseases are assumed to be not always correctly identified and/or notified , they represent one of the main cause of morbidity and mortality . in order to reduce their burden , and
the great number of risk factors for pneumococcal diseases and their interactions highlight the importance of immunoprophylaxis .
therefore , contrarily to other analyses , focused on age - based vaccination strategies , this study considered some risk groups as targeted cohorts for adult vaccination .
the inclusion of these groups followed indications of the italian ministry of health , which suggests the consideration of risk conditions for pneumococcal immunization in adults .
multimorbidity patterns were difficult to apprise , due to the absence of appropriate data collection systems ; this would produce artificial estimates and information far from adhering to reality .
the economic evaluation of pneumococcal vaccine for adult groups represents an essential instrument to support health policies .
in fact , due to the cost restriction , stakeholders should know the value for money of a new immunization strategy , but also its budget impact as in the short as in the long period .
as described above , this impact comes from the difference between cost of vaccination program and savings achievable through the vaccine - related reduction of cases .
the pharmacoeconomics evaluation performed in this study showed that both hypothesized immunization strategies could produce savings .
results showed that the vaccination of hr subjects of 5079 y could allow about 28 million euro of savings .
however , this strategy requires a high investment in a short period . on the contrary
it has to be noted that the present analysis did not consider the serotype coverage , which could be considered a limitation of the procedure .
however , the analysis considered only direct costs using the perspective of the national health service . therefore , the conservative nature of this evaluation disregards further implications that could be advantageous for the local health system .
moreover , the analysis takes into account only the pneumococcal pneumonia cases and does not include the other vaccine - preventable pneumococcal diseases , and it referred only to hospitalized cases however , the impact of vaccination may be wider , both clinically and economically , if non - hospitalized pneumococcal pneumonia cases are included .
furthermore , the infection in high - risk subjects is likely more severe than considered and , consequently , the economic impact of vaccination is more extensive .
lastly , vaccination advantages do not interest only immunized subjects but also the community , due to the herd immunity effects . also regarding this aspect ,
the present analysis is conservative , because further savings deriving from the increase of targeted cohorts were not considered . in the health care system
, policy - makers choices basically depend on 2 elements which are concatenated together . on the one hand , there are the financial resources available ; on the other , the epidemiological context of reference that will decree the priorities on the allocation of resources .
findings of this study support the vaccination for hr adults , as recommended by ema .
our findings could help to choice between the 2 proposed hypotheses , in order to better address economical resources .
this economic evaluation was developed to analyze for a 5-y period the impact of an adult pneumococcal vaccination program in the campania region .
the model considered 2 vaccination strategies : at first , the group of hr subjects aged 5079 ( about 1 million of individuals in the region ) , and later hr individuals aged 5064 , together with all those aged 65 y ( about 400 000 subjects ) .
hr subjects were considered those with chronic diseases such as diabetes , hypertension , nephropathies , copd and cardiovascular diseases .
the analyses were performed on the resident population in campania on january 1 , 2009 , as reported by the national institute for statistics ( istituto nazionale di statistica , istat ) , without considering sex and origin . on the base of the average national coverage for the last influenza immunization program in italy in the elderly , vaccination coverage of the targeted cohorts
the clinical outcome of the analysis were hospitalized pneumococcal cap cases in adult population ( 5079 y ) .
data were obtained from the hospital discharge forms ( scheda di dimissione ospedaliera , sdo ) of respiratory departments which participate to the training regional network . during the years 20102011 , 18 965 cap cases were reported on these sdos .
assuming that the overall rate of cap due to spn is about 40% , the number of pneumococcal cap per year among reported cases was estimated to be 3793 . on the basis of previous studies , the vaccine efficacy against pneumococcal pneumonia
was assumed to be 87.5% , then this value was used to calculate the number of avoided cases for each vaccination strategy .
expected cases were corrected for the global mortality rate , as obtained from 2010 istat data .
the economic model was based on the difference between the costs sustained with ( vaccine plus treatment for expected cases ) and without ( only expected cases ) a vaccination program .
as regard the first vaccination strategy , all the subjects at risk aged 5079 y were considered for immunization at first year ; since the second year only 50 y old were included in the vaccination program . as for the second strategy , individuals at risk aged 5064 y and those aged 65 y were considered for immunization at first year , while 50 y old and 65 y old were included for subsequent years .
the cost of the vaccine was 42.5 euro per dose ; the cost of a cap case due to spn was assumed to be the average of costs for complicated and non - complicated pneumonia cases , equal to 3809 .
costs were updated to a rate of 3% . to test the strength of results ,
a sensitivity analysis was applied by considering a + / 10% variation in the vaccine efficacy . | pneumococcal pneumonia has a high clinical burden in terms of morbidity , mortality and hospitalization rate , with heavy implications for worldwide health systems .
in particular , higher incidence and mortality rates of community - acquired pneumonia ( cap ) cases , with related costs , are registered among elderly .
this study aimed to an economic evaluation about the immunization with pcv13 in the adult population in campania region , south italy .
for this purpose we performed , considering a period of 5 y , a budget impact analysis ( bia ) and a cost - effectiveness analysis which considered 2 scenarios of immunization compared with lack of immunization for 2 targeted cohorts : first , the high risk subjects aged 5079 y , and second the high risk individuals aged 5064 y , together with all those aged 65 y. regarding the first group , the decrease of pneumonia could give savings equal to 29 005 660 , while the immunization of the second cohort could allow savings equal to 10 006 017.the economic evaluation of pneumococcal vaccine for adult groups represents an essential instrument to support health policies .
this study showed that both hypothesized immunization strategies could produce savings .
obtained results support the use of pneumococcal conjugate vaccine for adults .
this strategy could represent a sustainable and savings - producer health policy . |
extracorporeal membrane oxygenation ( ecmo ) , which can support gas exchange or hemodynamics in patients with severe respiratory or cardiac failure , has demonstrated considerable evolution over the last decade , with a steady rise since 2009 in the number of ecmotreated patients and number of centers providing ecmo support [ 2 , 3 ] . with more adult patients being placed on ecmo support , there is an increased need to understand the complex changes in drug pharmacokinetics and pharmacodynamics that occur with the addition of an ecmo circuit to the management of a critically ill patient .
the relationship between the dose of a drug and the elicited response may be altered in critically ill patients as a result of pharmacokinetic and pharmacodynamic changes .
the use of extracorporeal mechanical support , such as ecmo , can further increase the variability of pharmacokinetic alterations .
therefore , the combination of critical illness and ecmo presents considerable challenges to providing optimal pharmacotherapy .
the ability to anticipate alterations in pharmacokinetics and pharmacodynamics in this patient population is essential for providing an individualized therapeutic plan that maximizes therapeutic benefit while minimizing potential toxicity . despite improvements in extracorporeal technology and resurgence in its use in respiratory and cardiac failure
this chapter summarizes our current understanding of the effects of ecmo on the pharmacokinetics and pharmacodynamics of several drug classes commonly used to manage these critically ill patients .
pharmacokinetics encompasses the absorption , distribution , metabolism and elimination of a drug , ultimately influencing the concentration at the targeted site of action .
in contrast , pharmacodynamics denotes the relationship between the drug concentration and the physiologic and biochemical effects of the drug on the body , including the intensity of therapeutic and adverse effects .
the relationship between pharmacokinetics and pharmacodynamics is influenced by the drug , the underlying disease process , and the presence of extracorporeal factors .
the culmination of all of these factors can lead to considerable and unpredictable alterations in pharmacokinetics .
figure 1 summarizes the effects of serum drug concentrations and the resultant pharmacokinetic changes influenced by critical illness and ecmo.fig .
1changes in pharmacokinetics with critical illness and extracorporeal membrane oxygenation . increased 1acid glycoprotein and decreased albumin concentrations ; mostly affecting hydrophilic drugs
changes in pharmacokinetics with critical illness and extracorporeal membrane oxygenation . increased 1acid glycoprotein and decreased albumin concentrations ; mostly affecting hydrophilic drugs the elimination of drugs from the body is highly dependent on clearance of the drugs from the body and on volume of distribution . the patient s physiology and the specific physiochemical properties of drugs , such as protein binding , hydrophilicity , molecular weight , and degree of ionization at a given physiologic ph , may influence both clearance and volume of distribution .
the presence of ecmo frequently leads to additional alterations , including an increased volume of distribution and either increased or decreased drug clearance .
the liver and the kidneys are the two major organ systems responsible for drug metabolism and elimination , with less significant elimination occurring through the biliary system , gastrointestinal tract and lungs .
critically ill patients often develop organ insufficiency or failure during the course of their illness thereby altering drug elimination rates .
decreased renal blood flow or function will lead to a decreased glomerular filtration rate , affecting drugs that are dependent on this route of elimination ( mainly hydrophilic drugs ) , whereas decreases in hepatic perfusion or function may lead to toxicity through decreased enzymatic activity or decreases in extraction efficiency .
the distribution of drug throughout the body ( volume of distribution ) is largely dependent on the drug s hydrophilicity and its acidionization constant .
drugs that are hydrophilic will have a lower volume of distribution and their concentrations will primarily be influenced by fluidshifts and largevolume fluid resuscitation .
in contrast , lipophilic drugs penetrate into the tissues , leaving lower concentrations in the blood and increasing the apparent volume of distribution .
the ecmo circuit may increase the volume of distribution through either hemodilution or sequestration of drugs [ 5 , 6 ] .
an initial increase in volume of distribution that occurs at the initiation of ecmo from the introduction of priming solutions ( plasma , saline , or albumin ) , primarily affecting hydrophilic drugs , may result in decreased plasma concentrations and , potentially , therapeutic failure of a drug .
this increased volume may also lead to the dilution of plasma proteins , notably albumin , affecting drugs that are highly proteinbound , leading to potential toxicities as a result of an increase in the proportion of the unbound fraction of a drug .
modern extracorporeal circuits typically consist of cannulae , polyvinyl chloride ( pvc ) tubing , a centrifugal pump , and an artificial membrane for gas exchange ( often termed a membrane oxygenator ) . the membrane oxygenator and
pvc tubing comprise a large surface area for potential drug sequestration , which may lead to drug loss over time , particularly for lipophilic drugs [ 5 , 7 , 8 ] .
the composition of the tubing and membrane oxygenator may play a role in the amount of drug that is sequestered .
some studies have observed that both pvc tubing and the membrane oxygenators absorb drugs to a similar extent , whereas others have shown significant differences . this may be explained by both the age of the circuit and the type of pump used [ 812 ] .
albumin and 1acid glycoprotein , both synthesized in the liver , are the two major blood proteins that bind to drugs . in critical illness , albumin concentrations decrease in response to increased vascular permeability , decreased production and increased catabolism ; whereas , 1acid glycoprotein is an acutephase reactant that may increase in response to physiologic stress .
the changes in protein concentrations could affect both the amount of free drug available and the overall volume of distribution .
additionally the deposition of protein on the inner surface of the ecmo tubing may potentially increase the sequestration of drugs that have a high affinity for protein binding .
a recent ex vivo model tested the changes in concentrations over time of highly protein bound drugs .
highly protein bound drugs in this experiment included ceftriaxone , caspofungin and thiopentone , all of which had significantly lower mean drug recoveries at 24 h ( 80 , 56 , and 12% , respectively ) compared to drugs that were not highly protein bound . since lipophilic and highly protein bound drugs are more prone to sequestration in ecmo circuits , an understanding of the physicochemical properties of drugs can assist in determining the relationship between the dose administered and the anticipated blood concentration [ 5 , 15 ] .
the octanolwater partition coefficient or log p , is a common way to report the measure of a drug s lipophilicity .
drugs with high log p values ( around 2.0 ) will have a propensity to be very soluble in organic materials such as the pvc tubing used in the ecmo circuit .
however , to date , there has been no characterization of the drugcircuit interaction beyond 24 h and , as such , little is known regarding the adsorptive capacity of the circuit over longer periods of ecmo support .
table 1 summarizes the effects of critical illness and ecmo on pharmacokinetics of drugs based on degree of lipophilicity of the drug.table 1effect of critical illness and extracorporeal membrane oxygenation ( ecmo ) on pharmacokinetics ( pk ) of drugs , based on degree of lipophilicityhydrophilic drugslipophilic drugsvolume of distribution ( vd)lowhighprimary mode of clearancerenalhepaticlog p
lowhighpotential effect of critical illness on pkincreased vdno change in vdeffect of ecmo on pkincreased vdincreased vdno change in clearanceincreased clearance
log p : octanolwater partition coefficient ( measure of drug s lipophilicity )
effect of critical illness and extracorporeal membrane oxygenation ( ecmo ) on pharmacokinetics ( pk ) of drugs , based on degree of lipophilicity
log p : octanolwater partition coefficient ( measure of drug s lipophilicity )
patients receiving ecmo often require analgesia and sedation to reduce oxygen consumption , facilitate patientventilator synchrony , diminish patient stress and discomfort , and prevent patientinitiated device dislodgement or removal .
however , achieving the desired level of sedation and preventing delirium in the critically ill patient receiving ecmo remains a challenge in icus , owing to paradigm shifts in analgosedation regimens as well as pharmacokinetic alterations of commonly used analgesics and sedatives .
medication selection should be based on the patient s needs , with titration to a predetermined goal in accordance with recently published guidelines .
limited data exist on the most appropriate opioid and sedative medication regimen to use in ecmo patients to achieve the desired level of sedation while minimizing excess sedative exposure .
commonly used intravenous opioids include fentanyl , hydromorphone and morphine , while sedatives often include propofol , dexmedetomidine and benzodiazepines .
the choice of agent used is dependent on the desired physiological endpoint while balancing unwanted adverse effects of each individual agent .
three ex vivo studies using adult ecmo circuits composed of pvc tubing and a hollow polymethylpentene fiber membrane oxygenator demonstrated significant losses of commonly used opioids and sedatives [ 6 , 18 , 19 ] .
one investigation observed as much as a 93% loss in dexmedetomidine concentrations at 24 h .
another study , measuring concentrations of morphine , fentanyl , and midazolam over a 24hour period , demonstrated that the average drug recovery relative to baseline was lower with lipophilic drugs .
only 3% of fentanyl and 13% of midazolam were detectable at 24 h ; however , the ecmo circuit did not substantially alter morphine concentrations , with 103% recovery at 24 h. of interest , in the first hour of ecmo support , up to 70 and 50% of fentanyl and midazolam , respectively , were lost in the circuit .
the most recent ex vivo study using adult ecmo circuits primed with human whole blood observed a recovery of only 30% of baseline propofol concentrations at 30 min and negligible concentrations at 24 h .
additionally , midazolam concentrations were substantially reduced with 54 and 11% of baseline concentrations measured at 30 min and 24 h , respectively .
similar findings have also been observed in neonatal circuits composed of pvc tubing and earlier generation silicone membrane oxygenators .
these investigations observed up to a 68% loss of midazolam and a 98% loss of propofol within 40120 min and up to a 30% reduction in lorazepam concentrations [ 11 , 12 ] .
adult patients receiving ecmo for respiratory failure appear to have increased requirements of analgesia and sedation over time [ 20 , 21 ] .
the first case report demonstrating increased sedation requirements was in a 30yearold man with severe respiratory failure requiring venovenous ecmo as a bridge to lung transplantation .
over 19 days of ecmo support , his requirements of morphine and propofol to maintain deep levels of sedation increased significantly .
a small , singlecenter , retrospective study observed an increase in opioid and sedative requirements over time in 29 consecutive patients requiring ecmo ( 13 venovenous , 16 venoarterial ) where the sedation protocol was to keep patients heavily sedated during the first few days of ecmo followed by daily lightening of sedation when possible .
this study reported an increase in the average daily dose of midazolam and morphine of 18 mg ( p = 0.001 ) and 29 mg ( p = 0.02 ) , respectively .
interestingly , the authors did not find any significant increase in fentanyl requirements over time .
additionally , patients receiving venovenous ecmo had a significantly higher daily midazolam dose requirement than did patients receiving venoarterial ecmo ( p = 0.005 ) . a more recent singlecenter , prospective cohort study set out to determine the median daily dose of opioids and benzodiazepines in 32 patients receiving either venovenous or venoarterial ecmo . in this mixed cohort the median daily dose of opioids and benzodiazepines , 3875 micrograms and 24 mg respectively , was found to be lower than that reported from previous trials .
additionally , the authors did not find increasing requirements throughout the duration of ecmo support .
the lower opioid and benzodiazepine requirements observed in this study could be a result of the study institution s analgosedation approach , lower goal sedation scores , and use of other nonbenzodiazepine sedatives .
the only comparative trial to date is a recent retrospective cohort study that enrolled consecutive adult patients with severe respiratory failure with ( n = 34 ) or without ( n = 60 ) venovenous ecmo support requiring at least one sedative to maintain a level of wakefulness appropriate to maintain patient comfort and safety while optimizing oxygenation and ventilator support .
the authors found that the maximum median 6hour sedative exposure was nearly twice as high in the ecmo group and was reached nearly 3 days later when compared to the group not receiving ecmo .
however , there was no significant difference in 6hour sedative exposure in adjusted analyses . therefore , this study challenges whether the increased requirements of opioids and sedatives are a result of circuitrelated factors alone or whether other factors , such as tolerance , age or pharmacogenomics , play a central role .
existing data are sparse to guide the appropriate dosing of opioids and sedatives in adult patients receiving ecmo in the context of modern extracorporeal technology .
many of the firstline agents used in the management of pain and sedation are lipophilic and therefore have a high propensity to be adsorbed or sequestered by the extracorporeal circuit [ 6 , 11 , 12 ] .
one approach to achieving adequate sedation in patients receiving ecmo would be to start with continuous infusions of both an opioid and a sedative , anticipating requirements that exceed standard doses with the ultimate goal of minimizing the deleterious effects of sedative agents , especially benzodiazepines , with daily interruptions or down titrations .
additionally , clinicians should anticipate the need for significant dose reductions at the time of ecmo discontinuation given the likely rapid decrease in volume of distribution .
the reduction in dosing may be difficult to calibrate , so it is prudent to carefully monitor for signs of withdrawal and delirium .
consideration of adjunct agents , such as subanesthetic doses of ketamine , may help facilitate achieving sedation goals .
two uncontrolled studies demonstrated reductions in sedative rates with the addition of lowdose ketamine infusions [ 24 , 25 ] .
most recently , a randomized trial did not show any differences in opioid or sedative requirements with the addition of lowdose ketamine to standard sedation practices as compared to standard sedation practices alone in patients receiving venovenous ecmo for severe respiratory failure .
standard sedation practices consisted of infusions of fentanyl or hydromorphone and midazolam to achieve a richmond agitation sedation scale ( rass ) of 5 at the initiation of ecmo .
the median cumulative amount of fentanyl and midazolam equivalents in the lowdose ketamine group were almost twice and four times as high , respectively , when compared to the control group from ecmo initiation to the decision to achieve wakefulness .
however , patients receiving lowdose ketamine infusion had similar improvements in their rass scores over the 72hours after the decision to achieve wakefulness .
the incidence of delirium in patients receiving ecmo is not well characterized ; however , given the high use of benzodiazepines in these patients , it may be reasonable to presume that the rates are as high as those reported in the critically ill patient population not receiving ecmo .
when appropriate , the use of a regimented analgosedation approach , daily interruption of sedation , and early mobilization may help minimize opioid and sedative exposure and thus reduce the incidence of delirium associated with these drugs , as appears to be the case in critically ill patients in general .
infections are commonly encountered in critically ill patients and are associated with higher mortality . in a critically ill patient , source control , in addition to timely and appropriate antimicrobial administration , remains the cornerstone of successful treatment of infection . selecting the appropriate dose of an antimicrobial can be challenging given the potential effects of critical illness and ecmo on drug concentrations , particularly considering that most antimicrobial dosing regimens have been established in healthy adults with normal physiology .
changes in volume of distribution and clearance from critical illness and the ecmo circuit may affect pharmacodynamic parameters that ultimately determine the effectiveness of the antimicrobial agent .
inappropriate antimicrobial dosing may result in substantial drug losses , leading to therapeutic failure , development of resistance , and worse outcomes in patients with lifethreating infections whereas an empiric increase in dose may potentially lead to accumulation and toxicity .
vancomycin , a moderately protein bound , hydrophilic antimicrobial agent is commonly used to treat grampositive bacterial infections .
two in vitro ecmo studies observed steady vancomycin drug concentrations over 24 and 48hour periods [ 6 , 19 ] . in a matched cohort study of adult critically ill patients , those receiving ecmo
had a similar volume of distribution and clearance of vancomycin compared to those not receiving ecmo in the first 24 h of therapy .
all patients received a 35 mg / kg loading dose over 4 h , followed by a continuous infusion targeting a serum concentration of 2030 mg / l .
linezolid , an alternative grampositive antimicrobial , was studied in three adult patients receiving ecmo , the results of which suggest that therapeutic targets may not be achieved with standard dosing when the minimum inhibitory concentration ( mic ) is greater than 1
aminoglycosides , including gentamicin and tobramycin , are hydrophilic drugs with low protein binding and with increased volume of distribution in the context of critical illness , resulting in decreased maximal concentrations .
additionally , in the context of a higher volume of distribution from the ecmo circuit , standard or higher initial doses may be needed with normal or extended intervals in order to provide sufficient peak concentrations .
the study of aminoglycoside pharmacokinetic alterations during ecmo is largely limited to the neonatal population ; however , one observational study in adult patients demonstrated comparable pharmacokinetics with amikacin in critically ill patients with or without ecmo support .
therapeutic drug monitoring is readily available for this class of antimicrobial agents , making it feasible to target effective concentrations while limiting potential toxicity .
extendedspectrum penicillins , cephalosporins and carbapenems are commonly used in the treatment of gramnegative infections in the critically ill patient population . as a class , these antimicrobials
are generally hydrophilic , largely dependent on renal elimination , and have moderate to low protein binding ; however , variability exists with certain drugs such as ceftriaxone .
optimizing the timedependent , bactericidal effect of this class will be achieved by maximizing the time concentrations above the mic .
the use of extended or continuous infusions seems to be a reasonable approach to optimize the pharmacodynamics in critically ill patients receiving ecmo .
conflicting data have been reported on meropenem , a hydrophilic carbapenem with low protein binding .
while some studies suggest a significant loss of meropenem within the ecmo circuit [ 6 , 33 , 34 ] , other investigations have found no effect in vivo compared to other critically ill patients . a recent retrospective , casecontrol study observed no differences in pharmacokinetic paramenters with either piperacillin / tazobactam or meropenem in patients receiving ecmo compared to those not receiving ecmo . of interest ,
nearly 30% of all drug levels measured for the two aformentioned drugs were subtherapeutic , which may be a consequence of other pathophysiological disturbances not controlled for in this critically ill population .
mechanical circulatory support in general can induce a systemic inflammatory response , independent of the underlying critical illness , which , in turn , may augment renal clearance .
classes of antimicrobials in which direct correlations of augmented renal clearance and lower serum drug concentrations have been observed include betalactams , aminoglycocides and glycopeptides .
regarding other antimicrobial drug classes , azithromycin pharmacokinetics appear to be similar between patients receiving ecmo and nonecmo critically ill controls and tigecycline levels in one patient were similar to expected levels based on population pharmacokinetics .
concentrations of ciprofloxicin , a fluoroquinolone that is lipophilic with low to moderate protein binding , do not seem to be affected by ecmo .
to date there are no data on pharmacokinetic changes with polymyxin b or polymyxin e ( colistin ) that are used to treat multidrug resistant gramnegative infections .
ecmo does not appear to affect oseltamivir pharmacokinetics directly ; however , patients with renal dysfunction may experience impaired drug clearance [ 3941 ] .
data on the pharmacokinetic changes of antifungal agents in adult patients receiving ecmo are limited to in vitro studies or case reports .
while fluconazole does not seem to be affected by the ecmo circuit with a mean drug recovery of 91% at 24 h , voriconazole concentrations appear to be significantly affected , with up to a 71% loss at 24 h [ 14 , 42 ] . despite adequate serum concentrations of caspofungin at recommended doses , data suggest some sequestration by the ecmo circuit .
lipophilic formulations of amphotericin b as well as posaconazole and isavuconazole , both highly protein bound , may result in significant sequestration within the ecmo circuit .
when designing an appropriate antimicrobial dosing regimen for patients receiving ecmo , the biochemical properties of each drug should be considered , generally favoring a high initial concentration while monitoring for potential toxicities .
whenever possible , monitoring of drug concentrations , including peaks and troughs as appropriate , will help inform an effective dose and interval .
further insight into pharmacokinetic changes of antimicrobials in adult ecmo patients will be provided with the ongoing analgesia , sedation , and antibiotic pharmacokinetics during extracorporeal membrane oxygenation ( asap ecmo ) trial .
although there are known pharmacokinetic changes occurring as a consequence of ecmo support , there are very limited data addressing the clinical outcomes associated with these observations . for example , agents used for sedation or blood pressure control can be titrated to predetermined clinical endpoints , permitting the bedside clinician to use a dose for which the effects may be easily observed and measured
. however , in the absence of therapeutic drug monitoring , attainment of adequate antimicrobial concentrations may not be so readily observed , instead requiring the clinician to rely on surrogate endpoints , such as white blood cell counts or temperature curves , to assess effectiveness .
much of the existing data is limited to simulated circuits that do not account for metabolism or elimination .
additionally studies have not addressed how changing blood flow rates or using different ecmo configurations could impact the amount of drug sequestered within the circuit .
finally , many of the studies do not have control subjects , have not addressed the effects of circuits over longer time intervals , and have not studied the impact of additional extracorporeal circuits , such as continuous renal replacement therapy , on pharmacokinetic parameters .
there is potential for novel strategies , such as altering the materials used in ecmo tubing or creating polymeric micelles for drug delivery , to minimize drug sequestration within the ecmo circuit .
absorption of lipophilic drugs to traditional medical grade pvc tubing containing diethylhexyl phthalate , a plasticizer , has been well established over the years .
one in vitro study observed less adsorption capacity for lipophilic drugs when alternative materials , such as teflon or siliconecaoutchouc mixture ( srt 460 ) , were used .
another method would be to encapsulate intravenous lipophilic drugs within micelles , a concept that has previously been demonstrated with gene therapy .
the idea is to solubilize appropriate portions of lipophilic drugs into the hydrophobic core , allowing attraction of the lipophilic tail to the surface of the ecmo tubing and drug release with minimal adherence to the surface of the tubing .
the pharmacokinetics and pharmacodynamics of drugs administered to critically ill patients are influenced by several factors , including the physiochemical properties of the drugs , the etiology and severity of the underlying illness , and the function of the organs responsible for drug metabolism .
the presence of ecmo has an additional impact on drug distribution and metabolism , particularly due to increases in volume of distribution and sequestration by circuit components .
data are limited regarding the optimal regimen and dosing of sedatives and analgesics for critically ill patients receiving ecmo support , with the existing literature suggesting that , in many cases , higher amounts of analgosedation may be necessary to achieve therapeutic levels than would be expected for critically ill patients not receiving ecmo .
certain classes of antimicrobials may likewise be affected by ecmo , potentially leading to subtherapeutic drug concentrations if usual dosing regimens are used .
emerging data from the asap ecmo trial should help inform the appropriate administration of many commonly used antimicrobials , sedatives and analgesics in patients receiving ecmo . | this article is one of ten reviews selected from the annual update in intensive care and emergency medicine 2017 .
other selected articles can be found online at http://ccforum.com/series/annualupdate2017 .
further information about the annual update in intensive care and emergency medicine is available from http://www.springer.com/series/8901 . |
early reports of well - differentiated nonmedullary thyroid carcinoma clustering in families [ 17 ] , and studies utilizing population and hospital data bases [ 813 ] demonstrating that such cases occurred in families more often than could be explained by chance have led to the general acceptance of the existence of a familial form of the disease .
. a hereditary basis for this familial nonmedullary thyroid cancer is postulated , but no specific genetic defect has been established as yet [ 1517 ] , and the suggestion has been made that the disease may result from a heterogeneous form of inheritance or the interaction of susceptibility genes with unidentified environmental factors [ 1719 ] .
aspects of the natural history of familial papillary thyroid carcinoma ( fptc ) remain unclear , and specifically controversial is whether or not the familial form of the disease is more aggressive than the sporadic form . in certain reports , the familial form of the disease was associated with an earlier age at diagnosis [ 3 , 7 ] , a higher than usual ratio of male to female cases [ 14 , 20 ] , a greater incidence of spread outside the thyroid gland , [ 3 , 14 , 20 , 21 ] and a greater rate of persistence / recurrence [ 7 , 14 , 2023 ] .
however , a number of other studies did not confirm the presence of these measures of aggressiveness , including a recent retrospective study , and , in a review of published studies , loh did not find conclusive evidence for increased aggressiveness in the familial form of the disease .
charkes has pointed out that most reports consist predominantly of families with only two affected members , and in his analysis a high percentage ( 6269% ) of patients in two member families may suffer from the sporadic rather than the familial form of the disease .
hence , a number of reports may have included the data from a significant number of sporadic cases in their analysis of the characteristics of familial cases .
in contrast , kindred 's with three or more affected members overwhelmingly ( 96% ) represent the familial form of the disease , leading charkes to suggest that these kindred 's be used for clinical and genetic investigations .
further , as fptc may be a heterogeneous entity , there may be significant differences in the degree of aggressiveness between different families within any given report of cases , rendering comparisons in series with small numbers of families difficult . in this communication
, we describe a retrospective analysis of our experience with sporadic versus fptc cases seen at the university of western ontario . given charkes ' analysis and recommendations
, we chose a statistical approach designed to determine the presence or absence of a trend for the occurrence of more aggressive disease in families with three or more affected members by comparing the clinical and pathology findings of sporadic cases versus first degree families with two affected members versus first degree families with three or more affected members .
this approach was chosen to address the issue of mistaking sporadic cases for familial cases in two member families , and to address the possibility that abnormal genes causing a higher prevalence of disease may also result in more aggressive disease .
we further explored the possible presence of disease heterogeneity occurring between fptc families ( i.e. , the possible presence of an aggressive genotype clustering in certain families while other families contain members with a genotype resulting in more indolent disease ) .
patients attending our clinics at the university of western ontario with an established diagnosis of nonmedullary thyroid cancer were questioned regarding the presence or absence of similarly affected family members .
we attempted to confirm the diagnosis of identified additional affected members by obtaining their medical and pathology records and when possible reviewed these family members in our clinic .
the surgical pathology , therapeutic , and followup data on all cases were entered into an access database organized into four categories : ( 1 ) sporadic cases , ( 2 ) familial cases with affected second degree or higher relatives , ( 3 ) familial cases with first degree relatives in families containing only two affected members , and ( 4 ) familial cases with first degree relatives in families containing three or more similarly affected individuals .
for all cases , we entered into the data base details of the clinical course ( including evidence of recurrence / persistence of disease such as reoperation , requirement for additional radioactive iodine therapy , etc . ) and surgical pathology results including histological tumour type ; tumour number , size , and site ; the presence or absence of ( a ) tumour penetration of thyroid capsule and/or presence in perithyroidal tissue , ( b ) vascular or lymphatic invasion , ( c ) diffuse lymphocytic infiltration , ( d ) multifocal disease , ( e ) bilateral disease , ( f ) cervical nodal metastases , and ( g ) distant metastases .
the presence of distant metastatic disease was established by surgical removal or biopsy of thyroid cancerous tissue or by persistent radio - iodine uptake in abnormal sites ( e.g. , lung , bone , cns , etc . ) with no other known cause .
reoperation was defined as any surgery carried out to remove proven recurrent / persistent thyroid cancer after the initial surgical therapy of a total thyroidectomy ( one or two stage procedure ) .
deaths were attributed to thyroid cancer only if other causes of death could be ruled out .
radioactive iodine therapy was administered in accordance with the american thyroid association guidelines . within the ascertainment time of case acquisition , the data base contained 698 confirmed cases of nonmedullary well - differentiated thyroid cancer .
of these cases , 664 were papillary thyroid cancer ( table 1 ) , and all cases unequivocally established as occurring in a familial setting were classified pathologically as papillary thyroid cancer .
hence the term familial papillary thyroid cancer ( fptc ) is used in this paper .
none of the established fptc families had any evidence of additional tumours or clinical features to suggest the presence of a familial syndrome [ 18 , 27 , 28 ] characterized by a predominance of nonthyroidal tumours , for example , familial adenomatous polyposis , pten - hamartoma syndrome , carney complex , and so forth .
this study was approved by the ethics review board for human research at the university of western ontario .
sixty patients presenting in our clinic gave a history of first degree relatives being similarly affected with ptc ; by history , 22 families contained 3 or more similarly affected first degree relatives with ptc ( data was adequate for analysis on 52 patients ) , and 38 families had only 2 affected members ( data was adequate for analysis on 55 of these patients ) .
an additional 33 families with similarly affected second degree or higher relatives were identified ( data was adequate for analysis on 36 of these patients ) .
these patients form a data set derived from families classified by historical evidence alone without the necessity of having obtained confirmatory pathology on all family members .
this data set ( the history - defined data set ) was analyzed as described below , comparing four groups : first degree relative families with three or more affected members , versus first degree relative families with two affected members , versus second degree or greater relative families with affected members , versus 521 concurrent sporadic cases .
this history - defined data set was analyzed as described below , and the results are presented in table 5 .
on review of the pathology data obtained on additional identified family members , we were able to confirm ( in all members ) a pathology proven status of 14 first degree relative families with 3 or more affected members ( 41 members of this group had adequate data for analysis ) and 30 first degree families with only 2 affected members ( 50 members of this group had adequate data for analysis ) .
we performed an identical statistical analysis on this data set ( the pathology - defined data set ) comparing the presenting and clinical followup data on three groups : first degree families containing 3 or more affected members versus first degree family members containing 2 affected members versus 521 ptc patients with sporadic disease seen concurrently .
the results of this analysis on the pathology - defined data set are presented in detail in the text and in tables 24 . to assess the validity of classifying families based on historical evidence alone without requiring pathological confirmation of the presence of ptc in all additional identified members , we subjected the data sets obtained from both the history - defined families and the pathology - defined families to identical statistical analyses .
the results from the analyses of the two data sets yielded identical statistical results . for the sake of brevity , only the results of the pathology - defined data set are given in detail in the text and in tables 2 , 3 , and 4 .
table 5 displays the most relevant data from the history - defined data set together with a comparison of the statistical results derived from the analysis of both data sets . for statistical analysis , mean
ages were contrasted using analysis of variance , and distribution of gender in the groups was compared using the chi - square test for contingency tables .
the method of statistical analysis was chi square by trend for the frequency of the presenting surgical pathology parameters , metastases , and for the followup data including subsequent required treatments relative to the extent of familial incidence of papillary thyroid cancer ; the cochran - armitage test was used for evaluation . due to small counts for some events ,
the exact version of the cochran - armitage test was utilized . to examine for heterogeneity of disease between individual families in the first degree relative categories , the log linear model with poisson count
p values for the exact test were calculated using statxact software ( cytel software corporation , cambridge , mass .
tables 1(a ) and 1(b ) display the diagnostic details for all 698 patients with nonmedullary thyroid cancer in the database .
the overwhelming majority of cases , 664 patients ( 95.1% ) , were diagnosed with papillary carcinoma , 13 cases contained areas of the tall cell variant , 15 cases contained areas of the oxyphil cell variant of ptc , and 10 had areas of solid type growth patterns .
the non - ptc cases were classified as 19 follicular carcinomas , 14 hurthle cell carcinomas ( 11 of which were classified as minimally invasive ) , and one clear cell carcinoma .
three of the four sets had concordant diagnoses of ptc ( all made within one year of each other ) ; the fourth set has one twin with proven ptc , the other twin is currently under ongoing investigation for nodular disease , and a further sibling has treated ptc .
three of the four sets of twins occur in families with additional members with ptc , the fourth set occurs in a family with prominent nodular disease .
ten of the 13 cases with foci of the tall cell variant ptc occurred in the nonfamilial cases , one in a first degree relative ( 3 or greater ) family member who did not demonstrate any aggressive features at surgery nor on followup , and in two members of the second degree or greater relative families ( both of which had distant metastases ) .
there were 10 cases of ptc with foci of solid type growth pattern , all of which occurred in sporadic ptc cases .
the demographics of the nonfamilial and the ptc familial cases are shown in table 2 .
there was no statistically significant difference in the age at diagnosis between the nonfamilial and familial cases , and a predominance of females to males was seen in all groups with no statistically significant differences in sex distribution between the groups . similarly , no significant difference between the familial and nonfamilial cases was found in the percentage of patients aged 45 or older at diagnosis ( table 2 ) , nor was there any significant difference in the average length of followup .
there were no statistically significant differences seen for any of the surgical pathology parameters analyzed ( table 3 ) including tumour size greater than 3 cm , presence of tumour in perithyroidal tissue / penetration of thyroid capsule , multifocal or bilateral disease , vascular or lymphatic invasion , and lymphocytic infiltration .
similarly , there was no statistically significant difference in the presence of cervical nodal metastases between the groups ( table 4 ) .
in contrast , the presence of distant metastases was statistically significantly higher in the familial cases than in the sporadic cases ( p = 0.003 ) , the distant metastases being seen particularly in families with three or more affected members ( table 4 ) .
the familial group had statistically significant increased rates of recurrent / persistent disease as judged by the requirement for reoperation ( p = 0.05 ) , the requirement of additional radio - active iodine therapy administered at least two years after the initial therapy ( p = 0.03 ) , or a combination of these two therapeutic modalities ( p = 0.03 ) .
although the number of deaths due to thyroid cancer was small over the time period of case ascertainment , with the statistical approach used there was a statistically significant trend for greater mortality ( p = 0.01 ) in the familial groups .
the two deaths in the first degree relative families with 3 or more affected members group occurred in one family which had multiple members affected by the disease ( a third member is thought to have died of the disease but the cause of death could not be established with certainty ) ; both patients presented late in the course of the disease ( as was the case for the deaths occurring in both the familial and sporadic groups ) .
there were 24 cases with distant metastases ( table 4 ) , 16 in the sporadic disease group and 8 in the familial group .
the presence of distant disease was discovered close to or at the time of diagnosis of thyroid cancer in almost all of the patients ( a ) in the sporadic disease group , 12 cases had the distant spread discovered at the time of diagnosis of thyroid cancer , 3 cases within the first year , and in one case the precise time elapsed was uncertain , ( b ) in the 6 familial cases found in the three or more member families group , 3 had the distant spread discovered at the time of diagnosis of thyroid cancer , 2 patients within one year , and within two years in 1 patient , and ( c ) in the families with two affected members only , one patient was diagnosed with distant metastases within one year and one case eighteen years after the original diagnosis of thyroid cancer . an additional analysis , performed to explore for heterogeneity in our first degree families , did not find any statistical evidence for the presence of heterogeneity for either deaths ( p = 0.25 ) or distant metastases ( p = 0.54 ) .
however , it is noted that the number of events analyzed was small ; perhaps a greater number of events would be necessary to perform an adequate analysis .
table 5 presents the most relevant data from the history - defined family groups data set and compares the statistical results obtained in this group to the results obtained on the data set of the pathology - defined family groups . in agreement with the analysis of the pathology - defined family data set ,
there were no statistically significant trend differences seen in any of the demographic data ( age at diagnosis , sex distribution , mean followup time , and percentage of patients diagnosed after the age of 45 years ) .
there were no significant differences seen in any of the surgical pathology data although the history - defined data set analysis results approached significance in certain parameters : tumour in perithyroidal tissue / thyroid capsule penetration ( p = 0.08 ) , multifocal disease ( p = 0.06 ) , and bilateral disease ( 0.07 ) .
statistically significant trends also were seen for distant metastases ( 0.002 ) , deaths ( 0.02 ) , reoperation ( 0.03 ) , and requirement for additional rai ( p = 0.01 ) .
hence , the statistical results are consistent using either approach to defining the family groups .
despite the presence of a number of reports on the subject , it remains controversial as to whether the familial form of ptc is more aggressive than the sporadic form of the disease .
possible reasons for this uncertainly include the potential for ptc to reappear long after the initial therapy , the relatively infrequent occurrence of the familial form of the disease ( most studies report an incidence of approximately 5% of ptc cases , but one recent prospective study ( 32 ) reports an incidence of 9.4% of ptc being familial ) , the unrecognized inclusion of sporadic cases in the analysis of familial disease characteristics and the possibility of fptc being a heterogeneous disease resulting in varying degrees of aggressiveness occurring between different families . in this study , we used a different statistical approach , the chi square for trend analysis , comparing the characteristics of sporadic cases versus cases in first degree relative families with only 2 affected members versus cases in first degree families with 3 or more affected members . with this method of data analysis , our study found a statistically significant trend for the familial disease to exhibit more aggressiveness in certain disease characteristics and to require further intervention after the initial therapy .
we compared the data derived from using a historical approach ( history defined ) to defining the family groups ( i.e. , the number of affected patients in a family ) to that derived from family groups defined by requiring pathological confirmation of all included members ( pathology defined ) by subjecting both data sets to identical analyses .
this comparison yielded identical statistical results ; it appears that with an important disease entity such as ptc , families possess a good understanding of the family medical history .
the data for this study is derived from patients residing in an ethnically diverse population in which , similar to other regions , the incidence of ptc is clearly rising .
ptc accounted for 95.1% of our cases of well - differentiated thyroid cancer and the female - to - male ratio of cases is similar to that reported in other areas .
we did not see any significant difference in the proportion of female - to - male cases between the sporadic and familial cases in agreement with some reports [ 7 , 24 , 30 ] but in contrast to others [ 14 , 20 ] which found a higher than expected proportion of males in the familial cases .
similar to other reports [ 6 , 20 ] , we did not see a significant trend towards an earlier age at diagnosis of fptc , but other studies do report an earlier age at diagnosis of the familial disease [ 3 , 7 , 30 ] .
we did not find statistically significant differences between the nonfamilial and familial cases in any of the surgical pathology parameters .
it had been reported that the familial form of the disease has a greater incidence of cervical nodal metastases [ 3 , 2022 ] , but we did not find any such increase .
the multifocal nature of the disease was a very common finding in all of our cases ( present in 52.2 to 65.5% ) , as was the presence of bilateral disease in all groups , but there were no significant differences seen between the familial and sporadic cases ; a higher incidence of multifocality has been reported in certain series [ 6 , 7 , 14 , 20 , 27 ] .
we did not see a trend to a larger size of the primary tumour in the familial cases nor , somewhat surprisingly , any difference in the finding of vascular or lymphatic invasion by tumour .
although there was a greater incidence of tumour presence in perithyroidal tissues in the three or more member families , this was not statistically significantly different .
on analysis of the data on the clinical findings and course seen at or within a few years of the initial diagnosis ( table 4 ) , we found a statistically significant trend for the familial form of the disease to exhibit certain aggressive features .
a greater incidence of distant metastases ( p = 0.03 ) was seen in the familial form of the disease , being particularly prominent in the families with three or more affected members , the diagnosis being made in all of these cases within 2 years of the initial diagnosis of thyroid cancer . after the initial diagnosis and treatment , the familial cases also had a significantly greater requirement for reoperation ( p = 0.05 ) and , beyond 2 years of followup from the initial therapy , had a significantly greater need for further treatment with radio - active iodine ( p = 0.03 ) .
of note , as our mean followup times are short ( ranging from 4.22 to 5.37 years ) in all groups , these findings probably represent persistence of disease rather than recurrence . these measures of aggressiveness can not be readily explained by a greater incidence of tumours with histological features associated with a more aggressive clinical course .
there was only one first degree relative family member with the tall cell variant of the disease , and this patient did not display any aggressive features at surgery nor on clinical followup to date .
similarly , the presence of foci of the solid - type growth pattern was seen only in the nonfamilial cases .
nor can the trend for these measures of aggressiveness in the familial form of the disease be explained by an increased age at diagnosis since this factor was not statistically different between groups .
triponez et al . have reported that the survival rate of affected members of families with three or more members was significantly shorter than those in families with only two affected members ; this would be consistent with more aggressive disease being present in families with three or more affected members . in agreement with charkes analysis
, features of aggressive disease in this study were most frequently seen in the first degree relative families with three or more affected members , and these appeared to cluster in certain families ; the distant metastases were present in only 5 of the 14 families , and the two deaths occurred in one family .
an analysis to explore for the possible presence of heterogeneity in our families did not produce any statistical evidence for such heterogeneity .
however , the number of events in our data may not have been sufficient to yield an accurate appraisal of this possibility . with regard to the issue of heterogeneity , moses et al .
have recently published a prospective study which found a similar heterogeneity of numbers and types of somatic mutations in both the sporadic and familial forms of well - differentiated thyroid cancer cases ; no evidence was seen for a clear genotype - phenotype distribution based on hereditary disposition .
the cases with features of aggressive disease were most often present in the first member diagnosed in the family .
once the disease was recognized in the family , other affected members were usually diagnosed at an earlier stage of the process and have fared better on followup to date . for example , in one of our families , the initial case was diagnosed late in the disease with multiple distant metastases as well as inoperable local cervical disease .
two offspring in this family were screened for disease and both found to have micro - papillary carcinomas which had penetrated the thyroid capsule and spread to cervical nodes . following surgery and radio - iodine therapy , both
are currently considered disease - free ( negative stimulated thyroglobulin levels ) . similarly , in an earlier report by triponez et al . , it was found that survival times were significantly shorter for affected members treated before the familial nature of the disease was recognized .
. found a statistically significant earlier age at diagnosis in familial patients and attributes this to other family members being made aware of the familial nature of the disease and , hence , presenting at an earlier stage in the disease process .
we did not see a significantly earlier age at diagnosis , but , by focusing attention on the family history and encouraging family members to undergo screening for the disease , we well may have altered the outcomes in these patients for the better .
a potential problem biasing results in the opposite direction would result from familial patients being more motivated to present for followup procedures than patients with sporadic ptc .
if this occurred , less disease may have been discovered in our sporadic ptc patients .
this study is retrospective in nature , which would be expected to limit the completeness and accuracy of data collection in patients treated in the earlier periods of this study .
further , there have been significant technological advances over the period of data collection which has altered our ability to detect the presence of persistent / recurrent disease ( e.g. , advances in imaging , greater sensitivity and specificity of thyroglobulin assays , and the determination of stimulated thyroglobulin levels ) , rendering comparisons over time somewhat more difficult .
although the majority of our cases were seen in the recent past , a minority of our cases were first diagnosed and treated at a time predating current technical abilities .
our mean followup times are too short to allow the comparison of clinical outcomes beyond the period of a few years following the initial diagnosis .
the detection of persistent / recurrence of ptc may occur many years after the initial diagnosis and therapy ; microscopic disease below the limits of detection at the time of initial therapy may become apparent only with the passage of an appreciable period of time .
it is possible that differences in longer - term clinical outcomes between sporadic and familial disease may differ from those seen with the shorter time of followup in this study .
however , the logistics of carrying out a long - term prospective clinical research project to explore this question are quite daunting .
it is probable that ongoing research will yield a better understanding of the disease with the use of improved genetic diagnostic methods and imaging techniques .
a more precise approach to early diagnosis , treatment and followup , may render such long - term clinical studies unnecessary . in summary , using a statistical approach ( chi square by trend ) that compared ptc in sporadic cases versus first degree relative families with two affected members versus first degree relative families with three or more affected members , this study found evidence that the familial form of the disease exhibits significantly more aggressive features ( a ) the presence of distant metastases found at or shortly following the diagnosis of thyroid cancer and ( b ) the requirement for reoperation or further treatment with radio - active iodine following the initial treatment period . as these features were predominantly seen in the first degree families with three or more affected members ,
our results support the findings and the suggestion of charkes that genetic and clinical investigations should focus on families with multiple affected members . in agreement with triponez et al . , patients , diagnosed subsequent to the recognition of the familial nature of the disease , tend to have better outcomes .
this underlines the importance of early diagnosis and treatment for optimal outcomes . until reliable genetic testing becomes available , obtaining
a good family history is of great importance in the recognition of the familial nature of well differentiated thyroid cancer cases .
although the use of ultrasound as a routine screening device is controversial , it is well recognized that significantly more nodules are seen on ultrasound examination than are found on clinical examination , and that nodule size as a predictor of the presence or absence of malignancy is unreliable . in cases where the presence of fptc is suspected ,
the use of ultrasound for screening and the use of fna under ultrasound guidance on small lesions may result in earlier diagnosis and better outcomes .
although this may be a somewhat more aggressive diagnostic approach , it should be considered when investigating potential or known familial cases .
| background . whether or not the familial form of papillary thyroid carcinoma is more aggressive than the sporadic form of the disease remains controversial . methods . to explore this question and whether or not increased aggressiveness is more apparent in families with multiple affected members , we performed a chi square by trend analysis on our patients clinical and pathologic data comparing : first degree families with three or more affected members versus first degree families with two affected members versus sporadic cases of papillary thyroid carcinoma . results .
no statistically significant trends were seen for any presenting surgical pathology parameter , age at presentation , length of follow - up or gender distribution .
the familial groups exhibited significant trends for higher rates of reoperation ( p = 0.05 ) and/or requiring additional radioactive iodine therapy ( p = 0.03 ) , distant metastases ( p = 0.003 ) and deaths ( p = 0.01 ) .
these aggressive features were most apparent in certain families with three or more affected members .
conclusions . using the chi square by trend analysis ,
a significant trend was seen for the familial form of papillary thyroid cancer to possess more aggressive features than the sporadic disease .
prompt recognition of the familial nature of the disease may provide earlier diagnosis and treatment in similarly affected family members . |
it is the third - ranking cause of death , and therefore affects the health care system in thailand.1 one of the common consequences of stroke is spasticity .
it is defined as a motor disorder that is characterized by a velocity - dependent increase in tonic stretch reflexes ( muscle tone ) with exaggerated tendon jerks , resulting from hyperexcitability of the stretch reflexes.2 the prevalence of post - stroke spasticity has ranged from 19%3 to 39%4 of stroke patients at 3 months and 12 months after experiencing stroke , respectively . in thailand ,
the prevalence of post - stroke spasticity during rehabilitation was 41.6%.5 however , recent studies reported that the increased resistance of muscles with spasticity to passive movement is due not only to hyperexcitability reflexes , but also to altered properties of the muscle tissue.6,7 spasticity can cause pain , abnormal posture , and joint contracture
. it may interfere with functional recovery and the ability to perform daily activities , resulting in decreasing quality of life ( qol ) and increasing caregiver burden .
currently , many methods for decreasing spasticity are available , including pharmacological and nonpharmacological treatments . the pharmacological therapies , such as oral anti - spastic drugs,8,9 botulinum toxin injection,911 phenol injections,9,12 alcohol injection,13 and intrathecal baclofen,14,15 usually focus on reducing reflex hyperexcitability .
nonpharmacological treatments , which aim to inhibit neural activity , reduce muscle stiffness , and improve the surrounding connective tissue , include heat modalities , cryotherapy , electrical stimulation , stretching , splinting , acupuncture , and massage.16,17 the mechanisms of massage on spasticity vary depending on technique .
goldberg et al found that petrissage massage above the lumbar area can reduce h - reflex amplitude in the calf muscles of people with traumatic spinal cord injury.18 this result reflects a reduction in motor neuron excitability .
deep massage can stretch the muscle that experiences spasticity and reset sarcomere lengths to a more optimal state .
it helps to break down subcutaneous adhesions and prevent fibrosis,19 and may lead to improved sensory feedback from muscle spindle receptors .
moreover , the repetitive cutaneous stimulation provided by massage may reduce pain through the gate control theory.20 there have been a few study reports related to massage and spasticity .
only one randomized control trial was found , which reported the efficacy of rehabilitation exercise therapy in decreasing post - stroke hemiplegia limb muscle spasticity compared with traditional chinese massage therapy.21 their curative effects on patients were evaluated 4 weeks after treatment using the modified ashworth scale ( mas ) and limb motor function integration scale , and by measuring the changes in activities of daily living .
the researchers concluded , by examining total effective rates , that rehabilitation exercise therapy could improve muscle spasticity significantly more than traditional chinese massage therapy .
thus , it can not clearly be stated how the previous study measured the total effective rate and the details of the massage technique and dosage used .
currently , it is well recognized and regulated by the thai government . although massage is found worldwide in clinical practice , the scientific evidence that supports the effectiveness of massage in decreasing spasticity is limited .
therefore , a randomized controlled trial with a single - blinded assessor was performed to compare the effects of ttm and conventional physical therapy ( pt ) on spasticity , functional ability , anxiety , depression , and qol in stroke patients .
the study protocol was conducted in accordance with the ethical principles stated in the most recent version of the declaration of helsinki .
after the study protocol was approved by the institutional review board of siriraj hospital , stroke patients were recruited from the outpatient unit of the department of rehabilitation medicine , faculty of medicine siriraj hospital ( bangkok , thailand ) from august 2011 through july 2013 .
chronic stroke ( onset 3 months ) patients with moderate - to - severe spasticity of the elbow or knee in at least one limb ( grade 1 + ) , as evaluated by the mas , who were aged 50 years and able to communicate , were eligible to participate .
subjects who had contraindications for massage , eg , fever 38c or greater , uncontrolled blood pressure , bleeding tendencies , taking anticoagulant drugs , unhealed fractures , contact dermatitis , skin infections , or severe osteoporosis , were excluded . additionally , fixed joint contracture , a history of botulinum toxin injection within the last 6 months , a history of nerve block within the past year , severe dementia , or uncontrolled psychological disorders were excluded .
the study would be terminated if severe adverse events occurred , including fractures or hematomas in the muscle or soft tissue of the patients . after providing consent ,
a computer - generated randomization number was used , and the allocation codes were kept in opaque envelopes . an external investigator selected consecutive allocation envelopes for consecutive participants .
patients who were randomized into the treatment group received court - type ttm , two nonconsecutive days per week for 6 weeks . five certified personnel who performed massage for the duration of the study
therapists performed the massage with the same procedure over the hemiplegic side for 1 hour per session .
the basic massage lines and major signal points were the main massage treatment using only thumb and hand pressing at the point without traction or stretching .
the basic massage lines were pressed for 10 seconds and the major signal points were pressed for 30 seconds per point .
massage points included in this study were located in the leg , back , abdomen , arm , shoulder , and neck regions .
the pt group received a range of motion exercises for paralyzed limbs , strengthening exercises for the sound limbs , balance exercises for sitting and standing , and ambulation training in those who had potential for walking .
the program was administered by a trained physical therapist for 1-hour sessions , two times a week for 6 weeks . regarding the primary outcome ,
the percentage of patients who improved after treatment , ie , patients whose mas decreased by at least one grade after treatment , was measured .
the mas is a common clinical test used to determine spasticity , with ratings from zero ( no increase in muscle tone ) to four ( the affected part is rigid in flexion or extension).22 for secondary outcomes , the barthel index ( bi ) was used to measure functional abilities in self - care and mobility.23 possible scores range from 020 , with a higher score meaning better functions . to measure the patients psychological status , the thai version of the hospital anxiety and depression scale ( hads )
was used to screen for anxiety and depression.24 the scores for each subscale ( anxiety and depression ) range from 021 with scores of eleven or greater indicating the presence of a disorder.2426 additionally , the pictorial thai qol test was used to evaluate qol.27 it is composed of six dimensions : physical , cognitive , affective , social function , economics , and self - esteem .
scores range from 072 , where a higher score represents a better quality of life .
the adverse events that occurred in both groups were also recorded . at the end of the study
, the patients were asked about feelings of spasticity and the use of limbs functions as subjective outcomes .
the sample size of 186 patients ( 93 in each group ) provides a power of 80% to detect differences in the primary outcome ( ie , the percentage of patients whose spasticity decreased by at least one grade after treatment ) between a pt group of 30% and a ttm group of 50% , at a 5% two - sided significance level.28 demographic and baseline characteristics are presented as descriptive statistics in table 1 .
unpaired t - test was used to compare continuous data including age , bi , hads , and qol scores .
multiple logistic regression was used to adjust for age and sex for comparing the primary outcome .
mean changes in the scores of the bi , hads , and pictorial thai qol at baseline , compared with 6-week scores , were analyzed using paired t - tests , and the mean differences in those scores were compared between groups by using unpaired t - tests .
the number of adverse events and patients assessment of outcomes were compared using the chi - square test and fisher s exact test .
a p - value of less than 0.05 was considered statistically significant . for the primary outcome ,
intention - to - treat ( itt ) analysis , with the last observation carried forward method , was performed in addition to per protocol ( pp ) analysis .
the sample size of 186 patients ( 93 in each group ) provides a power of 80% to detect differences in the primary outcome ( ie , the percentage of patients whose spasticity decreased by at least one grade after treatment ) between a pt group of 30% and a ttm group of 50% , at a 5% two - sided significance level.28 demographic and baseline characteristics are presented as descriptive statistics in table 1 .
unpaired t - test was used to compare continuous data including age , bi , hads , and qol scores .
multiple logistic regression was used to adjust for age and sex for comparing the primary outcome .
mean changes in the scores of the bi , hads , and pictorial thai qol at baseline , compared with 6-week scores , were analyzed using paired t - tests , and the mean differences in those scores were compared between groups by using unpaired t - tests .
the number of adverse events and patients assessment of outcomes were compared using the chi - square test and fisher s exact test .
a p - value of less than 0.05 was considered statistically significant . for the primary outcome
, intention - to - treat ( itt ) analysis , with the last observation carried forward method , was performed in addition to per protocol ( pp ) analysis .
a total of 220 stroke patients were enrolled in the study , but 170 subjects were screened out owing to their spasticity grade ( mas ) being < 1 + ( 69 patients ) , inability to communicate ( 38 patients ) , complaints of inconvenience ( 43 patients ) , or refusals to change their treatment program ( 20 patients ) .
only 50 patients were randomized to receive ttm ( 24 patients ) or the pt program ( 26 patients ) twice weekly for the 6-week duration . at the end of study ,
five patients in the pt group were lost to follow - up owing to inconvenience ( four patients ) and having repeat hemorrhagic strokes ( one patient ) , as shown in figure 1 .
the demographic data of the 50 subjects in the ttm and pt groups did not differ except for sex , age , and bi scores ( table 1 ) .
patients in the ttm group were predominantly male ( 92% versus 58% ) , younger in age ( 60 years versus 66 years ) , and had higher bi scores ( 16.4 versus 10.9 ) compared with the pt group .
anxiety , depression , and qol scores did not differ between the groups at baseline .
the main outcome was the percentage of subjects whose mas score decreased by at least one grade after treatment .
the ttm group had a higher percentage of subjects whose spasticity improved by at least one grade than the pt group in both types of analysis ( 70.8% versus 61.9% in pp analysis ; 70.8% versus 50.0% in itt analysis ) . however , there was no statistically significant difference between the two groups , regardless of whether it was analyzed by pp analysis ( p=0.286 ) or itt analysis ( p=0.058 ) in muscles with maximal grade ( tables 2 and 3 , respectively ) .
figure 2 shows that , for secondary outcomes , bi scores increased significantly at week 6 compared with baseline in both groups ( p=0.008 and p=0.025 in ttm and pt groups , respectively ) , but no difference was found between the groups ( p=0.805 ) .
figure 3 illustrates the psychological aspects ; anxiety scores in the ttm group had decreased at week 6 compared with baseline scores ( p=0.083 ) , while the anxiety scores in the pt group had increased at week 6 ( p=0.624 ) . however , there was no difference in the scores when the two groups were compared ( p=0.144 ) .
the depression scores in the ttm group had decreased at week 6 compared with baseline scores but were not significantly different ( p=0.195 ) , whereas depression scores increased in the pt group ( p=0.747 ) .
figure 5 presents the qol of the ttm and pt groups at week 0 and week 6 . at the end of study ,
the total qol scores of both groups had increased significantly when compared with baseline scores ( p=0.041 in ttm ; p=0.001 in pt groups ) .
the number of patients in the ttm group who reported that their feelings of spasticity had decreased was greater than those who reported the same in the pt group ( 79.2% versus 52.4% ; p=0.057 ) .
however , the functions of their limbs improved only 37%38% with no difference between the groups ( p=0.967 ) . concerning patient satisfaction ,
90.5% of the patients were satisfied with the pt program compared with 75% of the massage patients .
the number of adverse events that occurred during the study did not differ between the groups ( p=0.807 ) , as shown in table 5 .
there were more patients who experienced adverse events of muscle stiffness , muscle pain , and muscle soreness in the ttm group compared with those in the pt group .
ttm is one of the methods of alternative medicine that is easily accessible in thailand .
studies examining whether ttm was effective in decreasing muscle spasticity in stroke patients were scarce .
the current study revealed that the percentage of subjects whose mas scores had decreased by at least one grade after treatment in the ttm group seemed to be greater than that of the pt program group . after using itt analysis
, the results displayed more significant differences than those from the pp analysis ; however , no significant difference was found in both analyses . additionally , the number of patients in the ttm group who reported that their feelings of spasticity decreased was greater than that of the pt group .
this subjective outcome was consistent with the number of patients whose mas ( ie , an objective outcome ) had decreased by at least one grade . however , these findings were not in line with chen and li s study .
they reported that rehabilitation exercise therapy can improve muscle spasticity in post - stroke hemiplegia significantly more than traditional chinese massage therapy.21 this might be due to the different style , as well as dosage , of massage .
although both ttm and pt programs increased the bi score by the conclusion of the study , there was no difference in the bi score between the two groups .
it was assumed that the decrease in spasticity in both groups may result in subjects using their affected limbs more easily and causing functional improvement after treatment .
additionally , the ability to perform daily activities can improve through spontaneous motor recovery in subacute stroke patients and functional adaptation after pt program.29 a decreasing trend in anxiety and depression scores at week 6 was only found in the ttm group , whereas those in the pt group seemed to exhibit an increasing trend instead .
this means that only the ttm group reported trends of improvement in anxiety and depression post - treatment .
the reasons for the differences in psychological outcomes between groups are possible from enhancing the parasympathetic nervous system30 and relieving muscle tension in the ttm group.31 these results were consistent with a meta - analysis of massage therapy research by moyer et al who reported that multiple doses of massage therapy can significantly reduce the traits of anxiety and depression.32 moreover , qol scores in both the ttm and pt group increased between baseline and week 6 , with no difference between the two groups .
ninety percent of the patients in the pt group were satisfied with the treatment program , compared with only 75% of the ttm group reporting satisfaction .
the reason why patients have been less satisfied with ttm may be due to the number of adverse events that occurred in the massage group , especially muscle stiffness , muscle pain , and muscle soreness .
this finding was consistent with a qualitative study by van der riet et al on ttm used in stroke patients.33 they reported that some stroke patients experienced pain after receiving massages , and the younger patients preferred to exercise rather than to receive massages .
though massage seems to be effective in decreasing spasticity in stroke patients , medical personnel should recognize that pressure during massage could adversely affect muscles .
therefore , standardized techniques of massage , in addition to the development of equipment for measuring pressure , are necessary .
a large number of patients were excluded for reasons such as communication difficulties , spasticity that was too mild , wishing to avoid inconvenience , and refusal to change their current treatment program .
some patients could not visit the hospital without assistance , so they declined to join the study .
for example , a low bi at baseline has been suggested as one of the predictors for the presence of severe spasticity .
thus , subjects who had a low bi score in the pt group may have a higher chance of developing spasticity34 and less potential for recovery.35 on the other hand , the bi is not sensitive for detecting change in stroke patients with a high bi score at baseline due to its ceiling effect.36 however , these variables were adjusted during analysis of the results .
finally , this study did not evaluate the long - term effect of massage on spasticity .
a large number of patients were excluded for reasons such as communication difficulties , spasticity that was too mild , wishing to avoid inconvenience , and refusal to change their current treatment program .
some patients could not visit the hospital without assistance , so they declined to join the study .
for example , a low bi at baseline has been suggested as one of the predictors for the presence of severe spasticity .
thus , subjects who had a low bi score in the pt group may have a higher chance of developing spasticity34 and less potential for recovery.35 on the other hand , the bi is not sensitive for detecting change in stroke patients with a high bi score at baseline due to its ceiling effect.36 however , these variables were adjusted during analysis of the results .
finally , this study did not evaluate the long - term effect of massage on spasticity .
this preliminary study could not demonstrate that ttm differs from conventional pt programs in decreasing spasticity or improving limb functions .
although no statistical significance was found , there were trends in decreasing spasticity , anxiety , and depression scores in the ttm group compared with the pt group .
massage may be an alternative method for stroke patients to decrease spasticity , improve psychological conditions and qol .
further studies employing an adequate sample size and evaluating the long - term effect of massage are needed .
step , area , and duration of traditional thai massage protocol notes : in steps 1 , 5 , 6 , and 7 , the subject was lying supine . in steps 24 , | purposeto study the efficacy of traditional thai massage ( ttm ) versus conventional physical therapy ( pt ) programs in treating muscle spasticity , functional ability , anxiety , depression , and quality of life ( qol ) in thai stroke patients.methodsthis randomized controlled trial with a blinded assessor was carried out at the department of rehabilitation medicine , siriraj hospital ( bangkok , thailand ) .
the study included 50 stroke ( onset 3 months ) outpatients experiencing spasticity at the elbow or knee muscles at a grade of 1 + on the modified ashworth scale who were 50 years old and able to communicate .
the subjects were randomly allocated to the treatment group receiving ttm ( 24 subjects ) or the control group receiving the pt program ( 26 subjects ) .
both groups received treatment ( either ttm or pt ) twice a week for 6 weeks .
spasticity grade , functional ability , anxiety , depression , and qol were measured at week 0 and week 6.resultsat week 6 , the percentage of patients whose modified ashworth scale score had decreased by at least one grade was not statistically significant between the two groups .
both ttm and pt groups experienced a significant increase in functional ability and qol , but no difference was found between the groups . anxiety and
depression scores showed a decreasing trend in the ttm group.conclusionthis preliminary report showed no evidence that ttm differed from the pt program in decreasing spasticity .
however , both interventions may relieve spasticity , increase functional ability , and improve qol after 6 weeks . only ttm can decrease anxiety and depression scores . further studies with adequate sample size are necessary . |
it has been reported that stress and anxiety stimulate adrenal gland and sympathetic nervous system which , in a sophisticated process , leads to an increase in bp , respiratory irregularities , dyspnea , an increase in the rate of respiration and hearth rate , and in case of frequent prolonged exposure to chronic stress and anxiety , it results in hypertension and other diseases .
restlessness , sorrow and sadness , loss of appetite , hypertension , an increase in respiratory irregularities , palpitation , and disturbance in concentration and doing daily activities are among the signs and symptoms of these depressive and stress - causing factors .
studies investigating anxiety , stress , and depression in patients families have been conducted , and it has been revealed that a disease creates a stressful situation for both the patients and their families .
research showed that hospitalization of a family member imposes high stress on other members and leads to depression , especially when their patient is to undergo invasive interventions . on the other hand , family , as the first social unit in iranian society , has a specific role and acts as a base for physical , cultural , and social health .
therefore , supporting the families whose patients undergo treatment is one of the main duties of nurses .
this can be given by nurses to families as psychological , mental , and social support .
as the families have a key role in maintenance of patients health after discharge , relieving their anxiety is helpful and of great importance .
one of the factors which play a major role in the reduction of anxiety , depression , and stress is individuals religious beliefs .
muslims always remember god when exposed to anxiety and stress and seek help from him . remembering god can be in the form of litany and prayer .
one type of litany is recommended azkar , as muslims believe zekr can relax , clear heart , treat pain , meditate the soul , and light wisdom .
it has been shown in muslim countries that prayer can decrease pain in addition to reducing anxiety , stress , and depression .
rosmarin ( 2009 ) showed that strong belief in god can also reduce stress and anxiety and increase happiness in non - muslims .
international studies reported that individuals , to seek help , refer to clergymen ( 42% ) , a general physician ( 29% ) , a psychologist or a psychiatrist ( 18% ) , and social workers institutes ( 11% ) . in some countries , there is a nurse at the operating room who tries to help families reduce their anxiety , stress , and depression through various methods .
meanwhile , where there are no such supporting systems , nurses should not forget the patients families and should try to find methods to reduce patients families anxiety , stress , and depression . with regard to high prevalence of anxiety , stress , and depression in families of patients before surgery and with respect to the importance of making peace for families , and the treatment property of recommended azkar as well as the socio - cultural conditions of iranian society ,
the present study was conducted to investigate the effect of religious recommended azkar on anxiety , stress , and depression of the families of the patients undergoing an open heart surgery .
this is a pre - experimental study conducted on 120 family members of the patients undergoing open heart surgery in shafa medical center in kerman , iran in the first half of 2011 .
the researcher referred to the surgical waiting room on different days when the families of the patients undergoing open heart surgery existed . after giving them information about the study and obtaining a written consent from them ,
it was such that all eligible individuals who fulfilled the inclusion criteria had equal chance to enter any of the groups ( study or control ) .
the inclusion criteria were no history of mental diseases , no history of intake of psychotropic medication including anti - depressants and anti - anxiety medication , no drug abuse , being a muslim , being an immediate family member of the patient ( father , mother , spouse , sister , or brother ) , being literate at least to the extent of reading and writing persian to fill the questionnaire , and being over 20 years of age ( in order to be mature enough to understand the meanings of recommended azkar ) .
the exclusion criteria was having open heart surgery for more than one time . to conduct the intervention , the researcher randomly selected a patient referring for open heart surgery to the cardiac surgery ward through lotto and assigned his / her accompanying person to the study group if he / she was an immediate family member of the patient . on another day ,
another patient was randomly selected ( through lotto ) and his / her accompanying person was assigned to the control group if he / she was an immediate family member of the patient .
the depression , anxiety , and stress scales ( dass 21 ) questionnaire was filled by both groups before intervention .
then , the study group underwent training about the use of azkar including hamd - va - soureh for seven times and la - ilaha - illallah for 100 times for 20 min at the session of selection . the importance of recommended azkar such as la - ilaha - illallah and
generally , muslims believe in two types of recommended azkar : 1 ) verbal recommended azkar , which is naming god and saying tasbih , tahmid , takbir , and tahlil ( la - ilaha - illallah ) and 2 ) internal recommended azkar , which is remembering god from the bottom of heart . among the recommended azkar causing relaxation and reducing anxiety and restlessness , hamd - va - soureh and la - ilaha - illallah ( no
so , the researcher used those recommended azkar , and 1 h after intervention , the questionnaire was filled again . in the control group ,
no training on recommended azkar was given and just the routine interventions were administered , and the questionnaire was filled after 1 hour . as an open heart surgery lasts for an average of 3 h and the highest level of anxiety , stress , and depression of patients families is observed in these 3 h , the researcher devoted the first hour in explaining to patients families , obtaining a signed consent form , and in initial filling up of the questionnaire .
the second hour was devoted to educating them on recommended azkar and making them say that , and the third hour was devoted to refilling the questionnaires . at the end of research , if any subject in the control group had accidentally used recommended azkar , he / she was left out of the study . before completing the questionnaire , an informed written consent was obtained from the subjects and the questionnaires were anonymous .
the subjects were assured that their acceptance or rejection to participate in the study had no effect on their patients treatment .
dass 21 questionnaire includes 21 questions through which signs of anxiety , stress , and depression are separately measured by seven questions , respectively .
each question is scored in a 4-point likert 's scale ( none many ) ranging from 0 to 3 , and the scores are summed up at the end . in this scale ,
scores 0 - 4 show normal level of depression , 0 - 3 show normal level of anxity and 0 - 7 show normal level of sress , 5 - 7 show low level of depression , 4 - 5 show low level of anxity and 8 - 9 show low level of sress , 8 - 11 show moderate level of depression , 5 - 7 show moderate level of anxity and 10 - 13 show moderate level of sress , 12 - 15 show severe level of depression , 8 - 9 show severe level of anxity and 14 - 17 show severe level of sress , 25 + show extremely severe level of depression , 10 + show extremely severe level of anxity and 18 + show extremely severe of sress , .
this questionnaire was suggested firstly by loviband ( 1995 ) and tested in a larger sample of humans .
it was adopted on numerous subjects in england and its reliability and validity were confirmed .
gylensten et al . , in a study titled investigation of the association between stress and work , used dass 21 questionnaire .
aghebati , in a study on the effect of touch therapy on the level of pain and mental signs of depression , anxiety , and stress in cancer patients , used this questionnaire and reported its reliability as 0.9 in iran .
one of the limitations was the contact between subjects and the families of the patients who had already undergone surgery , which could have an effect on the obtained results .
descriptive and inferential statistical tests ( chi - square , independent t - test , and paired t - test ) were used to analyze the data in spss .
scientific and ethical contents of the research project have been approved by kerman university of medical sciences , iran .
there was no significant difference in the demographic characteristics concerning age , gender , and level of education in the study and control groups ( p > 0.05 ) .
there was also no significant difference in anxiety , depression , and stress before intervention in the two groups ( p > 0.05 ) .
independent t - test showed a significant difference in the mean scores ( sd ) of anxiety , depression , and stress in the study and control groups ( p < 0.05 ) .
tables 1 and 2 compare the mean and sd values before and after intervention in both groups .
independent t - test ( based on relative changes ) was used to compare the mean ( sd ) of changes in the two groups with regard to neutrality of values before intervention .
this means that the values before the intervention decreased the value after the intervention intervention and then the calculated sum was divided to values before intervention .
mean ( sd ) values of relative change index have been presented in table 3 .
comparison of mean ( sd ) in the control group before and after intervention comparison of mean ( sd ) in the study group before and after intervention mean ( sd ) relative changes in the study and control groups paired t - test showed a significant difference in anxiety , stress , and depression in the study group before and after intervention ( p < 0.05 ) , but in the control group in which no extra intervention except routine interventions were administered , no significant difference was observed .
the findings showed that recommended azkar can reduce anxiety , stress , and depression of the patients undergoing open heart surgery .
so , this method can be used to reduce tension and to prevent anxiety , stress , and depression . in the past two decades
, numerous studies have been conducted on reduction of anxiety , but few studies have been carried out to reduce patients accompanying persons anxiety .
the present study showed that patients open heart surgery imposes anxiety , stress , and depression to their accompanying persons . in this
, in a study on the factors related to anxiety of accompanying persons of the patients hospitalized in icu , showed that 77% of patients families had anxiety and the families that encouraged the patients to surgery faced sixfold more of anxiety .
pochard et al . showed that observing a beloved person in a critical condition is stressful .
present study showed that askar can reduce anxiety , stress , and depression of patients accompanying persons .
nikbakht nasrabadi ( 2002 ) , in a study comparing the effect of benson relaxation method and recommended azkar on reduction of patients anxiety status before an abdominal surgery , concluded that recommended azkar had more effect on reduction of anxiety compared to benson relaxation method , which shows the effect of relying on god and its role in giving peace to humans .
hosini ( 2008 ) , in a study on the effect of prayer with emphasis on cognitive processes , showed that during praying , stimulation and activity starts in a special part of a human being 's brain .
this activity includes processing of the meaning of pray words , which causes recalling the saved information in the memory in addition to their related events and memories .
it consequently leads to lowering stress , bp , and heart rate , which gives us the ability to face the high - risk events appropriately .
peterson et al . , in a study on patients undergoing open heart surgery , found out that praying was effective on patients rapid recovery as well as control of stress signs .
vasegh , in a study on muslim students , showed that although religious beliefs lower anxiety , they can not reduce stress and depression . in a study conducted by rosmarin et al . , they concluded that remembering god increases the feeling of well - being in psychological and mental dimensions ( anxiety , stress , and depression ) .
the present study showed that recommendation of religious recommended azkar can reduce anxiety , depression , and stress in families of patients undergoing open heart surgery , and the nurses in muslim countries and in other countries where patients families believe in god can use this method to lower families anxiety . | introduction : admission of a family member to hospital would cause stress on other family members also .
one of the most stressful treatment interventions imposing high level of anxiety to the families of patients is when the patients are undergoing a surgery , especially a cardiac surgery .
so , we decided to investigate whether recommended azkar could reduce stress , anxiety , and depression in families of the patients.materials and methods : this study is a quasi - experimental study conducted on 120 immediate relatives of patients undergoing a cardiac surgery .
families of patients undergoing open heart surgery were randomly divided into two groups of study and control .
the depression , anxiety , and stress scales ( dass 21 ) questionnaires were completed by both groups .
then , some explanations about how to use recommended prayers were given to the study group , and an hour later , the questionnaires were completed again .
the data were analyzed by spss .
p < 0.05 were considered significant.results:stress , anxiety , and depression showed a significant difference in the study group before and after intervention .
similarly , there was a significant difference in the mean scores of anxiety , stress , and depression in the study and control groups after intervention.conclusions:findings of the present study showed that recommended religious prayers can significantly reduce anxiety , depression , and stress of families of the patients undergoing open heart surgery . |
twenty - one premature infants born between 30 and 34 wk ga with a birth weight > 1500 g were recruited for this study .
exclusion criteria included major chromosomal anomalies , intraventricular hemorrhage ( > grade ii ) , necrotizing enterocolitis , excessive drug / alcohol use , data missing regarding the date to full oral feeds , and/or the need for prolonged respiratory support ( i.e. , high - flow nasal cannula , continuous positive airway pressure , or ventilator support ) that would have delayed oral feeding trials .
saliva samples were taken at the closest approximate time ( within the 3234-wk period ) that an infant commenced oral feeding attempts .
there were a few infants ( see those indicated by an in table 1 ) that attained full oral feeds before the acquisition of the salivary sample .
two of these infants were capable of full oral feeds from birth , and the other took 5 d to attain full oral feeds , only briefly requiring nasogastric support before the attainment of a sample .
all infants in the tufts medical center nicu are subjectively assessed for oral feeding readiness based on the cue - based feeding algorithm described by ludwig and waitzman ( 2007 ) .
salivary samples were collected and processed with previously described techniques in order to simulate routine bedside care of the neonates ( dietz et al .
the neonate 's oropharynx was gently suctioned ( < 1 min ) and saliva was immediately stabilized in 500 l of rnaprotect saliva ( qiagen ) .
this stabilizing agent halts gene expression changes , inhibits microbial overgrowth , and destroys ubiquitous rnases .
one sample was analyzed for foxp2 expression ; the other sample was stored in a biobank for subsequent validation studies .
once collected , samples were stored for a minimum of 48 h at 4c before total rna extraction with the use of the rnaprotect saliva mini kit ( qiagen ) . on - column
every attempt was made to adhere to minimum information for quantitative real - time pcr experiments ( miqe ) guidelines established in 2009 to ensure proper and accurate reporting of rt - qpcr data ( bustin et al .
relative quantitative gene expression differences of salivary foxp2 were assessed on the applied biosystems quantstudio 7 flex real - time pcr instrument with the use of two reference genes ( gapdh and ywhaz ) previously shown by the maron laboratory to maintain their gene expression across pmas ( maron et al .
2012 ) . inventoried stock sequences of reference and target genes were provided by life technologies : gapdh ( hs03929097 ) , ywhaz ( hs03044281 ) , and foxp2 ( hs00362818_m1 ) .
for each salivary sample , foxp2 was run in duplicate , multiplexed one time each with the two reference genes with the path - id multiplex one - step rt - pcr kit ( life technologies ) . to ensure that multiplexing did not interfere with gene amplification , each gene was also run in singleplex one time with the use of a single sample .
negative controls with nuclease - free water were run on each plate to ensure that there was no primer
the rt - qpcr cycle profile was as follows : reverse transcription , 48c 15 min ; activation of dna polymerase , 95c 10 min ; 40 cycles of pcr , denaturing 95c 15 sec followed by annealing / extension at 60c 1 min .
if a sample failed to amplify both reference genes , it was deemed to be of insufficient quality and was not considered in the analysis .
the ct method was used for relative gene expression quantification in all samples ( livak and schmittgen 2001 ) .
the geometric mean of the ct values of both reference genes were used to calculate cts with the following equation :
ct=(meanctfoxp2[(ctgapdhctywhaz ) ] .
daily oral feed percentage was extracted from the nursing care notes and calculated across the eight daily feeds for all infants in the study .
total number of days required to reach full oral feeding was determined by subtracting pma on the day of the first oral feeding attempt from the pma on the day the nasogastric tube was successfully removed . infants who never required nasogastric tubes to sustain oral feeds were considered to have the developmental maturity of a successful oral feeder from birth .
a linear regression model was completed to examine the association between oral feeding and foxp2 gene expression with birth ga , sex , and weight at salivary sample entered as covariates into the model as they are known factors to influence feeding development ( maron et al . 2015 ) .
twenty - one premature infants born between 30 and 34 wk ga with a birth weight > 1500 g were recruited for this study .
exclusion criteria included major chromosomal anomalies , intraventricular hemorrhage ( > grade ii ) , necrotizing enterocolitis , excessive drug / alcohol use , data missing regarding the date to full oral feeds , and/or the need for prolonged respiratory support ( i.e. , high - flow nasal cannula , continuous positive airway pressure , or ventilator support ) that would have delayed oral feeding trials .
saliva samples were taken at the closest approximate time ( within the 3234-wk period ) that an infant commenced oral feeding attempts .
there were a few infants ( see those indicated by an in table 1 ) that attained full oral feeds before the acquisition of the salivary sample .
two of these infants were capable of full oral feeds from birth , and the other took 5 d to attain full oral feeds , only briefly requiring nasogastric support before the attainment of a sample .
all infants in the tufts medical center nicu are subjectively assessed for oral feeding readiness based on the cue - based feeding algorithm described by ludwig and waitzman ( 2007 ) .
salivary samples were collected and processed with previously described techniques in order to simulate routine bedside care of the neonates ( dietz et al .
the neonate 's oropharynx was gently suctioned ( < 1 min ) and saliva was immediately stabilized in 500 l of rnaprotect saliva ( qiagen ) .
this stabilizing agent halts gene expression changes , inhibits microbial overgrowth , and destroys ubiquitous rnases .
one sample was analyzed for foxp2 expression ; the other sample was stored in a biobank for subsequent validation studies .
once collected , samples were stored for a minimum of 48 h at 4c before total rna extraction with the use of the rnaprotect saliva mini kit ( qiagen ) . on - column dnase digestion
every attempt was made to adhere to minimum information for quantitative real - time pcr experiments ( miqe ) guidelines established in 2009 to ensure proper and accurate reporting of rt - qpcr data ( bustin et al .
relative quantitative gene expression differences of salivary foxp2 were assessed on the applied biosystems quantstudio 7 flex real - time pcr instrument with the use of two reference genes ( gapdh and ywhaz ) previously shown by the maron laboratory to maintain their gene expression across pmas ( maron et al .
2012 ) . inventoried stock sequences of reference and target genes were provided by life technologies : gapdh ( hs03929097 ) , ywhaz ( hs03044281 ) , and foxp2 ( hs00362818_m1 ) .
for each salivary sample , foxp2 was run in duplicate , multiplexed one time each with the two reference genes with the path - id multiplex one - step rt - pcr kit ( life technologies ) . to ensure that multiplexing did not interfere with gene amplification
, each gene was also run in singleplex one time with the use of a single sample .
negative controls with nuclease - free water were run on each plate to ensure that there was no primer
the rt - qpcr cycle profile was as follows : reverse transcription , 48c 15 min ; activation of dna polymerase , 95c 10 min ; 40 cycles of pcr , denaturing 95c 15 sec followed by annealing / extension at 60c 1 min . if a sample failed to amplify both reference genes , it was deemed to be of insufficient quality and was not considered in the analysis .
the ct method was used for relative gene expression quantification in all samples ( livak and schmittgen 2001 ) .
the geometric mean of the ct values of both reference genes were used to calculate cts with the following equation :
ct=(meanctfoxp2[(ctgapdhctywhaz ) ] .
daily oral feed percentage was extracted from the nursing care notes and calculated across the eight daily feeds for all infants in the study .
total number of days required to reach full oral feeding was determined by subtracting pma on the day of the first oral feeding attempt from the pma on the day the nasogastric tube was successfully removed . infants who never required nasogastric tubes to sustain oral feeds were considered to have the developmental maturity of a successful oral feeder from birth .
these infants achieved a full oral intake of 140 cc / kg / day within the first 7 d of life .
a linear regression model was completed to examine the association between oral feeding and foxp2 gene expression with birth ga , sex , and weight at salivary sample entered as covariates into the model as they are known factors to influence feeding development ( maron et al . 2015 ) .
the tufts medical center institutional review board approved this study and parental consent was attained prior to the start of the study .
we thank all of the parents and babies who participated in this study at the tufts medical center nicu .
we acknowledge support from the hearst foundations for the purchase of the quant - studio 7 flex real - time quantitative pcr system .
the tufts medical center institutional review board approved this study and parental consent was attained prior to the start of the study .
we thank all of the parents and babies who participated in this study at the tufts medical center nicu .
we acknowledge support from the hearst foundations for the purchase of the quant - studio 7 flex real - time quantitative pcr system .
| the objective of the study is to determine whether salivary foxp2 gene expression levels at the initiation of oral feeding attempts are predictive of oral feeding success in the premature newborn . in this prospective study , saliva samples from 21 premature infants ( 13 males ; birth gestational age [ ga ] : 3034 wk )
were collected around the initiation of oral feeding trials .
total rna was extracted and underwent reverse transcription - quantitative polymerase chain reaction amplification for foxp2 .
oral feeding success was denoted by the days required to attain full oral feeds . a linear regression model , controlling for sex , birth ga , and weight at salivary collection , revealed that foxp2 expression was significantly associated with oral feeding success ( p = 0.002 ) . the higher the expression level of foxp2 , the shorter the duration to feed .
salivary foxp2 expression levels are significantly associated with oral feeding success in the preterm infant .
foxp2 may serve as a novel and informative biomarker to noninvasively assess infant feeding skills to reduce morbidities and length of stay . |
to report an effect of the full tendon transposition augmented with posterior intermuscular suture and recession - resection surgery , for the patient with monocular elevation deficiency ( med ) and large exotropia .
full tendon transposition augmented with posterior intermuscular suture and recession - resection surgery was performed for a 26-year - old male patient had monocular elevation deficiency ( med ) and large exotropia .
preoperative angle of deviation was 56 prism diopters ( pd ) hypotropia and 45 pd right exotropia , compared with 18 pd left hypertropia and 10 pd right esotropia postoperatively .
essotropia persisted after 2.5 years , however , and so the right medial rectus was recessed after removal of the previous posterior intermuscular suture . at a three - year follow - up after the second surgery , alignment was straight in the primary position at near and far distances .
full tendon transposition augmented with posterior intermuscular suture and recession - resection surgery was effective for a patient with med associated with significant horizontal deviation , and a second operation was easily performed when overcorrection occurred .
a 26-year - old male patient had med and large exotropia from birth , and a marked limitation of supraduction in the affected eye . he had no significant medical or ophthalmic history of prior trauma .
an examination revealed best corrected visual acuities of 20/40 in his right eye and 20/20 in his left eye .
preoperative angle of deviation was 56 prism diopters ( pd ) hypotropia and 45 pd exotropia of the right eye ( fig .
the full tendon transposition of horizontal recti muscles to superior rectus muscle parallel to the spiral of tillaux was performed after lateral rectus muscle recession 9.0 mm and medial rectus muscle resection 5.0 mm ( fig .
the transposed horizontal recti muscles were augmented with a posterior intermuscular suture as we previously described.4 briefly , the posterior augmentation consisted of a 5 - 0 non - absorbable polyester suture with a t-5 spatulated needle ( dacron ; ethicon ) that was placed through 25% of the belly of each transposed and paralytic rectus muscle at 7 mm posterior from the original muscle insertion .
postoperative angle of deviation improved to 18 pd hypertropia of the left eye and 10 pd esotropia of the right eye ( fig .
binocular single visual fields improved to 65 from 0. 10 pd esotropia of the right eye persisted after 2.5 years , leading the patient to complain about the cosmetic problem .
accordingly , the right medial rectus was recessed 5.0 mm from the spiral of tillaux after removal of the posterior intermuscular suture . at a three - year follow - up , alignment was straight in the primary position at near and far distances .
there were no complications such as anterior segment ischemia during the surgery and postoperative follow - up .
surgery is indicated in med if there is vertical deviation in the primary gaze , deviation causing suppression and amblyopia , diplopia in the primary gaze , and contracted binocular fields . in patients with med
associated with a significant horizontal deviation , multiple extraocular muscle surgery or normal fellow eye surgery are generally necessary.1 - 3 most patients or their parents , however , do not allow surgery in the normal fellow eye , and surgery on the multiple extraocular muscles may cause anterior segment ischemia .
full tendon transposition augmented with posterior intermuscular suture and recession - resection surgery can effectively correct patients with med associated with significant horizontal deviation , while reducing the risk of anterior segment ischemia and avoiding an operation on the normal eye .
additionally , the intermuscular suture can be easily removed even when a second operation is needed . in this case report ,
full tendon transposition augmented with posterior intermuscular suture and recession - resection surgery was an effective and safe procedure in a patient with med associated with significant horizontal deviation . | purposeto report an effect of the full tendon transposition augmented with posterior intermuscular suture and recession - resection surgery , for the patient with monocular elevation deficiency ( med ) and large exotropia.methodsinterventional case report .
full tendon transposition augmented with posterior intermuscular suture and recession - resection surgery was performed for a 26-year - old male patient had monocular elevation deficiency ( med ) and large exotropia.resultspreoperative angle of deviation was 56 prism diopters ( pd ) hypotropia and 45 pd right exotropia , compared with 18 pd left hypertropia and 10 pd right esotropia postoperatively .
essotropia persisted after 2.5 years , however , and so the right medial rectus was recessed after removal of the previous posterior intermuscular suture . at a three - year follow - up after the second surgery , alignment was straight in the primary position at near and far distances.conclusionsfull tendon transposition augmented with posterior intermuscular suture and recession - resection surgery was effective for a patient with med associated with significant horizontal deviation , and a second operation was easily performed when overcorrection occurred . |
during the last decades , governments across many jurisdictions have been faced by the challenge of controlling increasing health care expenditure , partly due to new medical technologies , within a context of dwindling budgets , exacerbated by the recent economic and financial crisis . as a result ,
demand for evidence and justification of value are increasingly a requirement to support funding , coverage , and reimbursement decisions or price negotiation about health technologies.1 the use of health technology assessment ( hta ) to inform policy - making is established in north america , australia , in many european countries , and is rapidly growing in latin america and asia.26 hta has been described as the multidisciplinary process that summarizes information about several dimensions ( eg , clinical , ethical , economic ) related to the use of a health technology with the aim to achieve the best value in the allocation of public resources.7 in countries where hta is in place , third - party payers , pricing and reimbursement agencies , or hta bodies rely upon hta to determine the reimbursement status of a drug ( eg , listing in the local formulary ) , to issue guidance about the appropriate use of the technology within the health care system , and to support the price negotiation process.8 according to the remit of the organization and the structure of the health care system , this role is played at the central / national level or regional / provincial level.9 compared to licensing agencies that are focused on the risk - benefit profile of a product , evaluated in a rigorously controlled setting , typically randomized controlled trials , with high attention on internal validity , safety , efficacy , and manufacturing , hta agencies have different interests and , therefore , different evidence requirements.10 criteria for coverage or reimbursement decisions on pharmaceutical compounds vary across countries ; however , it is common to include , as part of the hta , a comparative effectiveness evaluation.11 demonstrating relative effectiveness in addition to efficacy means providing evidence that the intervention has beneficial health outcomes under real - world conditions and in comparison with the local standard of care
. the most stringent criteria of effectiveness versus efficacy are often met in a subgroup of the licensed - approved population ; therefore , the hta report has to focus on the appropriate target population identified from the reimbursement agencies perspective.12 in terms of health outcomes , final patient - relevant outcomes , which are outcomes that reflect how patients feel , function , or survive,13 are preferred over surrogate or intermediate outcomes ( eg , bone mineral density , ejection fraction ) by pricing and reimbursement agencies.14 this type of clinical data might go beyond that required for market authorization , thus creating an additional evidence gap between the regulatory and the reimbursement submission . in many cases ,
the information required at the market access stage also includes economic data , presented as cost - effectiveness analyses,12 budget impact analyses , or other types of economic evaluation .
although submissions to hta agencies do not have the same legal status as submissions to regulatory bodies , their relevance is consistently increasing in a pharmaceutical company s perspective , as market prospects are strongly influenced by third - party payers coverage . this is because market access , together with market authorization , is often regarded as an important goal of the drug development program,15 and technology appraisal is playing a significant role in opening or hindering market access for many innovative products .
a standard representation of the drug development process is given in figure 1 , where a simplified sequence of stages from basic research , discovery , and preclinical research , through clinical and postmarketing development is illustrated . in order to overcome the challenges posed by the
fourth hurdle,16,17 the manufacturers need to plan in advance for the evidence requirement and assessment for coverage and reimbursement .
moreover , while in the past postmarketing risk of failure of pharmaceuticals was sustained by payers , in recent years , managed market entry agreements ( mmeas ) have been introduced . under such agreements ,
the risk of a negative risk - benefit profile or excessive burden is , at least partially , covered by the industry ; therefore , the drug development cycle has to evolve to provide data for this purpose . in this study
, we aim to describe current hta activities , with comparative emphasis on the systems in place in italy and in the uk , with the potential to impact on the drug development path of pharmaceuticals .
we accessed the scientific literature as of june 2014 via pubmed , using two relevant keywords ( ie , hta and drug development ) , to build the background for this study . as we referred to institutionalized hta activities,18 we identified two hta bodies , the national institute of health and care excellence ( nice ) in the uk19 and the italian drug agency ( aifa [ agenzia italiana del farmaco ] ) in italy20 and accessed their institutional websites to collect information according to a predefined template
the two agencies were selected because , 1 ) they are operating in large and dynamic pharmaceutical markets , 2 ) they have both introduced an early advice program on hta with relevant guidance published on institutional websites , and 3 ) they present different models of price and reimbursement . in the uk
, there is no formal negotiation of price and reimbursement status between the agency and the manufacturer , which is instead indirectly regulated through the pharmaceutical price regulation scheme .
prioritized drugs are subject to an appraisal process based on clinical and cost - effectiveness data , managed by nice and the scottish medicine consortium , that produces a positive ( fully or restricted ) or negative recommendation . in italy , price and reimbursement are simultaneously negotiated by aifa and the pharmaceutical company : disease relevance ( ie , target population ) , place in therapy , added therapeutic value , and drug budget impact , together with prices in other countries , are the most important parameters considered , whereas cost - effectiveness does not play an important role.21
the development of hta to inform the coverage and reimbursement decisions about new drug therapies has necessarily contributed to the streamlining of the drug development program . in particular
, the emphasis on the incremental socioeconomic value to be established on new medical technologies has fostered the adoption and relevance of health economics and outcomes research through the drug development process.10 as outlined earlier , mainstream hta activities and reports demand more additional evidence than do regulatory dossiers .
effectiveness under real - world conditions and in comparison with the current standard of care , patient - relevant outcomes , patient - reported outcomes , and health - related quality of life , economic data , and , albeit with various definitions , innovation , are usually required in a value dossier for coverage and reimbursement agencies . in order to address these needs ,
manufacturers have strategically started to secure time and funding during the clinical development program to integrate health economics and outcomes research endpoints in clinical studies , to develop , validate , and use patient - reported outcomes tools ( eg , dermatology life quality index , psoriasis area , and severity index ) , to run burden of illness studies and build cost - effectiveness models or budget impact analyses .
most importantly , on top of mainstream hta , our review identified other activities , with lower levels of diffusion to date , that have the potential to influence the drug development process of a new medical technology .
horizon scanning systems ( hsss ) were introduced as part of the broad cycle of hta in order to systematically assess the potential impact of new and emerging technologies to anticipate policy development , access , and provision of health services.22 for this reason , they are also called early - warning or early - awareness systems . since 2006
, the vast majority of topics for technology appraisals at nice are identified by the national institute for health research horizon scanning centre , based at the university of birmingham.23 technologies are prioritized based on the expected health benefit or financial impact , inappropriate use , and variation of use across the country and effect on other health - related policies , such as reduction in health inequalities .
this process allows notification of relevant new drugs at nice , 20 months before the market authorization . in italy
this institution co - founded a project , named italian horizon scanning project ( ihsp ) , which is still part of the european network of horizon scanning ( hs ) centres.24 the ihsp produces an annual list of drugs that are expected to be launched within 3 years ; every 6 months , a report on the epidemiology of the target population , the clinical evidence , and the comparators of listed drugs is released and a more detailed report on prioritized drugs is released 12 months before their expected market launch .
prioritization depends on the burden of disease , the therapeutic value , and the expected impact on drugs expenditure . despite the fact that aifa has co - founded ihsp
, there is no evidence on whether and how it has relied on the results of this project .
murphy et al25 described an effective hss as one that identifies innovations likely to have a significant impact and disseminates in a timely manner the information relevant to the needs of the customers , so as to enable appropriate decision making , facilitate appropriate adoption , and identify further research requirements .
these characteristics are summarized in a set of five primary ( ie , relevance of information , independence , sufficient staff and funds , clearly defined pathway for the outputs to reach decision - makers , and defined customers ) and eleven secondary indicators ( eg , international collaboration , centrally coordinated , where tasks can be distributed among the participants , collaboration with industry to obtain information ) .
they were used to assess the 12 european network of hs centres member agencies,26 with some degree of variability in the presence of these desirable features . according to the definition , hsss
are primarily intended to support governments and health authorities decision - making and planning to anticipate the impact of emerging technologies within the health care system .
however , hsss may also contribute to support key decisions made by the manufacturers during the product development process .
although experiences have been reported mainly on diagnostics and devices,2729 hsss may help the drug developers to anticipate possible barriers to the downstream market access decision and to proactively work to overcome those by shaping the market and engaging stakeholders and key opinion leaders .
ijzerman and steuten30 proposed a conceptual framework for early hta as a tool to inform mainly the industry .
early hta is identified as the early assessment of safety , effectiveness , and cost - effectiveness profiles of new medical technologies based on evidence mainly derived from bench and animal tests , early clinical experience , and previous generations of the technology.31 this tool is certainly more suitable for the evaluation of medical devices than drugs32 and the extent to which it influences product development decisions is to date not known .
moreover , although its denomination seems to fall under the broad hta umbrella definition , it is rarely used by hta bodies and it is being developed by consultants or manufacturers to support industry and investors decisions on design and management of the technology , regulatory , and reimbursement strategies.30
horizon scanning systems ( hsss ) were introduced as part of the broad cycle of hta in order to systematically assess the potential impact of new and emerging technologies to anticipate policy development , access , and provision of health services.22 for this reason , they are also called early - warning or early - awareness systems . since 2006
, the vast majority of topics for technology appraisals at nice are identified by the national institute for health research horizon scanning centre , based at the university of birmingham.23 technologies are prioritized based on the expected health benefit or financial impact , inappropriate use , and variation of use across the country and effect on other health - related policies , such as reduction in health inequalities .
this process allows notification of relevant new drugs at nice , 20 months before the market authorization . in italy
this institution co - founded a project , named italian horizon scanning project ( ihsp ) , which is still part of the european network of horizon scanning ( hs ) centres.24 the ihsp produces an annual list of drugs that are expected to be launched within 3 years ; every 6 months , a report on the epidemiology of the target population , the clinical evidence , and the comparators of listed drugs is released and a more detailed report on prioritized drugs is released 12 months before their expected market launch .
prioritization depends on the burden of disease , the therapeutic value , and the expected impact on drugs expenditure .
despite the fact that aifa has co - founded ihsp , there is no evidence on whether and how it has relied on the results of this project .
murphy et al25 described an effective hss as one that identifies innovations likely to have a significant impact and disseminates in a timely manner the information relevant to the needs of the customers , so as to enable appropriate decision making , facilitate appropriate adoption , and identify further research requirements .
these characteristics are summarized in a set of five primary ( ie , relevance of information , independence , sufficient staff and funds , clearly defined pathway for the outputs to reach decision - makers , and defined customers ) and eleven secondary indicators ( eg , international collaboration , centrally coordinated , where tasks can be distributed among the participants , collaboration with industry to obtain information ) .
they were used to assess the 12 european network of hs centres member agencies,26 with some degree of variability in the presence of these desirable features . according to the definition , hsss
are primarily intended to support governments and health authorities decision - making and planning to anticipate the impact of emerging technologies within the health care system .
however , hsss may also contribute to support key decisions made by the manufacturers during the product development process .
although experiences have been reported mainly on diagnostics and devices,2729 hsss may help the drug developers to anticipate possible barriers to the downstream market access decision and to proactively work to overcome those by shaping the market and engaging stakeholders and key opinion leaders .
ijzerman and steuten30 proposed a conceptual framework for early hta as a tool to inform mainly the industry .
early hta is identified as the early assessment of safety , effectiveness , and cost - effectiveness profiles of new medical technologies based on evidence mainly derived from bench and animal tests , early clinical experience , and previous generations of the technology.31 this tool is certainly more suitable for the evaluation of medical devices than drugs32 and the extent to which it influences product development decisions is to date not known .
moreover , although its denomination seems to fall under the broad hta umbrella definition , it is rarely used by hta bodies and it is being developed by consultants or manufacturers to support industry and investors decisions on design and management of the technology , regulatory , and reimbursement strategies.30
some reimbursement agencies have set up in recent years early scientific advice ( sa ) services to pharmaceutical companies in order to outline , for specific submissions , the expected evidence base that could lead to optimal market access outcomes .
nice was the first agency to set up hta product - specific sa in 2009 ( table 1).33 the objective was to help product developers generate the evidence that is relevant for technology appraisal , with the aim of gaining more robust data for the reimbursement perspective , reducing the uncertainty at the time of the appraisal , and therefore , the length of decision - making and patients time to access the therapies .
the sa services given relates with a broad range of issues concerning the strategic development plans for postregulatory evaluation rather than advice on regulatory requirements or a preevaluation.34 the content of the sa pertains to research design considerations ( eg , study population , follow - up duration , appropriate comparators , endpoints , and type of study ) , approaches to economic analysis and generation of quality - adjusted life years , interpretation of guidelines , and other methodological issues . as the aim is that of making an impact on the strategic development and evidence generation plans , it is recommended to seek the advice when there is still time for the companies to amend the design of the pivotal trials , ie , when phase iii study design is ongoing .
at nice , the sa is available as a fee - for - service consultation to manufacturers , and it is provided through a structured process .
the company seeking advice has to contact the unit up to 9 months before the actual meeting and prepare a predefined briefing book with all relevant information for the advisors and a set of clearly stated questions accompanied by the respective manufacturer s position on them.35 after about 12 weeks of indirect exchange , a 4-hour face - to - face meeting is held where nice staff and companies representatives engage in a discussion around the questions on the agenda . after this meeting ,
a report is drafted and sent to the company for follow - up . as of october 2013 ,
77 sa requests from 26 companies were recorded at nice ; of these 17 ( 22% ) were withdrawn , possibly because final agreement on the contract between client and agency was not reached.36 of the remaining technologies , the majority ( 26 out of 60 , 43% ) were in the trial progression state , 17 ( 28% ) had completed the trials , and only one had completed the technology appraisal process , gaining a positive recommendation . however , as osipenko noted , hta advice was sought for that particular drug when the phase iii trial design was locked , thus limiting the likelihood of the favorable reimbursement decision being due to the hta advice provided.36 in addition to the standard bipartite ( ie , between hta body and manufacturer ) sa process , this agency also provides feedback on the drug development plan from the payers perspective alongside the medicines and healthcare products regulatory agency and the european medicines agency ( ema ) . in 2011 , aifa introduced both an sa and an hta advice ( sa - hta ) for regulatory and hta purposes in italy ( table 1).37 the guidelines for applicants to both processes were published in november 2012 and are available on the institutional website . as it is possible to ask for both advices jointly , this document refers to preclinical and clinical development for marketing authorization and production issues . the hta aspects are generally related to relative efficacy and relative effectiveness evaluation .
the sa - hta advice is generally asked for a single product in early stages of the product development , but it may be requested also for broader therapeutic classes .
issues subject to advice include the most appropriate comparator(s ) , the endpoints used , and the acceptability of indirect comparison . in addition , due to the relevance of disease severity and drug budget impact in price / reimbursement negotiation , the advice sought might often relate to the target population .
applicants wishing to apply should fill in a predefined form , the rationale for the request , whether other advices have been received by other european agencies , and the specific questions asked . within 20 working days , the sa coordinator notifies the applicant by email regarding whether the request for sa is acceptable or not . after 5 working days
, the aifa evaluation team is defined and the contract is drafted including the fee due to aifa , which ranges from 10k to 40k , according to the questions asked . the answers by the evaluation team are collected in a report due in 90 days from the signing of the contract between the agency and the company . a preliminary version of the report is discussed after 60 days with the company , within a meeting or a teleconference .
the minutes of the meeting are expected by the company and should be validated by the evaluation team .
the current uptake of this service by manufacturers in italy is currently not publicly available .
a representative of the agency reports that 21 advices have been processed in 20112013 : eleven ( 52% ) for cancer and autoimmune diseases , five ( 24% ) for neurologic diseases , four ( 19% ) for infectious diseases , and one ( 5% ) for hormone - related therapies.38 they generally refer to products in phase ii ( likely to be authorized after phase ii ) and phase iii .
the so - called tripartite early dialogue between regulators , hta organizations , and
first and foremost , it may reduce delays to patient access as the advice for different data needs for regulators and hta bodies is sought in parallel rather than in sequence .
second , it provides an opportunity to learn about hta requirements at an early stage and to discuss divergent data needs with all parties around the same table , with the aim of minimizing discrepancies and identifying trade - offs.39 for instance , when discussing the comparator for a novel therapy for chronic obstructive pulmonary disease during a parallel hta - ema sa session , the regulator agreed with the proposed licensed comparator while the hta agency wanted to compare the value of the new therapy to what it will replace in practice , even if the alternative was not licensed for that use .
the solution agreed was to introduce a new arm in the pivotal study to include both options.40 thirty procedures have undergone the parallel hta - ema sa process so far ; of those , 23 are now finalized .
they include drugs for several conditions : diabetes , alzheimer s disease , oncology , asthma , rheumatoid arthritis , osteoporosis , food allergies , and orphan conditions.40 the hta advice in the tripartite early dialogue may come from one or more reimbursement agencies , often represented through the european network for hta ( eunethta).41 as different hta bodies may focus on different dimensions ( eg , relative clinical effectiveness versus cost - effectiveness ) , this multi - hta dialogue aims at supporting the company in finding a balance between perspectives from licensing body and hta agencies in different countries as well as its own international product development program.42 most of the time , the hta body involved in the ema - hta parallel sa has been nice , followed by the german federal joint committee ( g - ba ) and aifa.43
linking between licensing and market access evidence requirements is also being extended to the postmarketing phase , with the aim of creating continuum evidence generation requirements relevant for registration , reimbursement , and postauthorization research.44 insufficient evidence and uncertainty at market launch and the need of limiting the budget impact of new drugs have increased the number of mmeas .
the taxonomy of these agreements has been illustrated in a recent review.45,46 mmeas may be 1 ) financial based or outcome based , when the price , the reimbursement status , or both depend on the financial and clinical impact , respectively , and 2 ) population based or patient based , when the performance is measured on the population as an aggregate or on each individual patient ( table 2 ) .
population- and outcome - based agreements , sometimes called coverage with evidence development , generally rely on new observational studies or randomized controlled trials , whereas individual- and outcome - based agreements are usually based on registries.47 both payers and the industry may take advantage of mmeas.48 in a situation where the alternative would be not reaching drugs reimbursement at the price requested by the industry , mmeas allow the product to be accessible to the patients , the companies to leave the list price in the price - corridor defined by the headquarters , and at the same time , to improve their reputation by making drugs accessible and sharing the risk of clinical failure and financial impact with payers .
on the other hand , payers have the advantage of prioritizing drugs , collecting real - world evidence , and applying , in the case of outcome - based agreements , value - based price and reimbursement .
the system as a whole takes the advantage of avoiding the risk that the drug is not available .
outcome - based agreements are more consistent with value - based pricing and reimbursement , and have the potential to influence the drugs development program differently from financial - based agreements .
the work of a dedicated task force at the international society of pharmacoeconomics and outcome research has recently disentangled important issues deriving from these agreements , including their desirability , design , governance , implementation , and impact evaluation,47 all essential factors for a successful contract .
for example , the coverage with evidence development contract on drugs for multiple sclerosis in the uk has partially failed49 due to important delays in patients recruitment and data analysis , and , on top of them , incompleteness of the contract.47 these agreements are highly desirable if the drug has an important budget impact ( high unit price and/or volume ) ; the level of uncertainty on benefits is significant , but the expected benefit is so high that the opportunity cost of delaying access to get more evidence would not be acceptable .
the uk has mainly adopted a dichotomous approach at market launch : the drug is recommended ( or recommended for a restricted target ) or not recommended . in order to make accessible drugs that are not cost - effective at list price , several strategies are in place : 1 ) companies have been allowed to require a patient access scheme : 31 of the 44 patient access schemes are discounts on list prices,50 2 ) thresholds on incremental cost - effectiveness ratio have been increased for some drugs categories ( eg , for end - of - life treatments , indicated for patients with a short life expectancy normally < 24 months small patient population and with sufficient evidence that the treatment offers an extension to life of at least 3 months , compared to current national health service treatment),51 3 ) a cancer drugs fund has been created to partially cover cancer drugs that have not been recommended ( eg , aflibercept as a second - line treatment of metastatic colorectal cancer ) or are waiting to be appraised ( eg , pertuzumab as first - line treatment of locally advanced or metastatic breast cancer).52 while in the uk , apart from patient access schemes , access is mostly driven by cost - effectiveness at market launch and many drugs have not been recommended , in italy most of the new drugs are approved with an mmea ( sometimes with multiple mmeas ) , and outcome - based contracts have been implemented , particularly on an individual basis , to oncological drugs : 25 drugs have been subject to such contracts to date . for example , everolimus has been approved with a performance - linked reimbursement contract that requires a payback to hospitals from novartis should patients not respond after 3/6 months of treatment for advanced renal carcinoma / human epidermal growth factor receptor-2negative advanced breast cancer and primitive neuroectodermal tumor , respectively .
finally , registries on 46 drugs have been created to carry out the 25 outcome - based contracts and , under a broader perspective , to review the prescribing behavior for expensive drugs.53
this study highlights particular hta activities that are spreading in most developed countries with the potential of significantly influencing the drug development paths of new pharmaceuticals . from early stages of clinical research up to postauthorization studies , there is a trend toward increased collaboration between parties,54 like regulators , developers , and hta assessors , to improve the evidence profile of new drugs , to anticipate patients access to innovative therapies , and to reduce clinical and economic risks deriving from an incomplete knowledge profile at the time of the launch.44 comprehensiveness and inclusiveness are acknowledged in a new paradigm , known as constructive hta , which , in addition to traditional hta , emphasizes the technology dynamics elements and the thorough assessment and involvement of multiple stakeholders in the production and evaluation of different types of new evidence.55,56 another emerging feature is the anticipation of market access evidence collection to early stages of development and the increasing importance of postmarketing real - world evidence .
it is accepted that the evidence required for marketing authorization and market access is different : comparative evidence and cost consequences are gaining importance in the eyes of payers , and payers are more and more important for market access . while licensing and regulatory postmarketing risk plans are quite homogeneous across regulators in different jurisdictions ( eg , ema , food and drug administration in the us ) , payers may have different preferences on comparative clinical and economic evidence , despite the effort of the european network for hta collaboration to develop common methodologies and practices for hta across european member states .
hence , the companies are offered the currently available opportunities for bipartite and tripartite early dialogue between regulators and hta assessors to identify product development plans that satisfy both interlocutors .
companies representatives who took part in the sa process generally agree that the advice given is reasonable and constructive ; some declare to have modified the development program as a result of the consultation.15,36,57 however , based on the current available experience and poor reporting of it , it is generally difficult to establish whether the modification and which modification on the drug development plan occurs as a direct consequence of the hta advice.57 beyond gaining a reimbursement agency s insight on the strategic development plan and optimizing the evidence generation program accordingly , other more subtle factors for the manufacturers to apply could be an expectation of a later - facilitated technology appraisal , the collection of an informal endorsement on the evidence program , or of the agency perspective on the development program , regardless of the intention to access that specific market.36,57 in terms of interactions between different players , models are still different in terms of the importance played by early interaction and postlaunch risk - sharing agreements , the level of transparency of this interaction , and the object of this interaction .
our research focused on two case studies that share a rather good level of transparency in the way premarketing advice is managed , but follow different appraisal and recommendation rules .
as already mentioned , heterogeneity of hta practices around europe increases the complexity of the market access environment and future european projects collaboration on early dialogue , such as the shaping european early dialogue program , are needed to address this issue.41 overall , there are concrete signals that market access departments and activities are gaining importance in the pharmaceutical companies , but there is still a lack of evidence and reporting on how the increasing relevance of hta has reshaped the way clinical development is designed and managed .
this may strongly depend on the importance played by market access issues compared with marketing approval and market access barriers : the more these barriers are diffused in countries where market potentials are high and growing , the more companies are expected to design clinical development considering market issues also.58 as our objective was to illustrate hta activities potentially influencing the drug development path of pharmaceuticals , we performed a review of information available on institutional websites or published documents .
an interesting extension of the present work would be the collection of primary data and insights from manufacturers to measure the actual extent of the impact such hta activities are having on their business . in conclusion ,
further research on how hta and regulatory authorities practices develop to manage evolving knowledge and on how manufacturers respond with an efficient and adequate drug development program , both with the ultimate interest of patients benefit , is welcome . | the use of health technology assessment ( hta ) to inform policy - making is established in most developed countries .
compared to licensing agencies , hta agencies have different interests and , therefore , different evidence requirements .
criteria for coverage or reimbursement decisions on pharmaceutical compounds vary ; however , it is common to include , as part of the hta , a comparative effectiveness evaluation .
this type of clinical data might go beyond that required for market authorization , thus creating an additional evidence gap between the regulatory and the reimbursement submission .
the relevance of submissions to hta agencies is consistently increasing in a pharmaceutical company s perspective , as market prospects are strongly influenced by third - party payers coverage . in this study
, we aim to describe current hta activities with a potential impact throughout the drug development process of pharmaceuticals , with a comparative emphasis on the systems in place in italy and in the uk . based on an extensive literature and website review , we identified three major classes of hta activities , beyond mainstream hta , with the potential to influence the drug development program : 1 ) horizon scanning and early hta ; 2 ) bipartite and tripartite early dialogue between manufacturers , regulators , and hta assessors ; and 3 ) managed market entry agreements . from early stages of clinical research up to postauthorization studies
, there is a trend toward increased collaboration between parties , anticipation of market access evidence collection , and postmarketing risk - sharing .
heterogeneity of hta practices increases the complexity of the market access environment .
overall , there are signals that market access departments are gaining importance in the pharmaceutical companies , but there is still a lack of evidence and reporting on how the increasing relevance of hta has reshaped the way clinical development is designed and managed . |
the brachial plexus supplies the upper extremity and is formed by the fusion of the anterior primary rami of the cervical ( c5 - 8 ) and the first thoracic ( t1 ) spinal nerves .
the five roots fuse to form three trunks which further branch into anterior and posterior divisions behind the clavicle .
these divisions reunite to form the medial , lateral , and posterior cords arranged around the axillary artery .
the medial pectoral nerve ( mpn ) arises from the medial cord ( c8 , t1 ) , usually pierces and supplies the pectoralis minor ( pmi ) .
a few branches of mpn also supply pectoralis major ( pmj ) . the lateral pectoral nerve ( lpn ) carries fibers from c5 - 7 to supply the pmj .
lpn communicates with the mpn to form a loop - the ansa pectoralis ( ap ) , in front of the thoracoacromial artery ( taa ) and hence lpn also supplies the pmi .
thus , pectoral muscles are normally supplied by lpn , mpn , and sometimes terminal branches from ap ( superior , middle , and inferior pectoral nerves ) may also innervate these muscles .
though some researchers have reported the ap to be absent , others have reported a 100% incidence in their studies .
if ap is present , it connects mpn with the lpn in front of the first or second part of axillary artery ; however , it is mostly distal to the lateral thoracic artery ( lta ) .
loukas et al . in 28% specimens found the ap directly connecting the lpn and mpn .
the mpn may be connected to the anterior divisions of the upper trunk ( 5% ) , middle trunk ( 25% ) or to the deep branch of the lpn ( 42% ) .
such anatomical variability in the formation of the ap are not known to affect the limb functions , but knowledge of such variations are essential in the evaluation of unexplained sensory and motor loss after trauma and surgical explorations .
during routine dissection of an embalmed 70-year - old male cadaver , a variant pattern of the brachial plexus was encountered in the left side .
the skin and subcutaneous tissue were dissected off , and the exposed pectoral muscles were reflected toward their insertion .
branches of the axillary artery were inspected and later trimmed for better visualization of the cords .
the lpn arose by a single root from the lateral cord , just distal to the origin of the taa , and measured 2.8 cm in length .
the mpn originated from the medial cord just distal to the lta and was 3.3 cm in length .
lpn and mpn united to form a loop - the ap , anterior to the second part of the axillary artery .
this conjoint nerve trunk pierced the deep surface of pmi to supply it ; thereafter it branched and supplied the pmj [ figure 1 ] .
concurrently , in the brachial plexus , a nerve communication ( nc ) existed between the lateral cord and the medial root of median nerve ( mrm ) .
this further communicated ( c ) with the conjoint trunk from the ap [ figure 2 ] . thereafter the fibers of mrm and the lateral root of median nerve ( lrm ) united at the junction of upper and middle one - third of the arm to form the median nerve ( mn ) .
nerve communication ( nc ) - between the medial root of median nerve ( mrm and lateral cord ( lc ) ; communication ( c ) - between ansa pectoralis ( ap ) and nerve communication ( mc : medial cord : mpn : medial pectoral nerve , lpn : lateral pectoral nerves , lrm : lateral root of median nerve , aa : axillary artery , cb : coracobrachialis , mcn : musculocutaneous nerve , un : ulnar nerve ) diagrammatic representation of the dissection highlighting the nerve communication ( nc ) receiving a twig from the conjoint trunk ( c ) of ansa pectoralis ( mc : medial cord , mpn : medial pectoral nerve , lpn : lateral pectoral nerve , lrm : lateral root of median nerve , aa : axillary artery , cb : coracobrachialis , mcn : musculocutaneous nerve , un : ulnar nerve ) the right side brachial plexus was found to be normal .
we describe a rare variant , wherein the entire lpn and mpn initially united to form the ap , which solely supplied the pectoral muscles through a common trunk .
we observed lpn to emerge from the lateral cord by a single root lateral to the second part of the axillary artery .
lpn may arise by a single root from the lateral cord or by two to three roots from the anterior divisions of upper and middle trunks .
mpn branched off the medial cord , arising just distal to the lta by a single root .
the mpn may arise from the medial cord ( 25% - loukas et al . ;
49.3% - porzionato et al . ) or from the anterior division of the lower trunk ( 43.8% - porzionato et al . ;
certain authors have observed ap to have 100% incidence in their studies while others have found it to be absent . in the present report ,
the mpn and lpn communicated in front of the second part of axillary artery to form the ap .
a single nerve trunk originated from the ap to supply the pectoral muscles and also communicated with the mn .
afshar and golalipour observed a similar single nerve trunk from the ap , but it supplied the muscular axillary arch of latissimus dorsi .
gupta et al . also reported a loop communication between mpn and lpn with a pseudoganglion like thickening .
variations of the brachial plexus may arise due to alterations in the migration of muscles derived from somites into the early upper limb mesenchymal primordium leading to modifications of the primitive segmental arrangement of nerves .
the anomalous innervation of the pectorals becomes particularly important while performing an axillary breast augmentation , wherein an implant is placed between the pectorals or during lymph node dissection procedures , though injury to few of the nerve branches of either pectorals usually leads to little consequence .
the pectoralis muscle island flaps are used for head , neck , and axillary reconstructive surgeries and currently neuromuscular blockade of pectoral muscles is being employed for pain relief by decreasing the muscle tone in orthopedic procedures such as shoulder dislocation .
such surgical procedures may be tricky in cases like ours , as an inadvertent sacrifice of the nerve by an unaware surgeon would lead to atrophy of both muscles because of a common nerve supply . in the brachial plexus ,
mn formation has been frequently reported to be variable with communications with other nerves being common . in our present study ,
this communication also received fibers from ap , which were probably destined to form the mn and hitch - hiked through one of the pectoral nerves to reach it .
injury to such rare communications of the brachial plexus could result in an unexplained atrophy of the pectorals and/or unexplained loss of power in few muscles supplied by mn .
| the knowledge of innervation of pectoral muscles is important to surgeons performing breast surgeries , axillary lymph node dissection , harvesting pectoralis major / minor for flaps , and during neurotization procedures where the pectoral nerves are particularly at risk .
the present case describes the innervation of the pectoral muscles solely by a nerve trunk arising from the ansa pectoralis ( ap ) - the loop of medial and lateral pectoral nerves .
interestingly , there was also a communication between the ap and roots forming the median nerve .
it is imperative for the operating surgeons to be aware of these rare variations in order to prevent denervation and subsequent atrophy of the pectoral musculature . |
meningioma has been known to be a common intracranial tumor , accounting for 13% to 19% of all primary brain tumors .
in contrast , extracranial meningioma are uncommon , and particularly those with extension into a middle ear .
especially , ear and temporal bone meningiomas have been reported to show a variety of clinical findings such as cochear and vestibular symptoms , moreover cranial nerve palsy .
we describe a case of meningioma extending to the middle ear and , based on the past history , clinical findings , audiogram , computed tomography ( ct ) , magnetic resonance imaging ( mri ) and operative findings , the symptoms of our case appeared to result from the extension of an extracranial meningioma from the sphenoid ridge to the middle ear via the petrous pyramid .
a 74-year - old female presented at our hospital with a complaint of right ear bleeding during the process of cleaning earwax two weeks earlier . in her past history , she underwent craniotomy 4 years before for the sphenoid ridge meningioma with right visual impairment . in physical examination
, a dark - red mass was seen through the right tympanic membrane with a perforation ( figure 1 ) .
a pure tone audiogram showed a mixed hearing loss with a 3040 db air - bone gap in right ear , and the left and right bone conduction were almost equal ( figure 2 ) .
she had no other ear symptoms such as tinnitis , vertigo / dizziness and facial nerve palsy .
mri ( figure 3 ) and ct ( figure 4 ) showed that the right sphenoid ridge meningioma had invaded widely into the right middle cranial fossa and involved the extension along the petrous apex and bilatearal optic nerve canals , but the internal auditory canal , facial nerve canal , jugular fossa , cochlea , vestibule , semicircular canal , facial nerve canal and ossicles were intact . the nasal fiberscopy revealed that nasal cavity and nasopharynx were intact , but did not show any tumors .
the patient underwent only partial extirpation with decompression for optic nerve , rather than total extirpation including the middle ear and temporal bone , due to the wide invasion of the middle cranial fossa and cavernous sinus .
figure 1a dark - red mass ( asterisk ) was observed behind in anterior portion of the right tympanic membrane with a perforation ( arrow ) .
a dark - red mass ( asterisk ) was observed behind in anterior portion of the right tympanic membrane with a perforation ( arrow ) .
figure 2pure - tone audiogram .
figure 3magnetic resonance imaging ( a : t1 coronal and b : t2 axial ) showed that the right sphenoid ridge meningioma ( arrow ) had invaded widely into the right middle cranial fossa and involved the extension along the petrous apex and bilatearal optic nerve , but the internal auditory canal , cochlea , vestibule , semicircular canal and jugular fossa were intact .
magnetic resonance imaging ( a : t1 coronal and b : t2 axial ) showed that the right sphenoid ridge meningioma ( arrow ) had invaded widely into the right middle cranial fossa and involved the extension along the petrous apex and bilatearal optic nerve , but the internal auditory canal , cochlea , vestibule , semicircular canal and jugular fossa were intact .
figure 4computed tomography ( a : axial and b : coronal ) showed that the meningioma had invaded the petrous apex ( arrow ) and occupied the middle ear space , but the cochlear and vestibular organs , facial nerve canal and ossicles were intact .
computed tomography ( a : axial and b : coronal ) showed that the meningioma had invaded the petrous apex ( arrow ) and occupied the middle ear space , but the cochlear and vestibular organs , facial nerve canal and ossicles were intact .
figure 5histopathology of meningioma showed proliferating spindle cells with small oval nuclei and fibrous stroma ( h&e staining x400 ) .
histopathology of meningioma showed proliferating spindle cells with small oval nuclei and fibrous stroma ( h&e staining x400 ) .
the most common sites of meningiomas are the sphenoid ridge and the dome of the cerebrum , followed by the falx cerebri and parasagittal sinus . in the arising processes of the extracranial meningioma ,
hoye reported that extracranial meningiomas could be subdivided into the following four main groupings : i ) primarily intracranial tumors with extracranial direct extension .
ii ) tumors originating from arachnoid cell rests of cranial nerve sheaths with extracranial growth .
iii ) extracranial growth without any apparent connection with foramina or cranial nerves presumably from embryonic rest of arachnoid cells .
pathways for meningioma extension into the middle ear as suggested by rietz were the tegmen tympani , greater superficial petrosal groove , anterior and posterior surfaces of the petrous pyramid , internal auditory canal , and jugular fossa .
various clinical findings can present according to the route of by which the tumrs extend .
goel , thompson et al . reported that ear and temporal bone meningiomas could show variety of clinical findings as coc - hear and vestibular symptoms or cranial nerve palsy .
reveled that hearing loss was the most common clinical prsenting feature in temproral bone meningiomas .
the compliant of our patient was hearing loss alone , but no tinnitis , vertigo / dizziness and facial nerve palsy . a pure tone audiogram showed a mixed hearing loss in right ear , and almost equals left and right bone conduction .
her imaging findings showed that the right sphenoid ridge meningioma had invaded widely into the right middle cranial fossa and involved the extension along the petrous apex and bilatearal optic nerve canals , but the internal auditory canal , facial nerve canal , jugular fossa cochlea , vestibule , semicircular canal , facial nerve canal and ossicles were intact .
therefore , bone conductive loss would be caused by aging change rather than tumor invasion for internal auditory canal , cochlea vestibule and ossicles . in her past history
, she previously underwent craniotomy 4 years before for the sphenoid ridge meningioma with right visual impairment . from the above findings , past history and literatures , the symptoms of
our case would appeared to result from the extension of an extracranial meningioma from the sphenoid ridge to the middle ear via the petrous pyramid .
the patient underwent only partial extirpation with decompression for the optic nerve , rather than total extirpation including middle ear and temporal bone , due to the wide invasion of the middle cranial fossa and cavernous sinus .
some hospitals reported that sphenoid wing and temporal meningiomas involved caversinus sinus or optic nerve like as our case .
therefore , they recommend the optic canal decompression in all patients to ameliorate or preserve visual function . | extracranial meningioma with extension into a middle ear is very uncommon .
a 74-year - old female was admitted to our hospital with right ear bleeding when removing earwax . in this case ,
magnetic resonance imaging , computed tomography , her past history and operative findings would consider as infiltrative growth from the right sphenoid ridge meningioma to the right middle ear via the right petrous pyramid and bilateral optic nerve .
she underwent only partial extirpation with decompression for optic nerve , rather than total extirpation including middle ear and temporal bone , due to wide invasion of the middle cranial fossa and caversinus sinus . |
gold catalysts , both homogeneous and heterogeneous , have seen a meteoric rise in importance in the last three decades , and interest continues unabated .
this has been amply documented , for example , in special editions of major review journals . for organic transformations , gold in the oxidation state
+ i is overwhelmingly used as the catalyst , as documented in recent reviews concerning synthetic applications .
important aspects in homogeneous gold(i ) catalysis include , inter alia , the role of oxidation states , silver activators , ligand structure and the role of couteranions , the formation of heterocycles , the nature of gold carbene intermediates , the influence of aurophilic interactions on au(i ) catalysis , silver - free gold catalysts , as well as the important role of counterions and their effect on chemo- and regioselectivity and of chiral brnsted acids in gold catalysis .
gold(iii ) catalysts have also found widespread applications in synthetic methodology , most commonly in the form of simple gold salts such as aucl3 as catalysts or catalyst precursors . while such catalysts can be fast and efficient ,
as schmidbaur pointed out in a recent review , the nature and oxidation state of the catalyst is in most cases difficult to determine and remains a matter of conjecture .
it is the most electronegative metallic element , with a pauling electronegativity value close to that of carbon and hydrogen ( au : 2.54 ; c : 2.55 ; h : 2.20 ) . as gold(i ) , with d configuration , it is akin to a main group element , while as gold(iii ) it behaves as a transition metal and displays a coordination chemistry which mirrors that of other heavy - metal d ions .
the 3 row elements platinum , gold and mercury are strongly affected by relativistic effects , which cause an expansion of the 5d orbitals and a contraction of the 6s shell and reach their maximum for gold .
there are certainly many chemical and structural similarities between the d systems platinum(ii ) and gold(iii ) , which have led to platinum and gold catalysts often being compared side - by - side .
numerous reaction mechanisms and intermediates proposed for gold catalysed reactions have been drawn up in analogy to organometallic palladium or platinum catalysts . however , there are also significant differences between those two neighbours in the periodic table .
most prominent are the redox potentials , which are very much more positive for gold than for platinum ( values under standard conditions , 1 m aqueous acid , 25 c):au / au 1.52 vau / au 1.36 vau / au 1.83 vpt / pt 1.19 v gold(iii ) is therefore very much more readily reduced than platinum(ii ) , to either au(i ) or au(0 ) , and the formation of purple gold nanoparticles during the course of unsuccessful reactions is only too familiar to practitioners of gold chemistry .
the redox chemistry of gold is illustrated , for example , by the ability of naked solvated au ions to oxidatively cleave benzylic c h bonds of arenes with an oxidation potential of up to 1.82 v , and even the c
c bonds of diarylmethanes , to give carbocation intermediates , with metallic gold being generated in the process .
oxidation catalysts based on mixtures of aucl and the diketiminate anion [ hc(mec
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
1111111111111111111111111111111111
1111111111111111111111111111111111
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
1111111111111111111111111111111111
1111111111111111111111111111111111
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
nc6h3pr
2)2 ] have been reported , the assumption being that under such conditions au(i ) diketiminato chelate complexes are generated in situ as catalytically active species .
however , it was shown subsequently that even at 0 c au(i ) and [ hc(mecnar)2 ] anions lead to the immediate reduction to metallic gold , which would seem to suggest that the observed oxidation catalysis is in all probability effected by gold nanoparticles .
in other organic transformations , reduction of au(iii ) vinyl intermediates to au(i ) could be demonstrated , while the formation of gold nanoparticles could be excluded by x - ray absorption spectroscopy ( xas ) methods .
on the other hand , there are also notable cases where au(iii ) vinyl reaction intermediates of the type [ ( vinyl)alcl3 ] and [ ( vinyl)2aucl2 ] , could be isolated and structurally characterised .
the particular bonding and redox characteristics of gold are of course reflected in the reactivity of the types of intermediates that , by analogy to other metal - catalysed reactions , are assumed to be involved in catalysis : complexes with alkenes and alkynes , co , metal hydrides and alkyls are all quite reasonable proposals , but , in the case of gold , how much is really known about such compounds ? for which ligands or ligand combinations , and for which gold oxidation states , is it realistic to assume the existence , or indeed non - existence , of such species ?
gold , as the embodiment of a noble metal , had long been regarded as unreactive and chemically uninteresting ; it is therefore a late - comer to catalysis .
this means that many aspects of fundamental organometallic chemistry , which after all forms the basis of our understanding of catalytic mechanisms , remained unexplored for gold .
the most dramatic example of this are olefin complexes : whereas platinum(ii ) provided the first alkene complex of any transition metal , zeise 's salt k[ptcl3(-c2h4 ) ] , reported in 1827 , ethylene complexes of the isoelectronic au(iii ) ion are conspicuous by their absence ( scheme 1 ) .
similarly , hydride complexes of platinum(ii ) have been known for over 50 years , whereas those of au(iii ) remained unknown .
this brief perspective presents some recent results on the identification and characteristics of some key types of catalysis - relevant gold complexes and tries to highlight the similarities and differences in bonding and reactivity of species with au
as will be seen , in some cases these gold complexes display a reactivity pattern that is rather different from that of most other transition metal compounds .
the chemistry of gold hydrides and compounds with hydrogen bonds to gold have recently been the subject of a comprehensive review .
early attempts to generate gold hydride complexes and to stabilise gold(iii ) with complex anions like bh4
and alh4
at 120 c were reported by wiberg ; the result in all cases was reduction to metallic gold .
the formation of gold hydrides and their stability is of course particularly interesting in connection with catalytic cycles since h - transfer , -h elimination or m c bond hydrogenolysis are common reaction steps in transition metal catalysis .
the first examples of isolable gold hydrido species were binuclear metal complexes ( co)5m(-h)aupph3 ( m = cr , mo , w ) and lau(-h)mln [ mln = irh2(pph3)3 or pt(c6cl5)(pet3)2 ] ; all these bimetallic compounds show strongly shielded hydride nmr resonances between 2 and 6 ppm ( in contrast to terminal au
hydrides of gold in oxidation states i , ii and iii could be identified by vibrational spectroscopy in the reactions of laser - ablated gold atoms condensed into frozen gas matrices at 5 k or below , including ( h2)auh , ( h2)auh3 , auh2 , auh4
and auh2
.
evidence for more thermally stable hydride complexes was provided by mass spectrometry , to give ions of type 1 , and by nmr studies of the reactions of ( p^p)aucl with phme2sih , which gave binuclear h - bridged compounds 2 and 3 ( scheme 2 ) ; such hydrides are thought to be involved in gold - catalysed alcohol dehydrosilylation .
the h nmr hydride signal of 2 was found at 8.29 ( j
ph 49.5 hz ) , and while the h signal of 3 could not be detected , the h resonance occurs at 7.0 ppm ( j
pd 8.4 hz ) .
the first example of a mononuclear gold(i ) hydride was isolated by sadighi in 2008 , ( ipr)auh ( 4 ) , either by the reaction of ( ipr)aucl with lihbet3 , or by treating ( ipr)auobu with hsi(ome)3 ( ipr = 1,3-bis(2,6-diisopropylphenyl)imidazole-2-ylidene ) .
the hydride ligand gives rise to a signal with positive chemical shift , at 5.11 in c6d6 ( 3.38 in cd2cl2 ) , with an au h ir stretching frequency of 1976 cm (
au - d 1407 cm ) . the same compound was also made from ( ipr)auoh .
this family of gold hydrides stabilised by n - heterocyclic carbene ( nhc ) ligands was recently increased by ring - expanded carbenes to give 5 and 6 (
h 3.57 and 3.15 , respectively , in c6d6 ) ( scheme 2 ) .
protonation of 5 or 6 with [ h(oet2][b{c6h3(cf3)2}4 ] at 80 c gave -h cations of type 1 , indicated by a high - field shift of the hydride h nmr signal by 45 ppm ; the suspected intermediate gold dihydrogen complexes [ ( nhc)au(h2 ) ] could not be detected .
hydridic character . and indeed , solid 4 is stable to air and moisture at ambient conditions , does not react with protic solvents like t - butanol , and reacts only very slowly with benzoic acid .
on the other hand , dimethyl acetylenedicarboxylate gives a trans - insertion product , and ethyldiazoacetate reacts under c h activation ( scheme 3 ) .
the hydride ligand in 4 can however be abstracted by cph3
to give a binuclear hydride - bridged cation of type 1 , indicated by the high - field shift of the h nmr signal to 0.42 .
the mesityl - substituted analogue of 4 , ( imes)auh , shows low reactivity in the hydrodefluorination of perfluoroarenes , but the reaction is accelerated by p - dimethylaminopyridine ( dmap ) and involves a spectroscopically detectable -stacked intermediate ( scheme 4 ) . under 19 bar o2 ,
complex 4 undergoes slow dioxygen insertion into the au h bond to give the hydroperoxide 7 .
product 7 readily decomposes to ( ipr)auoh ( 8) , followed by condensation of 7 with 8 to give the structurally characterised peroxide 9 ( scheme 4 ) .
the reaction of 4 with stable radicals was used to estimate the au h bond energy . whereas 4 does not react with tempo (= 2,2,6,6-tetramethyl-1-piperidyl - n - oxide ) , the hydride ligand is abstracted by galvinoxyl , with formation of the crystallographically characterised galvinoxide salt 10 ( scheme 5 ) . since the o
h bond dissociation energies ( bdes ) of tempoh and galvinoxyl - h are 291 kj mol and 329 kj mol , respectively , this provides an estimate of the au
gold(i ) hydrides could , in principle , also be formed by -h elimination of gold(i ) alkyls , and conversely , the hydrogenation of unsaturated substrates is usually thought of as involving the -insertion of a coordinated alkene or alkyne into a metal
gold(i ) is of course a d system , without suitable energetically accessible vacant orbitals for interacting with a -c h bond , nor for the coordination of an olefinic substrate .
it is therefore consistent that for ( ipr)auet a high barrier for -h elimination was found , such that this pathway becomes kinetically competitive only above the decomposition temperature of the complex ( > 200 c ) . as bertrand
has shown , both -h and , much more slowly , -h abstractions from gold alkyls are possible , but only by using an external electrophile such as cph3
, not however by intramolecular h - transfer reactions . in line with this , calculations have suggested that for alkene hydrogenations with homogeneous gold(i ) catalysts in polar solvents like ethanol , a heterolytic , solvent - assisted h2 activation pathway prevails over a -bond metathesis - type au c bond cleavage by an h2 molecule ( scheme 6 ) .
alkene hydrogenation is thought to follow an ionic mechanism , in which a proton is delivered from the solvent to the alkene .
the rate of alkene hydrogenation increases therefore as the stability of the resulting carbocation increases , e.g. cyclohexene > ethene .
whatever the detailed energetics of these solvent - assisted mechanisms , it is clear that for gold catalysts the ionic reaction profiles are always energetically favoured over the homolytic pathway .
in addition to gold(i ) catalysts , gold(iii ) catalyst precursors are also frequently employed in organic transformations . however , in the large majority of cases the fate of the gold species , the oxidation state and the coordination environment of the resulting gold species under such conditions are a matter of conjecture . the facile reduction of au(iii ) to au(i ) suggests that the active species formed in situ are most probably gold(i ) , particularly if catalysts with labile ligands are used , such as aucl3 .
the inadvertent formation of catalytically active gold nanoparticles is also not easy to rule out .
hydride complexes such as haucl2 are occasionally invoked in catalytic cycles , but as yet no such species has actually been identified .
schiff - base gold(iii ) complexes , supported and unsupported , proved to be potent catalysts for the hydrogenation of diethyl itaconate , comparable in activity to palladium catalysts .
the process of hydrogen activation ( heterolytic , ethanol - assisted ) and the likely intermediacy of au(iii ) hydride species was explored by computational methods .
square - planar au(iii ) hydride and hydrido - ethylene complexes are energetically favoured ; the alternative formation of a 5-coordinate [ ( o^n^n)auh(c2h4 ) ] species was disfavoured by an increased energy barrier ( scheme 7 ) .
this confirms the general picture that , although au(iii ) complexes with coordination numbers 5 and 6 are not unknown , the square planar geometry is strongly preferred .
there was , however , no experimental evidence that the postulated species could actually be detected .
polydentate ligands offer the possibility of stabilising complexes kinetically and may also shift the redox potentials sufficiently to allow isolation and characterisation of such elusive hydride species . cyclometallated mono- and diarylpyridine ligands provide particular stability ; e.g. 2,6-diphenylpyridine derivatives form c^n^c bonded pincer complexes with au(iii ) .
an example is the gold hydroxide 11 which has proved to be a highly versatile starting material ; for example , it undergoes aryl transfer reactions with boronic acids in toluene under neutral conditions and c h activates fluoroarenes , to give photoluminescent gold aryl complexes in essentially quantitative yields .
surprisingly , the reaction of 11 with superhydride , lihbet3 , at 78 c followed by stirring at room temperature proceeds without reduction of the metal centre and gives the gold hydride 12 as a yellow crystalline complex , the first example of an isolable au(iii ) hydrido complex ( scheme 8) .
the complex was crystallographically characterized and the gold - bound h atom was located in the electron density map . the infrared spectrum of the compound shows a sharp
au
this is higher than the value for ( ipr)auh ( 1976 cm ) but comparable to the frequencies assigned to auh and ( h2)auh in an argon matrix ( 2226.6 and 2173.6 cm , respectively ) . in the h nmr spectrum
the hydride ligand gives rise to a broad singlet at 6.51 in cd2cl2 and at 5.73 in c6d6 .
the deuteride shows a h nmr resonance at 6.58 ( cd2cl2 ) .
the observed au
h h nmr chemical shift for 12 is in line with theoretical predictions ( taking relativistic effects into account ) , which show that systems with fully filled d - shell are dominated by deshielding spin orbit coupling and signals
are therefore found at low field , ( e.g. the au(i ) complexes 46 ) , whereas for d systems the spin
orbit coupling is strongly shielding , resulting in hydride resonances high - field of tms , as is indeed observed .
the chemical shift therefore does not allow any conclusions to be drawn concerning the polarity or
the au(iii ) hydride 12 is stable to air and moisture at ambient conditions , an indication of the highly covalent character of the au
the compound was found to be moderately light sensitive , and exposing ch2cl2 solutions to sunlight for a few hours leads to chloride abstraction from the solvent , with formation of ( c^n^c)aucl .
complex 12 does not reduce co2 or benzaldehyde in the dark or under photolytic conditions , and attempted insertion reactions with ethylene , 3-hexyne , phenylacetylene , and even dimethyl acetylenedicarboxylate were unsuccessful .
however , it does react with allenes under 1,2-insertion to give isolable au(iii ) vinyl complexes .
however , treating dichloromethane or toluene solutions with the strong brnsted acid [ h(oet2)2][h2n{b(c6f5)3}2 ] at 70 c led to the disappearance of the hydride resonance , together with signals that resembled the product of cleavage of one of the au phenyl bonds , [ as for example in ( hc n^c)au(c6h4f)(o2ccf3 ) ] .
similarly , upon reacting the deuteride 12-d with [ h(oet2)2][h2n{b(c6f5)3}2 ] or trifluoroacetic acid at 70 c gave no evidence of the characteristic triplet of hd .
c(phenyl ) bond in preference to the hydride , in agreement with the covalent nature of the au
a more unusual reaction of 12 occurs if excess ( c^n^c)auoh is present : water is eliminated in a slow reductive condensation reaction , to give the gold
gold bonded dimeric au(ii ) complex 14 , a rare example of a thermally stable au(ii ) complex with an unsupported au au bond . for comparison ,
the recently reported dimer [ au(cf3)2(py)]2 follows the thermodynamic trend and spontaneously disproportionates in solution to give au(i ) and au(iii ) products .
by contrast , 14 can be heated to 120 c without decomposition , is stable to air and moisture in the solid state , and in some reactions turns out to be the unreactive end product . on uv irradiation
no reaction was observed between 14 and elemental sulfur in toluene ( 60 c , 12 h ) or with o2 under 9 bar pressure , even though calculations suggest that o2 insertion into the gold
the lack of reactivity is most probably a reflection of the steric shielding provided by the four t - butyl substituents that envelope the au au bond . on the other hand ,
the reduction of the hydride 12 to the au(ii ) dimer 14 was studied by cyclic voltammetry .
12 shows three reduction waves with peak potentials at 2.00 v ( corresponding to a metal - centred au(iii ii ) reduction ) , 2.25 v and 2.60 v vs. fc , respectively .
the 2.25 v wave was tentatively assigned to the reduction of a hydride - bridged species , such as [ ( c^n^c)au h
au(c^n^c ) ] , formed from the reaction of 12 with a ( c^n^c)au radical ( scheme 9 ) .
the reduction wave at 2.60 v corresponds to ligand based multi - electron processes . the reduction process of 12 differs from that of the hydroxide 11 , where no such binuclear intermediate could be identified , which suggested that under these conditions the au o bond is more readily cleaved than the au
h bonds in 11 and 12 ( neglecting differences in solvation between hydride and hydroxide ) , which showed that the au
this energy difference has a number of consequences and makes gold chemistry distinctive from other metals ( vide infra ) .
the oxidation of the dimer 14 ( + 0.59 v vs. fc ) was found to be irreversible at all voltage scan rates .
this seems to suggest that the 1-electron oxidation product of the au(ii ) dimer is unstable and likely undergoes rapid follow up chemistry .
there was no evidence for detectable amounts of a mixed - valence radical cation intermediate [ ( c^n^c)au au(c^n^c ) ] in the oxidation process .
electrochemistry also allowed the determination of the au au bond energy of 14 . neglecting entropic contributions , the au
au bond enthalpy was estimated as 198 1 kj mol , in reasonably good agreement with dft calculations ( 225 kj mol ) . for comparison ,
pyykk described the au au bond as a 6s6pz5dxy hybrid and calculated a bond energy of 255 kj mol for [ au(cf3)2(py)]2 .
this suggests that the stability differences between this complex and 14 is due to kinetic rather than thermodynamic factors .
heterogeneous gold catalysts are involved in a number of oxidation reactions , and the reaction pathways and the surface species that are likely to be involved have been the subject of numerous experimental and computational studies .
selective oxidations and the mode of o2 activation on oxide - supported gold catalysts for co oxidation have been reviewed .
a number of studies are concerned with oxygen activation and the role of au(-o)au species in co oxidation and at the au / oxide support interface .
au moieties have been proposed as key species in o2 activation of supported gold catalyst supported on a variety of metal oxides .
gold hydroperoxides are likely involved in the catalytic synthesis of h2o2 , as gold surface species and in catalytic methanol oxidation .
evidence has also been provided for peroxide and superoxide adsorbed on anionic and cationic gold clusters , and a variety of species including hydrides , hydroxides , peroxides and hydroperoxides are thought to be involved in propene oxidation . with few exceptions these proposed mechanisms and surface intermediates
key to rationalising all these processes is a detailed understanding of the gold oxygen bond , including possible -bonding contributions and preferred coordination geometries .
oxide bond has been the subject of considerable debate , notably the possibility ( or impossibility ) of forming terminal oxo species lnmo with metals in groups 911 ( the oxo wall ) .
indeed , the only structurally confirmed example of a noble metal oxo complex is wilkinson 's oir(mes)3 ( 15 , d ) ( scheme 10 ) , and there is indirect evidence for pt
o ( d ) .
earlier claims of an octahedral au(iii ) ( d ) oxo species with an auo double bond proved to be based on error .
the gold oxygen bond in au(i )
complexes has been the subject of some detailed theoretical investigations , e.g. ( nhc)au ocf3 ( 16 ) which , with an au o c angle of 118.3(4) , shows little evidence of -overlap . for ( nhc)au o aryl complexes of type 17
it has been argued that there is interaction between the phenolate lone electron pairs and the au c antibonding orbital .
c(aryl ) angles are indeed much wider than expected for sp hybridisation [ in the range of 121.2126(3) ] , but significant steric repulsion between the bulky nhc and aryloxide ligands must be taken into account .
in contrast to gold(i ) , which prefers coordination to soft donors like phosphorus and sulfur , gold(iii ) has long been known as a hard , lewis acidic metal ion .
this is probably best exemplified by the square - planar tetrahydroxoaurate anion , [ au(oh)4 ] ( 18 ) which forms numerous salts , as well as by the classical work of tobias concerning the identification of dimethylgold aquo ions [ me2au(oh2)2 ] , na[me2au(oh)2 ] and dimethylgold hydroxide 19 ( scheme 11 ) .
the latter is a dimer in solution but forms a tetrameric structure in the crystal , based on square - planar me2au(-oh)2 building blocks .
vicente showed that gold complexes with aryl ligands form similar compounds with au o bonds , including phenolate ( 20 ) and hydroxide ( 21 ) derivatives .
the structure of the hydroxide 22 has also been determined .
like other lewis acids , gold(iii ) readily forms adducts with pyridine and related n - donors , such as 2,2-bipyridyl and terpyridyl .
the formation of cyclometallated ortho - arylpyridine derivatives has proved a particularly successful strategy for the preparation of gold(iii ) complexes with oxo and hydroxo ligands .
scheme 11 shows a number of representative examples . while terminal auo complexes are unknown , gold(iii ) compounds with bridging oxo
ligands are well established . in 23 , for example , the square - planar metal centres form part of a planar au2o2 core , with au
o single - bond distances of 1.976(3 ) and 1.961(3 ) ( for r = ch2bu ) .
tridentate ligands form more stable pincer complexes and have been useful in providing dicationic terpyridyl - ligated gold hydroxides such as 26 , as well as the cyclometallated n^n^c - bonded complexes 27 .
however ,
as of 2012 there were no reports of isolable gold complexes containing the types of o - ligands that were being postulated in heterogeneous gold - catalysed oxidations , such as superoxides au - oo , peroxides au
we were therefore interested in the synthesis and characterisation of such compounds . given that the hydroxide ( c^n^c)auoh ( 11 ) had proved an ideal starting material for hydride synthesis ( scheme 8) , we chose this compound as the starting point for reactions with peroxides .
the reaction of 11 with t - buooh readily affords the alkylperoxide 28 . similarly ,
treatment of 11 with h2o2 resulted in the rather delicate hydroperoxide 29 , which has a tendency to condense with excess 11 to give the peroxide 30 .
such a dehydration reaction also became apparent when 11 was stirred in dry solvents ( e.g. dry acetone ) , which led to the formation of the -oxide 31 ( scheme 12 ) .
subsequently a gold(i ) t - butylhydroperoxide has also been prepared by an analogous route .
the crystal structure of the -oxide 31 also answers the question concerning possible -bonding contributions to the au
o -bonding leads to a widening of the m o m angle up to 180 and m
the au o au angle in 31 , on the other hand , is 113.5 , only slightly less acute than the tetrahedral angle of 109.5 , a rather small steric effect given the bulkiness of the two ( c^n^c)au fragments .
there is therefore no evidence for any interaction between the au(iii ) centres and the lone pairs on the bridging o ligand .
this is further borne out by dft calculations on 11 , which show that the lobes of the gold(iii ) d - orbitals and of the oxygen lone pairs in the relevant mo ( here homo1 ) are out of phase ( fig .
attempts to generate these au peroxide species directly from o2 failed : neither the hydride 12 nor the au(ii ) dimer 14 insert o2 , even under forcing conditions , in spite of the calculated exothermic character of the reaction .
the reactivity of these ( c^n^c)au(iii ) pincer complexes differs therefore from the behaviour of palladium and platinum , where o2 insertions into m
the reactivity of these gold(iii ) hydride and oxide species is in keeping with the au x bond energies .
treatment of the hydroperoxides with a phosphine par3 led to selective and stepwise o - atom abstraction , with formation of the phosphine oxide ( ar = 4-c6h4r ; r = h , f , me , ome ) ( scheme 13 ) .
remarkably , even the hydroxide 11 could be deoxygenated , to give the hydride 12 .
h > au o . the transformation of a metal hydroxide into a metal hydride ( in the absence of a hydride transfer agent ) is unique to au(iii ) and is not known for any other metal . for platinum(ii ) , the order of bond energies is reversed , pt h < pt o , and the hydroxide - to - hydride transformation is thermodynamically unfavourable .
this is therefore an example of a reaction which gold can do but platinum can not .
oh deoxygenation works for au(iii ) , gold(i ) hydroxides like ( ipr)auoh do not react . the hydroxide to hydride conversion is , in principle , a key step in a water splitting cycle which does not require oxidation state changes of the metal ( scheme 14 ) .
however , so far it has not been possible to close the cycle because the gold hydride is too stable and does not react with acids under h2 elimination .
the kinetics ( over the temperature interval 10 to 55 c , toluene ) show a secondary kinetic isotope effect k
h / k
d = 1.45 , consistent with an au o
dft studies confirmed that the oxygen is abstracted via an external attack by phosphine ( pme3 as model ) ; intermediates involving a 5-coordinate phosphine adduct of gold could not be identified .
o - labelling confirmed that the phosphine oxide formed was derived from the gold hydroxide .
the reaction is 1 order in both [ au ] and [ pr3 ] ; the kinetic parameters ( e
a = 31.4 1.7 kj mol ; h
= 35.02(67 ) kj mol ; s
= 105.9(3 ) j mol k ) show a strongly negative entropy of activation , in agreement with an associative mechanism .
the rates for different phosphines with electron donating and withdrawing para - substituents follow a hammett relationship ( = 3.15 ) , with electron - donating groups such as me or ome accelerating the rates substantially .
the negative value ( 1.05 per aryl ring ) suggests that positive charge is built up on the reaction centre and is in good agreement with a zwitterionic transition state ( scheme 15 ) .
in summary , studies in gold(iii )
oxide systems have confirmed the absence of double - bond character and of -bonding contributions in the au o bond .
both au(i ) and au(iii ) hydrides are highly covalent species , stable to air and moisture and surprisingly resistant to weak acids
. one interesting consequence of these bond energy differences was the facile o - abstraction from a gold(iii ) hydroxide to give the corresponding gold hydride , a reaction that is so far unique to au(iii ) and found neither for pt(ii ) nor for au(i ) .
the chemistry of -complexes of gold in various oxidation states with alkenes and alkynes has recently been the subject of a very detailed and comprehensive review .
the first alkene adducts of aucl were reported in the 1960s by chalk and httel ; the latter developed this chemistry throughout the 1970s and 80s .
the advent of very non - coordinating anions made it possible to isolate complexes where au(i ) is exclusively ligated to alkenes , as in the trigonal - planar [ au(c2h4)3 ] ( 32 ) , [ au(norbornene)3 ] and tetrahedral [ au(cod)2 ] cations ( cod = 1,5-cyclooctadiene ) ( 33 ) ( scheme 16 ) .
the reaction of the gold(iii ) oxide [ au2(-o)2(bipy)2 ] with alkenes proceeds with reduction to give the au(i ) bipyridyl - stabilised alkene complexes of type 34 ( bipy = variously 6,6-substituted bipyridyl ) , with alkene = styrene , p - methoxystyrene , -methylstyrene , cis - stilbene ; the complexes with norbornadiene , cod and dicyclopentadiene are alkene - bridged binuclear species .
tris(pyrazolato)borates act as bidentate n - donor ligands in the zwitterionic complex 35 .
strongly donating phosphine and nhc ligands
complexes with alkenes carrying 14 substituents are now known , including the crystallographically characterised isobutene ( l = ipr ) and tetramethylethylene derivatives ( l = bu
2pc6h4 - 2-ph ) .
structurally similar complexes have been reported for 1,3-dienes , which are -coordinated , as well as for cyclic dienes , allenes , and vinyl ethers .
an -cyclopentadiene complex of type 36 has also been characterised ( l = bu
2pc6h4 - 2-ph ) , and mixtures of ph3paucl and agsbf6 were found to polymerise cyclopentadiene and 1,3-cyclohexadiene .
a mechanism was not suggested , although au or h initiated carbocationic diene polymerisation seems most likely .
the structural chemistry of gold(i ) alkyne complexes follows the pattern set out for alkene adducts and was recently discussed in detail by schmidbaur .
the involvement of au(i ) alkyne compounds in homogeneous gold catalysed alkyne reactions has been reviewed .
two - coordinate adducts prevail , and complexes of cyclic alkynes tend to be the most stable due to the stronger -interactions of strained c
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
1111111111111111111111111111111111
1111111111111111111111111111111111
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
1111111111111111111111111111111111
1111111111111111111111111111111111
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
1111111111111111111111111111111111
1111111111111111111111111111111111
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
0000000000000000000000000000000000
c bonds with metal centres
it has proved possible to crystallographically characterise the 3-hexyne complex aucl(etccet ) , which shows a cc bond length of 1.224(6 ) , only marginally longer than that of a typical non - coordinated alkyne ( around 1.202 ) . in line with this very small degree of bond elongation , structural and dft studies on au(i ) complexes with cyclic alkynes
l bond ( l = nhc ) is 34 times larger than d(au ) *(cc ) back - donation .
these observations explain the electrophilic activation of alkynes by lau fragments in gold(i ) catalysed reactions .
with silyl alkynes , desilylation has been observed , which leads to a number of structurally characterized oligonuclear complexes ( 37 ) , not dissimilar to the interactions postulated for di - gold activation of alkynes .
nmr spectroscopic studies ( h f hoesy experiments ) showed that the relative cation anion positions in gold(i ) alkyne complexes [ ( l)au(alkyne)]bf4
have been found to vary , depending on the ligand structure ; the anion is located close to gold in the phosphine complex 39 but has close interactions with the alkyne ligand in the nhc derivative 40 ( scheme 18 ) .
anions also play an important role in the intermolecular alkoxylation of alkynes catalysed by carbene - based gold(i ) salts , ( nhc)aux ( x = [ b{c6h3(cf3)2}4 ] , bf4
, otf , ots , tfa , or oac ) .
the anion is involved in alcohol deprotonation , and the intermediate basicity of the ots anion gave the most efficient catalysis .
in addition to the mono - alkyne adducts of aucl and [ ( l)au ] , the bis- and tris - alkyne compounds 4143 have also been characterised ( scheme 19 ) .
the structures are analogues of alkene adducts , but the tris - cyclooctyne complex 42 shows a slight propeller - type twist of the ring ligands , with torsion angles of 3.0 , 6.3 and 43.9. the au
c bond lengths to the cyclooctyne with the largest twist are significantly larger than the au
this indicates weaker bonding of one of the alkyne ligands , and indeed on recrystallization some alkyne is lost and crystals of the bis - alkyne adduct could also be obtained . in the [ au(cyclooctyne)2 ] cation 43
the cc bonds in these compounds are slightly elongated compared to the corresponding free alkynes , with the largest elongation being displayed by the neutral aucl adduct , which can be expected to display the strongest back - donation .
interestingly , in nmr studies on the reaction of the thermally unstable gold(iii ) diaryl complex [ ( c6f5)2aucl]2 at 80 to 0 c laguna et al .
were able to detect some tantalising indications for the formation of an alkyne complex with the tentative structure ( c6f5)2aucl(-phcch ) (
h 3.76 , toluene - d
8) .
however , on removal of the solvent above 50 c the compound decomposed to metallic gold .
by contrast to alkynes , the first examples of alkene complexes of gold(iii ) have recently been reported , by two very different routes .
initial attempts seemed to confirm the susceptibility of gold(iii ) alkene species to reduction ; for example , efforts to displace the ether ligands in uson 's bis(pentafluorophenyl)gold(iii ) cation [ ( c6f5)2au(oet2)2 ] with norbornene or 1,5-cyclooctadiene were unsuccessful , while chloride abstraction from [ ( c6f5)2aucl2 ] with agsbf6 in the presence of norbornene led to reductive elimination of perfluorobiphenyl and formation of [ au(norbornene)3 ] .
the strategy of taking advantage of the stability of the c^n^c pincer ligand framework proved more successful .
the trifluoroacetate ligand in ( c^n^c)au(oac ) ( 44 ) is easily abstracted by b(c6f5)3 in dichloromethane , and adding alkenes to this solution at 40 c resulted in the clean formation of the corresponding -alkene complexes 4547 ( scheme 20 ) .
dichloromethane solutions of the cyclopentene and ethylene complexes are stable up to about 0 and 20 c , respectively , whereas solutions of the norbornene complex 47 can be handled at 20 c .
removal of volatiles afforded the complexes as yellow powders which can be stored indefinitely under nitrogen .
once isolated , these complexes proved stable in the solid state for hours at room temperature and in air with minimal degradation . in the absence of excess ethylene , complex
45 shows a sharp h nmr singlet at 6.29 , downfield shifted from the 5.38 signal of free c2h4 ( cd2cl2 , 40 c ) , in contrast to au(i ) and pt(ii ) ethylene complexes , which show an upfield shift of the ethylene resonance ( table 2 ) , suggestive of -electron donation which is poorly compensated by back - bonding . in the presence of excess ethylene
the h nmr signals broaden on warming from 70 to 10 c , as expected for ligand exchange .
the c2h4 complex shows a c{h } nmr resonance at 108.9 , up from 122.8 for the free alkene ; j
ch increased on coordination from 156 to 166 hz .
similar j
ch values have been found for gold(i ) ethylene complexes , although in those cases the changes in the c nmr chemical shifts ( c ) are much larger , of the order of 60 + ppm , for both au(i ) and pt(ii ) .
the au(iii)alkene bond is evidently weaker , particularly the back - bonding contribution , than the pt(ii)alkene bond .
in cd2cl2 unless indicated otherwise .
this remarkably low au(iii)ethylene back - bonding contribution , compared to pt(ii ) , is further underlined by the calculated rotational barriers .
the energy difference for the in - plane and perpendicular ethylene orientations for 45 was estimated to be only 2.4 kj mol , less than 10% of the value for isostructural cationic platinum pincer compounds ( scheme 21 ) .
this agrees with the donation / back - donation ( d / b ) ratio obtained by charge decomposition analysis , which gives a value of d / b = 3.41 for au(iii ) but d / b = 1.70 for pt(ii ) , i.e. back - donation is much more important for platinum than for gold .
this general picture , i.e. the remarkably small contribution of back - bonding for au(iii ) , has since been confirmed for other systems , including chelating olefin and co complexes ( vide infra ) .
cyclopentene coordination in 46 was indicated by a downfield shift of the olefinic proton signal from 5.74 to 6.3 ppm , as well as by the inequivalence of the methylene protons .
there was no indication of ligand exchange , which would have resulted in the interchange of the methylene - protons ha and hb ( scheme 20 ) ; the ch2 grous of -cyclopentene are diastereotopic , and coordination is via one -face only .
unlike the cyclopentene and ethylene compounds , the chch signal of the norbornene complex 47 shows only a small upfield shift from 6.02 to 5.8 ; in this case a clearer indication of cc coordination is provided by the bridgehead ch signals , which are shifted from 2.84 for free norbornene to 3.49 .
a different route to an au(iii ) alkene complex was developed by tilset and co - workers , by reacting the cyclometallated dimethylgold complex 48 with trifluoroacetic acid at 78 to 0 c in the presence of cod . under these conditions
the phenylpyridine ligand is displaced , to give cis-[me2au(cod ) ] ( 49 ) ( scheme 22 ) .
the olefinic h nmr signal is found at 6.39 , 0.8 ppm higher than the signal of free cod . the au
cod bonds in the analogous platinum complex ( 1,5-cod)ptme2 ( average au : 2.389 ; pt : 2.228 ) , while the cc bond lengths in both complexes are identical within experimental error ( average au : 1.356(5 ) ; pt : 1.355(17 ) ) ; both show slight elongation compared to free 1,5-cod [ cc 1.340(3 ) ] .
the au c bond length to the two c - atoms of the same cc bond differ by over 0.04 , a consequence of conformational strains in the cod backbone .
dft modelling showed that back - bonding from the metal dxz and dyz orbitals is substantially weaker for au(iii ) than for pt(ii ) ; this reinforces the bonding picture suggested by the spectroscopic results for compounds 4547 .
the norbornene complex 47 was found to react with traces of moisture or with alcohols to give the corresponding hydroxy- or alkoxyalkyl complexes 50 , respectively ( scheme 23 ) .
the reaction involves the protolytic cleavage of one of the au phenyl bonds of the pincer ligand .
an unusual reaction that was discovered in the course of this work is the direct insertion of ethylene into the au - trifluoroacetate bond , to give the crystallographically characterised 2-acetatoethyl complex 51 ( scheme 24 ) .
the insertion proceeds slowly ( 1472 h ) at room temperature and is quantitative ; it is accelerated by agoac impurities which catalyse the acetate abstraction .
the reaction is yet another expression of the thermodynamic trend in au o and au c bond strengths ( cf .
the mechanism of ethylene insertion into gold(iii)trifluoroacetate bonds was subsequently investigated in detail by tilset and co - workers . in trifluoroacetic acid as the solvent the ethylene insertion is fast ( 530 min ) .
the reaction is reversible , and there is ethylene exchange with c2d4 . with z - c2h2d2
the threo isomer 52-d2 is formed , arising from external anti attack by acetate on ethylene in the likely intermediate [ ( c^n)au(oac)(-c2h4 ) ] ; this ethylene complex itself could however not be detected . in trifluoroethanol as solvent the corresponding 2-alkoxyethyl complex 53 is formed ( scheme 25 ) . whereas alkene insertions into au x bonds lead to functionalised alkyls , the formation of new c c bonds requires alkene insertion into a gold alkyl bond .
these are generally not at all facile . as amgoune and bourissou were recently able to show , such a pathway becomes feasible if a coordinatively unsaturated , formally three - coordinate gold(iii ) species can be generated .
this was successful in the case of the cyclometallated phosphinonaphthalene complex 54 ( scheme 26 ) .
while cold ( 80 c ) mixtures of 54 with b(c6f5)3 as methide abstractor failed to react with ethylene or styrene and gave au c6f5 species on warming , the reaction with norbornene led to the first observation of an alkene insertion into a gold(iii ) alkyl bond .
the addition of neutral n - donors allowed the isolation of 55 , while chloride addition afforded the crystallographically characterised complex 56 .
the syn-1,2 character of the insertion process suggests an intramolecular migratory pathway via an ( undetected ) alkene complex [ ( c^p)aume(-norbornene ) ] as the intermediate .
the picture that has emerged is that au(iii ) alkene complexes can indeed be isolated and characterized , over 180 years after the first preparation of the famous pt(ii ) analogue , zeise 's salt .
they differ however substantially from the alkene complexes of both au(i ) and pt(ii ) in terms of bonding and reactivity and rely almost entirely on the ( cc)au(iii ) donor interaction , with very little back - bonding contribution and very low rotational barriers .
this makes gold(iii ) alkene compounds highly susceptible to nucleophilic attack , even by traces of moisture or weak nucleophiles such as trifluoroacetate in acidic media . as
yet there are no examples of isolable gold(iii ) alkyne adducts , most likely because alkyne complexes rely for stability even more on -back - donation .
the first carbonyl complex of gold was manchot 's aucl(co ) , first reported in 1925 . since then a number of neutral and cationic co complexes of gold(i ) have been described .
the bonding of co to gold in compounds of the type [ ( l)au(co ) ] ( l = phosphine or nhc ) has recently been analysed in detail .
these complexes are linear . in gold co complexes the donation of the non - bonding electron pair on carbon dominates over back - donation , which leads to an increase of the co stretching frequency to a value higher than that of free co ( 2143 cm ) and more akin to the formyl cation hco ( 2184 cm ) , a situation fund for quite a number of highly electrophilic metal ions ( often referred to as non - classical co complexes , a term that is not without objections ) .
for linear gold complexes [ ( l)auco ] it was shown that the
co stretching frequencies do not correlate readily with the familiar tolman electronic parameters of the l ligands ; instead the
co frequencies are mainly determined by the flux of -electron density from the l au bond .
a three - coordinate co complex with a chelating carboranyl bis(phosphine ) ligand , [ ( p^p)au co ] ( 57 ) , has also been reported ; here the increased electron density provided by the chelating phosphine increases the -back - bonding sufficiently to give the first
classical gold co complex (
co = 2113 cm ) ( scheme 27 ) .
the situation resembles that in the three - coordinate styrene complex 34 , which also shows stronger back - bonding than two - coordinate alkene complexes .
since the metal has a full complement of d - electrons and there are no energetically accessible orbitals cis to co , au(i ) carbonyls do not participate in carbonylation catalysis . on the other hand , au
co adducts are likely involved in heterogeneous gold catalysis , including the much - studied catalytic oxidation of co and of methanol .
this includes surface species of tio2-supported co oxidation catalysts where gold is thought to be present as au ions ; and co adducts with
co = 2158 cm have been identified .
however , isolable co complexes of gold in the oxidation state + iii were unknown .
indeed , as early as 1905 it was reported that treating haucl4 with co leads to reduction and is in fact an excellent method for the preparation of colloidal gold nanoparticles ; this method continues to be used for this purpose .
given that au(iii ) and pt(ii ) are isoelectronic and pt(ii ) provided the first transition metal carbonyl complex ever made , ptcl2(co)2 ( 1868 ) , the lack of isolable au(iii ) co complexes is rather surprising .
redox potentials can of course be shifted by suitable ligands , and in spite of the reducing power of co , we found recently that co complexes of au(iii ) are in fact readily accessible .
thus , treating mixtures of ( c^n^c)au(oac ) ( 44 ) and b(c6f5)3 in dichloromethane at 30 c with co generates [ ( c^n^c)au co][(c6f5)3boac ] ( 58 ) ( scheme 28 ) .
the complex can be isolated by precipitation with light petroleum as a yellow microcrystalline solid .
[ ( c^n^c)au co ] ( 58
-
c ) was obtained similarly and gives a c nmr resonance at 167.6 ( cf .
the same product was obtained when [ ( c^n^c)au(c2h4)][(c6f5)3boac ] ( 45 ) was exposed to co. the co complex is thermally labile , and solutions must be kept < 10 c
. the
co stretch of 58 was observed at 2167 cm ( 58
-
c : 2143 cm ) .
this value is rather close to that of co bound to au centres on titania - supported gold mentioned above ( 2158 cm ) , which may suggest that the co bonding in 58 resembles that found in these supported catalysts .
these are prominent in the lumo which , however , is separated from the homo by a large energy gap ( fig .
c interaction is a single bond , according to natural bond orbital analysis , with the major contributions from gold derived from the 6s , 6py and 5dx y molecular orbitals .
the low - temperature solution ir spectra of 58 and 58
-
c were always accompanied by bands at 2338 cm for co2 and 2273 cm for co2 , respectively ( ch2cl2 ) .
since the co2 must have originated from the co complex , this observation pointed to a gold - mediated water
gas shift reaction due to the presence of traces of moisture condensation under the recording conditions ( 20 c ) .
the water - gas shift ( wgs ) reaction ( co + h2o co2 + h2 ; exothermic , h
r = 41.2 kj mol ) is catalysed by heterogeneous gold and platinum catalysts , with gold exhibiting a significantly lower activation energy than platinum .
at least three mechanisms are being discussed : ( 1 ) the redox mechanism assumes reduction of the oxide support , which is then re - oxidised by h to generate h2 ; ( 2 ) the formate mechanism assumes co insertion into surface - bound au
oc(o)h , which then decomposes to give auh + co2 , and ( 3 ) in the carboxylate mechanism co and auoh give au
the nature of the catalytically active species is equally uncertain , and the debate concerns whether the active species are dispersed and mononuclear , whether they are gold nanoparticles or in fact metal ions , and if so , in which oxidation state .
there are arguments in favour of nanoparticles and gold clusters , as well for dispersed gold ions .
very recent proposals for active sites have included models such as au(o)x(oh)y(na)9 ( e.g. x = 6 , 7 ; y = 2 ) with octahedrally coordinated gold in remarkably high oxidation states , with little apparent account of the coordination chemistry and ionisation potentials of gold .
the isolation of a well - defined au(iii)co complex 58 has now allowed the role of au(iii ) in the wgs reaction to be explored . by contrast , for none of the well - known au(i ) and pt(ii ) co complexes has wgs activity been reported . in order to monitor the reaction by nmr spectroscopy , ( c^n^c)auoh ( 11 )
was used as a surrogate for water ; in which case the wgs product would be ( c^n^c)au h ( 12 ) instead of h2 , which of course has an easily recognisable nmr signature . exposing 11 to co
did indeed generate 12 , via a hypothetical carboxylate intermediate ( c^n^c)au cooh ( 59 ) ( scheme 29 ) .
reactions a , b and c are part of a wgs cycle according to the carboxylate mechanism .
calculations showed that overall steps a and b are 181 kj mol more favourable for gold than for platinum . once again , the high stability of the hydride 12 prevented the closure of the cycle , since it does not react with h2o or acids to liberate h2 .
although intermediate 59 could not be spectroscopically identified , support for its formation comes from the analogous reaction of co with the methoxide ( c^n^c)auome . the product , ( c^n^c)au coome ( 60 ) , is thermally stable and was isolated as white crystals ( scheme 30 ) . in an effort to explore possible formate formation as observed in heterogeneous wgs catalysis , attempts were made to hydrogenolyse 60 under 4 bar h2 .
however , methylformate was not observed ; the reaction was in any case calculated to be almost thermoneutral .
in addition , the reaction of 11 with co also produced the co2-bridged complex 61 , which is most probably formed in an off - cycle reaction by condensation of intermediate 59 with excess hydroxide .
compound 61 was structurally characterised ; it represents the first co2 complex of gold in any oxidation state . on heating to 80120 c , 61 was found to release co2 in a reductive elimination process , to give the thermally stable au(ii ) complex 14 ( scheme 31 ) .
the results show that there is a very significant difference in terms of susceptibility to nucleophilic attack between co complexes of au(iii ) and pt(ii ) .
this is in spite of the fact that 58 and , for example , cis - ptcl2(co)2 have very similar
co frequencies , which could be taken to mean that the c atoms in the co ligands should possess similar degrees of electrophilicity .
drastic differences were however revealed in the donation / back - donation ratio ( d / b ) , as estimated by charge decomposition analysis .
[ ( c^n^c)au(co ) ] shows a d / b ratio of 2.26 , compared to a value of only 0.65 for ptcl2(co)2 ( scheme 32 ) .
evidently back - donation , even for co complexes with high frequencies , is much more prominent in pt(ii ) than in au(iii ) .
the study also demonstrates that it is the d / b ratio , rather than the ir frequency , which provides the more informative guide to chemical reactivity .
in summary , this perspective has highlighted some recent advances in gold chemistry , alongside some of its peculiarities .
the characteristics that set gold apart from other metals , and even from its neighbour in the periodic table , platinum , are the high electronegativity , with the resulting highly covalent character of the au h and au c bonds , and the order of bond strengths : au
h > au c au o , which runs counter to experience with other transition metals .
these special characteristics need to be kept in mind in mechanistic proposals for gold catalysed reactions .
the last few years have also seen the isolation of some long sought - after representatives of important compound classes ; in particular , the first examples of gold(iii ) hydride , olefin and co complexes .
bonding studies have highlighted the absence of -bonding contributions to and from the metal centre , which appears to be the overriding bonding characteristic of gold(iii ) .
this applies to au o bonds ( absence of ligand - to - metal donation ) as well as to alkene and co complexes ( absence of metal - to - ligand back - bonding ) . in the case of co complexes
this has remarkable consequences for reactivity , and illustrates an unexpectedly drastic difference between gold and platinum .
these advances notwithstanding , many types of organometallic complexes that are commonplace for other d metals are still missing , such as complexes with labile sites cis to h , co or alkenes , also dihydrides , dicarbonyls and alkyne complexes , as well as cationic and anionic hydrides and carbonyls , hydrido and carbonyl phosphine complexes , and so forth . the synthesis of these much more reactive species will be both challenging and instructive , not least since these are the types of compounds that are likely to be realistic intermediates in catalytic cycles . | gold has seen a remarkable transformation from inert noble metal to highly reactive catalyst .
however , its organometallic chemistry contains many unknowns .
how certain can we be about the species involved in catalytic cycles ?
this perspective summarises recent discoveries . |
brown - mclean syndrome ( bms ) is an infrequently diagnosed condition , defined by peripheral corneal edema that spares the center .
clinically , bms is most often seen after cataract extraction but has been described after other surgeries as well ( e.g. , corneal transplantation ) [ 2 , 3 ] .
nonsurgical insults such as lens subluxation [ 2 , 4 ] , angle closure glaucoma , myotonic dystrophy , and keratoconus have also been described . here ,
his past ocular history was significant for bilateral congenital cataracts which required bilateral cataract extraction by phacoemulsification with posterior chamber intraocular lens ( pciol ) implantation at 2 months of age .
there was no other relevant medical ( topical or systemic medication ) or surgical history . despite this intervention ,
the patient developed bilateral amblyopia and had severe nystagmus when either eye was occluded . at the time of presentation , his visual acuity was hand motion in both eyes .
slit lamp examination of the right eye revealed a clear cornea with a fine pigmented line involving 360 degrees of the peripheral endothelium and a clear pciol .
the left eye revealed normal conjunctiva , a clear central cornea with 360 degrees of peripheral stromal corneal edema with microbullae without epithelial defect , or vascularization , and a clear pciol ( fig .
these findings were consistent with incipient bms in the right eye and established bms in the left eye .
bms , first described in 1969 , is a condition defined by the presence of peripheral corneal edema with sparing of the central cornea . in its initial phase , a pigmented line on the peripheral endothelium may be seen . on progression ,
our patient 's findings were consistent with early bms in the right and more advanced disease in the left eye .
one previous report described a 12-year - old girl with pigmented endothelial precipitates in the inferior peripheral cornea in the setting of congenital cataract removal 11 years earlier .
however , the diagnosis of bms was not certain given the lack of corneal edema .
bms typically occurs , on average , 17 years after surgery , with a widely published range of 1 month to 34 years [ 3 , 4 , 6 , 7 , 8 ] . a recent randomized , controlled study in 114 patients who underwent cataract extraction for congenital cataracts evaluated outcomes after lens implantation versus no lens implantation .
overall , complications were low . with regard to corneal edema , no patients in the aphakia group and 1 patient in the iol group developed central corneal edema that lasted more than 30 days .
the pathophysiology , which leads to peripheral corneal edema as opposed to central changes , is still unclear .
bms has been thought to be associated with altered dynamics of the iris and aqueous that can occur after surgery and/or trauma .
bms may have a genetic influence as it was reported in three members of an extended family , with a strong family history of low vision and ruptured tendons .
it has also been reported in two pairs of sisters , one pair of which had marfan syndrome .
interestingly , the condition is typically stable without progressive central corneal decompensation , and , therefore , patients usually remain asymptomatic . although specular microscopy may be performed to monitor the condition , it may not predict the clinical course , as the central cornea usually remains transparent , even when the central cell density is low .
however , transient central edema associated with increased intraocular pressure , foreign body sensation , infectious keratitis , bullous keratopathy , and corneal endotheliitis in patients with bms has been reported . as our patient was asymptomatic , at this stage , our plan is to observe him . however , prior reports of patients with bms described various treatments to alleviate ocular surface discomfort including topical corticosteroids , hypertonic solutions , and/or therapeutic contact lenses .
other reported options include stromal micropuncture of the peripheral cornea and annular amniotic membrane transplantation [ 12 , 13 ] . in summary
, our case highlights that bms may develop in children as well as in adults .
all children who undergo ocular surgery or experience ocular trauma should be monitored on a regular basis for the development of this and other potential ocular complications .
| the purpose of this manuscript is to report the case of a 12-year - old patient who presented for routine ophthalmic examination after congenital cataract surgery performed at 2 months of age .
the patient was diagnosed with bilateral brown - mclean syndrome by slit lamp examination .
no treatment was required because the patient was asymptomatic and had a clear central cornea .
this is the first described case of brown - mclean syndrome in a pediatric patient , representing the importance of clinical examination in the pediatric age group after cataract surgery because of the risk for patients of developing peripheral edema . |
acute poststreptococcal glomerulonephritis ( psagn ) is a common glomerular cause of gross hematuria in children .
it commonly presents as nephritic syndrome with sudden onset of gross hematuria , edema , hypertension , and renal insufficiency .
varied presentations have been reported such as asymptomatic , mild syndrome , or with complications , such as myocardial dysfunction , acute renal failure , or encephalopathy .
we herein report a case of an 8-year - old boy with psagn , presenting as posterior reversible encephalopathy syndrome ( pres ) .
an 8-year - old boy presented with complaints of headache and vomiting since 3 days .
he had one episode of generalized tonic clonic convulsion on the day of admission lasting for about 5 min .
there was no history of oliguria , drug intake , head injury , or koch 's contact .
the child was unconscious with a pediatric glasgow coma scale score of 8/15 ( e2 v2 m4 ) .
his heart rate was 110/min , respiratory rate was 24/min , and blood pressure was 160/120 mm hg in right upper arm ( > 95 percentile for age and sex ) .
all his peripheral pulses were felt normally . there were no neurocutaneous markers on examination .
his pupils were equal and reactive to light and fundoscopy was normal . there were no meningeal signs , focal neurological deficit , or cerebellar signs .
laboratory studies revealed : hemoglobin 9.7 g / dl , white blood count 17,200/cumm ( neutrophils 70% , lymphocytes 30% ) , and platelet count 3.6 lac / cumm .
blood urea nitrogen was 34 mg / dl and serum creatine was 1.2 mg / dl .
urine microscopy showed 3 - 4 pus cells / hpf and 100 - 120 red blood cells / hpf .
serum complement c 3 level was 26 mg / dl ( normal : 90 - 180 mg / dl ) .
the child was started on nifedipine ( 0.3 mg / kg / dose , 6 hourly ) and intravenous furosemide ( 1 mg / kg / dose , 8 hourly ) . intravenous valproic acid ( 10 mg / kg / day ) and anticerebral edema measures ( i.e. intravenous mannitol and dexamethasone ) were also started . in view of the convulsion , severe hypertension , and persistent altered sensorium a magnetic resonance ( mr ) imaging with fluid - attenuated inversion recovery ( flair ) sequence was done .
it showed hyperintense lesions in bilateral frontal and parietooccipital parenchyma on flair images suggestive of pres [ figure 1 ] .
the blood pressure was controlled and he regained full consciousness within 48 h of admission .
furosemide was omitted at the time of discharge and oral nifedipine was continued . on follow - up after 2 weeks , the child was asymptomatic with his blood pressure well - controlled .
his nifedepine was also omitted and a repeat serum complement c3 done after 8 weeks was normal , that is , 110 mg / dl .
magnetic resonance imaging brain with fluid - attenuated inversion recovery sequence shows bilateral symmetric hyperintense lesions in parieto - occipital parenchyma
pres was first reported in 1996 by hinchey et al . it is a rare clinico - radiologic condition manifesting with headache , vomiting , seizures , confusion , visual disturbances , and occasionally focal neurological deficit .
it is described mainly in conditions associated with an abrupt increase in blood pressure . in children , it has been reported in association with takayasu arteritis , ganglioneuroma , henoch - schnlein purpura , acute lymphoblastic leukaemia , steroids , hemolytic uremic syndrome , addison 's disease , hypertension , intraabdominal neurogenic tumours , porphyria , and bone marrow transplant .
psagn typically follows infection of the throat or skin by nephritogenic strains of group - a beta hemolytic streptococci .
the pathogenesis of psagn is immune - complex - mediated and results in depression in the serum complement ( c3 ) levels .
hypertension is found in up to 60% of patients with psagn and may be associated with hypertensive encephalopathy in 10% of the cases .
encephalopathy may also result from the toxic effects of the streptococcal bacteria on the central nervous system .
the pathophysiology of pres in hypertensive encephalopathy has been explained by the hyperperfusion injury " theory
. a sudden rise of blood pressure overcomes the normal autoregulation of cerebral blood flow and causes dilatation of the cerebral arterioles , resulting in brain hyperperfusion .
this increased perfusion pressure can overcome the blood - brain barrier , resulting in opening up of endothelial tight junctions , and leakage of plasma and red cells into the extracellular space , leading to cerebral edema ( vasogenic edema ) .
the relative paucity of sympathetic innervation in the posterior brain results in increased susceptibility to hyperperfusion and vasogenic edema during acute blood pressure elevations . computed tomography ( ct ) scan reveals nonenhancing hypodensities of cerebral white matter in bilateral parietooccipital lobes .
sparing of the calcarine fissure and paramedian occipital lobe structures helps differentiate pres from bilateral infarction of the posterior cerebral artery territory .
involvement of brain stem , cerebellum , basal ganglion , and frontal lobes has also been reported .
t2-weighted mr images , at the height of symptoms , characteristically show diffuse hyperintensity selectively involving the parietooccipital white matter .
children develop pres at a lower absolute pressures than adults owing to the relative left shift of their range of cerebral blood flow autoregulation
. early recognition of the condition , adequate control of hypertension , and appropriate anticonvulsant treatment are important in the management .
anticonvulsant therapy is usually given for a short period due to the reversible nature of the condition .
however , irreversible cytotoxic edema , ischemia , and infarction in brain can occur due to delay in the diagnosis and treatment . this can result in long - term sequelae including neurodevelopmental delay , neurological deficits , epilepsy , persistent vision abnormalities , or even death . in conclusion , we highlight this unusual presentation of psagn as posterior reversible encephalopathy syndrome .
also , physicians should maintain a high index of suspicion and consider psagn in their differential diagnosis when treating patients with posterior reversible encephalopathy syndrome . | posterior reversible encephalopathy syndrome ( pres ) is a rare clinicoradiologic condition manifesting with headache , seizures , altered sensorium , visual disturbances , and characteristic lesions on neuroimaging predominantly affecting the posterior regions of the brain .
we report a case of an 8-year - old boy with poststreptococcal glomerulonephritis , presenting as pres .
a magnetic resonance imaging showed hyperintense lesions in bilateral frontal and parietooccipital parenchyma on fluid - attenuated inversion recovery and t2-weighted images , suggestive of pres .
patient made a complete neurological recovery without any deficit . |
in recent years , it has been established that in patients maintained on chronic hemodialysis ( hd ) there is a strong association between markers of inflammation , nutritional status , cardiovascular disease and overall morbidity and mortality .
it was found that 3060% of hd patients have evidence of activation of inflammation [ 24 ] .
the stimuli for such a response have not been clearly defined , however , several have been proposed , such as reduced renal clearance of cytokines , accumulation of age s , occult inflammatory processes or infections , bioincompatibility of dialysis membranes and exposure to endotoxins , bacterial dna fragments , and unspecified pro - inflammatory substances present in dialysis fluid . nonetheless , activation of inflammation , even if low grade , is linked to malnutrition .
an important role in the innate immune system is played by a cd14 pattern - recognition receptor .
cd14 is a receptor for endotoxin , and binds components of gram - positive and gram - negative bacteria .
cd14 protein is present both in soluble ( scd14 ) and membrane - bound forms .
increased scd14 levels have been described in hd patients [ 68 ] and linked to increased mortality risk , inflammation and protein - energy wasting . in the promoter region of the cd14 gene polymorphism ( dbsnp : rs2569190 ) was identified at position -159 designated according to the transcription start site that corresponds to -260 from the aug start codon , which is associated with the intensity of cd14 gene product expression .
the aim of the present study was to investigate the distribution of cd14 promoter gene c-159 t polymorphism in hd patients , and to determine whether this polymorphism in the cd14 gene is associated with indices of nutritional status .
the study population consisted of 185 caucasian patients ( 81 females and 104 males ) undergoing hd in 3 centers in warsaw , 1 in bydgoszcz and 1 in bialystok , poland .
patients had been maintained on chronic hemodialysis for at least 3 months before entering the study .
causes of crf were as follows : uncertain ( n=36 ) , chronic glomerulonephritis histologically not examined ( n=54 ) , primary glomerulonephritis ( 9 ) , chronic pyelonephritis / interstitial nephritis ( n=31 ) , various cystic kidney diseases ( n=25 ) , renal vascular diseases and ischemic renal diseases ( n=16 ) , amyloidosis ( n=6 ) and other ( n=8 ) .
dietary recommendations during the study were as follows : protein 0.81.0 g / kg of ideal body mass ( with at least half of the protein being of high biologic value ) , 30% of calories derived from fat , and 62% of calories from carbohydrates .
the control group consisted of 94 apparently healthy volunteers ( 34 males , 60 females ; aged 45.717.2 y ) .
patients were eligible if they were between the ages of 18 and 90 years , had been receiving hd treatment for at least 3 months before entering the study , and agreed to participate in the study .
exclusion criteria included : recent acute inflammatory disease , history of allergy , diabetes mellitus , lupus erythematosus , wegener s granulomatosis , colitis ulcerosa , crohn s disease , history of juvenile rheumatoid arthritis , history of neoplastic disease , and treatment with steroids and immunosuppressive drugs .
subjects were weighed in light clothing to the nearest 0.5 kg and their height recorded to the nearest 0.5 cm .
skinfold thickness ( sft ) was measured using harpenden skinfold calipers at the triceps , biceps , and subscapular sites , using defined anatomical landmarks .
the waist and hip circumference were measured to the nearest 0.5 cm at the umbilicus and greater trochanter .
nutritional status was assessed using the modified sga scale ( mis ) proposed by kalantar - zadeh and colleagues .
each mis component has 4 levels of severity from 0 ( normal ) to 3 ( very severe ) .
the sum of all 10 mis components ranged from 0 to 30 , denoting increasing degree of severity .
nutritional status was also evaluated by anthropometric methods , including triceps skinfold thickness , mid - arm circumference ( mac ) and mid - arm muscle circumference ( mamc ) .
mamc was calculated as follows : skinfold measurement was performed by trained investigators ( as , tp , ap ) .
blood samples for analysis of crp , albumin , creatinine , urea , cholesterol and triglycerides were taken before scheduled hemodialysis , as a part of routine protocol .
genomic dna was isolated from blood , using a commercially available kit ( nucleospin blood , macherey - nagel , germany ) .
genotyping was performed after amplification of the target sequence using specific primers ( f 5-gtgccaacagatgaggttca -3 ; r 5-cgcagcggaaatcttcatc-3 ) .
the ethics committee of central clinical hospital of the ministry of home affairs approved the study protocol , and informed consent was obtained from all the patients and control subjects .
results are expressed as means standard deviations for normally distributed variables , or as median ( interquartile range ) .
univariate statistical analysis was performed using chi - square test , fisher s exact test or student s t - test as appropriate .
associations between variables were analyzed using pearson s correlation coefficient or spearman rank correlation , as appropriate .
genotype distributions between the study groups were compared by 22 and 23 contingency table and chi - square analysis .
stata software , version 9.2 ( stata corporation , college station , tx , usa ) was used for statistical computations .
the study population consisted of 185 caucasian patients ( 81 females and 104 males ) undergoing hd in 3 centers in warsaw , 1 in bydgoszcz and 1 in bialystok , poland .
patients had been maintained on chronic hemodialysis for at least 3 months before entering the study .
causes of crf were as follows : uncertain ( n=36 ) , chronic glomerulonephritis histologically not examined ( n=54 ) , primary glomerulonephritis ( 9 ) , chronic pyelonephritis / interstitial nephritis ( n=31 ) , various cystic kidney diseases ( n=25 ) , renal vascular diseases and ischemic renal diseases ( n=16 ) , amyloidosis ( n=6 ) and other ( n=8 ) .
dietary recommendations during the study were as follows : protein 0.81.0 g / kg of ideal body mass ( with at least half of the protein being of high biologic value ) , 30% of calories derived from fat , and 62% of calories from carbohydrates .
the control group consisted of 94 apparently healthy volunteers ( 34 males , 60 females ; aged 45.717.2 y ) .
patients were eligible if they were between the ages of 18 and 90 years , had been receiving hd treatment for at least 3 months before entering the study , and agreed to participate in the study .
exclusion criteria included : recent acute inflammatory disease , history of allergy , diabetes mellitus , lupus erythematosus , wegener s granulomatosis , colitis ulcerosa , crohn s disease , history of juvenile rheumatoid arthritis , history of neoplastic disease , and treatment with steroids and immunosuppressive drugs .
subjects were weighed in light clothing to the nearest 0.5 kg and their height recorded to the nearest 0.5 cm .
skinfold thickness ( sft ) was measured using harpenden skinfold calipers at the triceps , biceps , and subscapular sites , using defined anatomical landmarks .
the waist and hip circumference were measured to the nearest 0.5 cm at the umbilicus and greater trochanter .
nutritional status was assessed using the modified sga scale ( mis ) proposed by kalantar - zadeh and colleagues .
each mis component has 4 levels of severity from 0 ( normal ) to 3 ( very severe ) .
the sum of all 10 mis components ranged from 0 to 30 , denoting increasing degree of severity .
nutritional status was also evaluated by anthropometric methods , including triceps skinfold thickness , mid - arm circumference ( mac ) and mid - arm muscle circumference ( mamc ) .
mamc was calculated as follows : skinfold measurement was performed by trained investigators ( as , tp , ap ) .
blood samples for analysis of crp , albumin , creatinine , urea , cholesterol and triglycerides were taken before scheduled hemodialysis , as a part of routine protocol .
genomic dna was isolated from blood , using a commercially available kit ( nucleospin blood , macherey - nagel , germany ) .
genotyping was performed after amplification of the target sequence using specific primers ( f 5-gtgccaacagatgaggttca -3 ; r 5-cgcagcggaaatcttcatc-3 ) .
the ethics committee of central clinical hospital of the ministry of home affairs approved the study protocol , and informed consent was obtained from all the patients and control subjects .
results are expressed as means standard deviations for normally distributed variables , or as median ( interquartile range ) .
univariate statistical analysis was performed using chi - square test , fisher s exact test or student s t - test as appropriate .
associations between variables were analyzed using pearson s correlation coefficient or spearman rank correlation , as appropriate .
genotype distributions between the study groups were compared by 22 and 23 contingency table and chi - square analysis .
stata software , version 9.2 ( stata corporation , college station , tx , usa ) was used for statistical computations .
the frequencies of the c and the t allele in hd subjects were 61.3% and 38.7% , respectively .
sixty - seven ( 36.0% ) were homozygous for the c allele ( cc ) , 89 were heterozygous ( ct , 48.1% ) , 26 patients ( 14.1% ) had the genotype tt , and in 3 cases ( 1.6% ) it was not possible to determine genotype due to technical reasons . in the control group , frequencies of the c and the t allele were 55.9% and 44.2% , respectively .
twenty - eight patients ( 29.8% ) had cc , 49 ( 52.1% ) had ct , and 17 ( 18.1% ) had tt genotype .
neither allele ( p=0.225 ) nor genotype ( p=0.460 ) frequencies differed between hd patients and controls .
allele and genotype frequencies did not show a significant departure from the hardy weinberg equilibrium in hd patients ( p=0.630 ) or controls ( p=0.580 ) .
there were no differences in investigated biochemical ( with the exception of triglycerides ) , clinical and anthropometrical parameters , between groups defined by the presence of non - c allele status ( tt ) or c allele presence ( ct , cc ) in the cd14 promoter gene 159 site ( table 2 ) .
interestingly , carriers of the c allele had lower triglyceride levels in comparison to tt genotype ( p=0.007 ) .
due to technical reasons , il-6 level determinations were possible only in 115 patients , and therefore are not given in table i. its concentration was 8.5 pg / ml ( 5.414.3 ; n=98 ) 8.2 pg / ml ( 5.211.2 ; n=17 ) in c allele ( ct / cc ) and non - c allele ( tt ) allele carriers , respectively ( ns ) .
the frequencies of the c and the t allele in hd subjects were 61.3% and 38.7% , respectively .
sixty - seven ( 36.0% ) were homozygous for the c allele ( cc ) , 89 were heterozygous ( ct , 48.1% ) , 26 patients ( 14.1% ) had the genotype tt , and in 3 cases ( 1.6% ) it was not possible to determine genotype due to technical reasons . in the control group , frequencies of the c and the t allele were 55.9% and 44.2% , respectively .
twenty - eight patients ( 29.8% ) had cc , 49 ( 52.1% ) had ct , and 17 ( 18.1% ) had tt genotype .
neither allele ( p=0.225 ) nor genotype ( p=0.460 ) frequencies differed between hd patients and controls .
allele and genotype frequencies did not show a significant departure from the hardy weinberg equilibrium in hd patients ( p=0.630 ) or controls ( p=0.580 ) . there were no differences
in investigated biochemical ( with the exception of triglycerides ) , clinical and anthropometrical parameters , between groups defined by the presence of non - c allele status ( tt ) or c allele presence ( ct , cc ) in the cd14 promoter gene 159 site ( table 2 ) .
interestingly , carriers of the c allele had lower triglyceride levels in comparison to tt genotype ( p=0.007 ) .
due to technical reasons , il-6 level determinations were possible only in 115 patients , and therefore are not given in table i. its concentration was 8.5 pg / ml ( 5.414.3 ; n=98 ) 8.2 pg / ml ( 5.211.2 ; n=17 ) in c allele ( ct / cc ) and non - c allele ( tt ) allele carriers , respectively ( ns ) .
we found no association between cd14 c-159 t polymorphism and indices of nutrition in patients on maintenance hemodialysis therapy .
genotype and allele frequencies in our study were not different from the control group , and were similar to previously described results from central europe . additionally , allele and genotype frequencies did not show a significant departure from the hardy weinberg equilibrium . in recent papers , groups from the usa and sweden have show in 2 separate cohorts that scd14 levels correlate positively with levels of several inflammatory markers , and independently predict mortality in hd patients .
there is a growing body of evidence that inflammatory response in chronic hd patients is at least in part induced by cytokines .
the simultaneous combination of malnutrition and inflammation has been referred to as malnutrition - inflammation complex syndrome ( mics ) or malnutrition - inflammation - atherosclerosis syndrome .
mics appears to play a central role in poor clinical outcome , including the high rate of mortality and hospitalization and diminished quality of life seen in dialysis patients .
reverse epidemiology of cardiovascular risks in these patients , where a low , and not a high , bmi or serum cholesterol is associated with poor dialysis outcome several putative factors were proposed as triggers of this reaction . from previous studies it appears , however , that endotoxin is one of the most active .
lipopolysaccharide ( lps ) , after interacting with lps - binding protein , binds to cd14 , tlr4 and md4 - 2 , making lipopolysaccharide - recognition complex .
this complex activates nuclear factor-b , which moves into the nucleus and induces the transcriptional activation of inflammatory and immune - response genes , including tumor necrosis factor- ( tnf- ) .
in addition , caspase-1 is activated , resulting in the activation of the pro - inflammatory cytokine il-1 .
cd14 is upregulated by bacterial stimuli , ifn- and tnf- , and is downregulated by th2-type cytokines such as il-4 .
low concentrations of lps induce inflammatory genes , including il-10 , il-12 p35 , tnf- , and irf-1 through cd14 and tlr4-dependent pathways in macrophages .
pretreatment of healthy subjects with a cd14-specific monoclonal antibody prior to lps attenuates lps - induced fever , clinical symptoms , and leukocyte activation and degranulation , and inhibits release of tnf - alpha , il-6 and il-10 , and delays the release of stnfr(i ) and il-1ra .
wang et al . reported that anti - cd14 antibody can alleviate the progression of acute necrotizing pancreatitis in mice .
sharif et al . found that , in mice lacking either tlr4 or cd14 receptors , the severity of acute experimental pancreatitis was ameliorated .
cd14-deficient mice are resistant to shock induced by either gram - negative bacteria or lps .
tt homozygotes appear to have significantly higher scd14 serum levels , and higher cd14 density on the surface of monocytes than do carriers of both the cc and ct genotypes , although some authors dispute this .
the cd14 - 260 polymorphism is also associated with il-1 beta levels , and higher values were found in c homozygotes .
no association was found between the cd14 - 260 genotypes or the tnf-308 cd14 - 260 genotypes and the leukocyte tnf- and il-1 synthesis capacity upon endotoxin stimulation . in clinical reports ,
cd14 promoter gene c-159 t polymorphism was reported to be associated with decreased kidney function , progression of iga nephropathy , carotid artery disease in hd patients , coronary heart disease , septic shock susceptibility and mortality , and may affect susceptibility to allergic asthma . although rahman et al . found that soluble cd14 receptor expression was associated with severity of acute pancreatitis , c-260 t cd14 genotype was not .
we have found significant association of cd14 c-159 t polymorphism with serum triglyceride levels , but this must be interpreted with caution .
this finding was not confirmed , however , by a group from europe in a population closely related to ours ethnically .
it should be mentioned , however , that age of our subjects was more similar ( mean 61.2 ) to the report by shin et al .
( mean 64.8 years ) than to hubacek s paper ( mean 48.9 years ) .
firstly , and most important , is the small sample size of the study , which therefore may be regarded rather as hypothesis - generating .
secondly , our patients were relatively well nourished , and this might have obscured a weak relationship with genotype cd14 c-159 t polymorphism .
while our study focused on the impact of only 1 polymorphism , a combination of different polymorphisms , as well as environmental factors , very likely might have influenced the investigated parameters .
in conclusion , the results of this study indicate that cd14 promoter gene c-159 t polymorphism does not seem to be associated with nutritional status parameters in hd patients .
it may , however , influence triglyceride blood levels . the possible influence of other genetic polymorphisms on nutritional status and inflammatory response in chronic kidney disease patients should be investigated . | summarybackgroundcd14 is a membrane glycoprotein that acts as a co - receptor for the detection of bacterial lipopolysaccharide ( lps ) .
mutual interaction between cd14 and lps plays an important role in the innate immune system .
increased serum soluble cd14 levels have been described in hemodialysis ( hd ) patients , and linked to increased mortality risk , inflammation and protein - energy wasting .
the expression of cd14 may be influenced by cd14 promoter gene c-159 t polymorphism.this study aimed to clarify the possible association between cd14 promoter gene c-159 t polymorphism and nutritional status in hemodialysis patients.material/methodsthe study population consisted of 185 ( 104 males ; 81 females ) long - term hd patients treated in 5 dialysis centers .
the control group consisted of 112 apparently healthy volunteers ( 32 males and 80 females ) .
nutritional status was assessed using a modified sga scale , and anthropometric methods ( bmi , whr , waist , hip and mid - arm circumferences , biceps , triceps , subocular and subscapular skinfolds ) .
biochemical parameters evaluated included : crp , albumin , creatinine , urea , cholesterol , triglycerides and tibc .
cd14 promoter gene c-159 t polymorphism was determined by restriction fragment length polymorphism , after digestion of the pcr product with hae iii restriction endonuclease.resultsgenotype and allele frequencies were similar to controls and compliant with hardy - weinberg equilibrium .
no between - group differences were detected in measured variables with the exception of lower triglyceride levels in carriers of c allele in comparison to tt genotype.conclusionscd14 promoter gene c-159 t polymorphism does not seem to be associated with nutritional status parameters in hd patients . it does seem , however , to influence triglyceride blood levels . |
it is believed that physical activity for the prevention of falls should primarily aim to
increase the strength of large groups of muscles , mainly in the lower limbs , as well as
improve gait , balance , and coordination parameters1 . among the organized exercise systems
only tai - chi has been
mentioned in this context , and the importance of physiotherapy is emphasized2 .
however , it has not been determined which
specific forms of rehabilitation activities should be undertaken in order to achieve
satisfactory results in this field .
it involves marching
using poles , and is an adaptation of cross - country skiing .
the main purpose of using the
poles is to involve the muscles that are not used during normal walking .
this enables high
intensity exercises to be performed at a relatively low level of perceived exertion .
nw is
also a form of physical activity which is recommended for the elderly .
there is evidence of
its importance in the prevention and physiotherapy of musculoskeletal disorders , vascular
diseases , cardiovascular diseases and parkinson s disease3 .
reported that nw improved all health - related
components of fitness of the elderly based on functional tests , however , no improvement of
gait parameters was observed4 .
takeshima
et al . have confirmed the efficacy of a 12-week nw training program and its positive impact
on selected fitness parameters on the basis of a physical fitness test and a test on a force
platform5 . however , gait parameters were
not assessed . in another study
it was demonstrated that nw training improved both the
strength and walking speed of the elderly , compared to the traditional walking training6 .
exercise is recommended to improve abnormal posture control
during walking , and the use of exercises focused on performing two independent tasks has a
particular impact on the improvement of gait parameters and postural control7 . in this study , a set of objective tests were used to evaluate the fitness elements related
specifically to postural control , especially those used during daily routines , such as
reaching in various directions or bending the body and gait parameters .
this study used a
force platform to evaluate performance in specific functional tests the forward reach test
( frt ) and the upward reach test ( urt ) to evaluate the changes in postural control during
reaching .
moreover , kinematic gait analysis was performed in order to assess the potential
impact of nw training on selected gait parameters .
our hypothesis was that a long - term nw
training would improve gait symmetry , lengthen the gait cycle , and decrease the frequency of
the strides made eliciting greater economy of energy and improvement in incorrect gait
patterns of the elderly .
gait analysis of this study mainly focused on assessment of
coordination and symmetry of gait .
the aim of the study was to evaluate the effects of 12
weeks nordic walking training on selected gait parameters and elements of fitness related to
postural control .
the subjects were randomly
assigned to 2 groups : the experimental group ( nwg ) and the control group ( cg ) ( fig .
the tests were conducted by
blinded assessors , in a single blind experiment ; the person making the measurements did not
know to which study group a given subject belonged .
simple random sampling was used , with
unmarked envelopes and a 1:1 chance of drawing a group .
the flow of participants through the trial sixty - seven volunteers who met the inclusion criteria , were enrolled in the study .
the
inclusion criteria were : age between 65 and 74 years , no regular physical activity in
leisure time , and a willingness to participate in the study .
the exclusion criteria were :
age , regular forms of physical activity undertaken in leisure time , professional activity ,
musculoskeletal dysfunctions preventing exercise and walking , acute inflammations possibly
associated with a disease , previous episodes of cardiac arrest , uncontrolled arrhythmias ,
chronic heart failure ( nyha 3 and 4 ) , uncontrolled and untreated high blood pressure ( over
140/90 mm / hg at rest ) , uncontrolled asthma , diabetes with insulin treatment , liver or renal
failure , neoplastic disease or medical advice against taking exercise .
the level of physical activity of all subjects was assessed using a physical activity
questionnaires the 7-day physical activity recall ( 7par)8 , in order to compare the initial level of physical activity between
the groups at baseline .
all the patients were informed about the purpose and procedures of the study in detail and
gave their consent to participation in the study .
the study received the approval of the
local bioethical committee of university of medical sciences no .
the subjects of the experimental group and the control group performed the same set of
tests at the beginning and at the end of the 12-week training period .
the functional equilibrium tests performed on the force platform aimed to evaluate postural
control while reaching .
the tests were performed with the subjects standing on a force platform ( cq stab
poland ) placed against a wall .
measuring tapes were stuck to the wall , in accordance with
the diagram presented in the study by row et al9 .
the assessed parameters included the length of forward reach
( measured tape affixed horizontally ) and upward reach ( centimetre tape affixed at a
45-degree angle ) , and the forward displacement of the centre of pressure ( cop ) in relation
to the initial position of the patients , labeled point 0 . point 0 was determined as the
vertical projection from the centimetre tape stuck to the wall onto the furthest point of
the foot on the force platform .
these points were marked with a special tape , which the
subjects were not allowed to cross , and were identical for all measurements and subjects .
the position of the platform in the baseline and post - intervention tests was identical
( marked with a tape stuck to the floor ) , the position of the feet and the upper limbs also
remained unchanged ( the tape stuck to the platform , the measuring tapes stuck to the wall ) .
the evaluated parameters included the forward displacement of the cop ( the furthest point in
the computer diagram ) , and the forward and upward reach ( forward or upward movement of the
fingers on the centimetre tape ) .
both in the baseline and post - intervention tests , the
patients performed the test 3 times and the best result , in which the patient reached the
furthest , was chosen for use in the analysis .
gait parameters were evaluated on a zebris fdm - t treadmill ( zebris medical
gmbh , germany ) .
this treadmill has often been used as a research tool to examine the
elderly , and patients with neurological deficits10 ,
11 .
the subjects performed two trials
at baseline and post - intervention at a rate of 4 km / h , which reflected their average walking
speed .
the first trial lasted for 30 seconds and was supposed to prepare the patient for
walking on the treadmill , the second lasted for 5 min and the results were used in the
analysis .
a training session lasted for 75 minutes : 10 minutes of warm - up
activities with stretching , 60 minutes of walking and 5 minutes of cool - down
activities
with stretching . each session was performed in a group and was supervised by two
instructors . the walking distance and pace of the groups
were gradually increased by the
instructors who took into consideration both the period of the intervention and the level of
perceived exertion of the participants ( mean pace : 1st week : 4.20.2 km / h ; 12th week :
5.60.2 km / h ) .
the pace of walking was monitored by the instructors , using handheld
equipment ( polar gps sensor g5 , polar electro oy , kempele ) . in each session ,
when
participants reached half the distance , they had short break during which they performed
breathing exercises .
the participants in both groups received the recommendation , that they
should perform exercise , no less than 12 and no more than 14 points on the perceived
exertion scale12 .
the subjects of cg
received detailed instructions regarding walking training , which they performed unsupervised
over the next 12 weeks .
moreover , each cg subject was given a gps sensor with instructions
to turn it on before starting a walk , and to turn it off after finishing the walk .
once a
month the data concerning the amount of training and the times of individual training were
checked .
the subjects of both groups ( nwg and cg ) were told not to take up any additional
forms of physical activity , and not to change their current eating and motor habits during
the course of the study . before the start of the training , the subjects in nwg were taught
to the proper technique of walking with poles ( a 2-hour instruction ) .
as data
were not normally distributed , comparisons between the two study groups were performed using
the mann - whitney test . for paired variables ,
the subjects of both groups were similar in terms of the basic descriptive characteristics
and the level of physical activity as assessed by 7par ( table 1table 1.the descriptive characteristics of the subjects ( mean sd)featurenordic walking group ( n=28)control group ( n=29)age ( years)70.5 3.771 2.9genderfemale 100%female 100%weight ( kg)67 9.966.4 11.2bmi ( kg / m)27.13 3.726.9 4.5height ( cm)157.2 5.9159.5 5.8the time spent in physical activity during leisure in
the week preceding the study ( 7par hours : min)rest : 45:52rest : 46:00light pa : 84:12light pa : 80:16average pa : 31:02average pa : 30:01high pa : 05:51high pa : 06:45very high pa : 0:00very high pa : 0:00there were no significant differences in any of the parameters between the nordic
walking group and the control group . ) .
there were no significant differences in any of the parameters between the nordic
walking group and the control group .
the results obtained in the urt and frt tests demonstrate that after nw training , the nwg
subjects showed significant improvement in the range of reach in both tests , compared to the
baseline .
also the difference between the urt results at the baseline and post - intervention
tests proved to be significantly different from that of the control group . neither group
showed a statistically significant improvement in the maximum forward cop displacement
( table 2table 2.the results of the forward reach test and the upward reach test performed on a
stabilometric platform ( mean sd)testnordic walking groupcontrol groupbaseline test frt ( cm)114.83.4115.93.8end test frt ( cm)115.84.6116.74.1baseline test frt cop ( mm)89.0510.384.813.2end test frt cop ( mm)85.109.480.513.2baseline test urt ( cm)105.55.9105.23.7end test urt ( cm)107.85.5105.94.4baseline test urt cop ( mm)87.1310.384.613.8end test urt cop ( mm)82.012.281.912.8frt : forward reach test , urt : upward reach test , cop : centre of pressure .
p < 0.05 in the wilcoxon test.p<0.05 for differences
between the groups in the changes from baseline to post - intervention ) .
frt : forward reach test , urt : upward reach test , cop : centre of pressure .
p < 0.05 in the wilcoxon test.p<0.05 for differences
between the groups in the changes from baseline to post - intervention gait analysis failed to identify any significant changes during the evaluation of a single
stride and any of its phases .
the only parameter which showed a noticeable change was the
slightly reduced percentage difference from baseline in the swing phase of the control group
at post - intervention .
however , the analysis of the full gait cycle showed it was longer ( cm )
at post - intervention in nwg , and this was accompanied by prolongation of the time of strides
and a reduction in their frequency compared to cg ( table 3table 3.the treadmill walking parameters ( mean sd)gait parametersnordic walking groupcontrol groupbaselineendbaselineendwidth ( cm)6.21.65.91.65.91.85.81.8the difference in stride length between the left and
the right leg ( cm)2.42.32.91.82.41.63.11.3 the difference in stride - making time between the
right and the left leg ( sec)0.010.010.010.010.090.010.010.01the percentage difference in the total time of the
stance phase between the left and the right leg ( % ) 0.80.50.70.71.10.70.70.4 load response ( % ) 0.90.70.80.91,00.50.70.6 single support ( % ) 0.81.30.60.61.11.10.70.4 pre - swing ( % ) 0.90.70.840.91.00.60.70.5the percentage difference in swing phase time between
the left and the right leg ( % ) 0.81.30.70.61.11.10.70.4the percentage of double support time ( % ) 25.02.324.12.425.52.324.82.0the length of the cycle ( cm)101.89.01112.411.2106.212.4108.910.5the time of the cycle ( sec)1.000.11.10.11.00.11.00.1cadence ( strides / min)60.66.955.67.157.75.256.94.3p < 0.05 in the wilcoxon test .
p<0.05 for differences
between the groups in the changes from baseline to post - intervention ) .
p<0.05 for differences
between the groups in the changes from baseline to post - intervention
the study attempted to evaluate the impact of nordic walking training , on some parameters
of functional fitness of women over 65 years of age .
the evaluated parameters were
associated with postural control , balance and gait , i.e. the elements of fitness which may
play important roles in the prevention of falls . in most cases , the obtained results were
consistent with the research hypothesis and showed that nordic walking has potential as a
form of exercise for the elderly .
postural control is defined as the ability
to control the body s position in space for the purposes of stability and orientation , and
is critical during standing balance and walking tasks13 .
postural control was analyzed using a stabilometric platform ,
which is often used in studies of the elderly and in neurology14 , 15 .
however , in
the present study it was used as an auxiliary tool to evaluate the displacement of the
centre of pressure ( cop ) during two functional tests , urt and frt .
moreover , urt has been defined as
the more demanding test , reflecting everyday - life activities better than frt9 .
drawing on the experience of the authors ,
the measurements were performed in a similar way , but facilitated by the placement of
coloured sticky tapes on the floor and the stabilometric platform , and measuring tapes on
the wall , which were not removed throughout the study in order to maintain a uniform
standard of research .
the results suggest that only nwg subjects improved their range of
reach the both in the frt and urt tests , however only significant found between nwg and cg
was in the post - intervention changes from baseline in the urt test . no significant changes
in the forward displacement of the centre of pressure
moreover , the average results indicate that there was a decreasing trend in
this result , which may suggest that during trials , the nwg subjects moved cop to their heels
and at the same time tried to reach forward and upward more boldly .
it is possible that nw
training did not increase body control while moving cop forward during the urt and frt
tests , but it could have improved muscle strength and trunk stability , thereby improving
postural control .
the urt or frt tests help to evaluate the ability to maintain balance during a functional
task16 , 17 , and perhaps , in the future , these tests should be incorporated
into a comprehensive assessment of dynamic balance and postural control .
parkatti et
al.4 , demonstrated a statistically
significant improvement in all the components of the fitness test for the elderly , apart
from the backscratch test .
similar results were reported by lee et al.18 , and by kukkonen - harjula et al . in a study
in which the tested women showed improvements in most tests evaluating physical fitness , but
each time on the basis of a different set of tests19 . in that study measurements of postural control
were not taken into
account , and balance was assessed using the standing on one - leg time and walking backwards .
it seems that the strength of the trunk muscles plays a vital role in maintaining balance
when crossing the circumference of the support base , and during each phase of walking the
erector spinae and multifidus muscles are engaged20 .
these muscles probably work in a similar way regardless of whether
we use poles or not , especially since opinions about the reduced burden on the legs and the
trunk during walking with poles were premature21 ,
22 .
the evaluation of the involvement
of individual trunk muscles during nw calls for further investigation .
studies suggest that gait parameters should be evaluated at least once a year23 , since selected gait parameters may become
an important index for determining the risk of falling .
our study results are promising ,
because they demonstrate that in the nordic walking group the length ( cm ) and time ( sec ) of
the gait cycle were increased and the frequency of strides decreased .
earlier studies have
demonstrated that walking training performed once a week , can improve gait speed and
performance during the timed up & go test24 .
moreover , the results of our study are consistent with other
findings that show that the use of different forms of unconventional walking ( e.g. with
music or with exaggerated upper - body involvement ) improves gait parameters25 .
the assumptions of the present study seem similar to those made by takeshima et al5 . in their study
, it was suggested that nw
should be the recommended form of physical activity for improving fitness and reducing the
risk of fall in the elderly . in our study
however , it seems that the intervention had a
greater efficacy among elderly persons with high risk of falls or a history of falls .
the subjects of this study were only women ( no man volunteered ) ,
therefore , it is difficult to draw conclusions for the entire population of the elderly and ,
in order to draw definitive conclusions , the size of the experimental group needs to be
increased .
for this reason , an objective and universally recognized functional test should be used at
the later stage of the study for the comparison of results . at this stage of the study there
was no supervised control group and it was difficult to determine whether walking would not
affect the results in a way similar to the nordic walking training . therefore , at a further
stage of the study , a group will be created which , instead of the nordic walking training ,
will perform traditional walking in a supervised form .
finally , it is in our opinion that 12-week nw training has a beneficial impact on selected
gait parameters and may improve postural control , on the basis of results of selected
functional tests performed by women between the ages of 65 and 74 . | [ purpose ] to assess the effect of 12-weeks nordic walking training on gait parameters and
some elements of postural control .
[ subjects and methods ] sixty - seven women aged 65 to
74 years were enrolled in this study .
the subjects were divided into a nordic walking
group ( 12 weeks of nordic walking training , 3 times a week for 75 minutes ) and a control
group . in both study groups , a set of functional tests
were conducted at the beginning and
at the end of the study : the forward reach test ( frt ) and the upward reach test ( urt ) on a
stabilometric platform , and the analysis of gait parameters on a treadmill .
[ results ] the
nw group showed improvements in : the range of reach in the frt test and the urt test in
compared to the control group .
the length of the gait cycle and gait cycle frequency also
showed changes in the nw group compared to the control group .
[ conclusion ] a 12-week nw
training program had a positive impact on selected gait parameters and may improve the
postural control of women aged over 65 according to the results selected functional
tests . |
cardiac rehabilitation ( cr ) is a multidisciplinary multifaceted intervention aiming at physical , psychological and social recovery of cardiac patients after a cardiac event or therapeutic intervention .
traditional indications for cr are related to coronary artery disease ( cad ) , including patients with myocardial infarction , angina pectoris and patients who underwent coronary artery bypass grafting or percutaneous transluminal coronary angioplasty .
previous studies showed that cr is highly effective in these patients , both in terms of improving quality of life and in reducing the risk of future events [ 13 ] . in the last decade , it has become clear that cr is not only beneficial for cad patients but also for patients with chronic heart failure , congenital heart disease , arrhythmias , cardiac neurosis and patients who underwent heart transplantation , valvular surgery or implantable cardioverter defibrillator ( icd ) implantation [ 47 ] . because of this rise in referral indications and the increasing prevalence of cardiovascular patients due to ageing of our population , it is crucial that cr is widely available and well integrated in daily clinical practice . in 2004 ,
the netherlands society of cardiology released the current cr guideline ( richtlijn hartrevalidatie 2004 ) .
given the complexity of this guideline and the involvement of many healthcare disciplines ( cardiologist , nurse / nurse practitioner , physical therapist , dietician , social worker , medical psychologist , sports / rehabilitation physician ) , it was decided by the cr committee that a clinical algorithm had to be developed serving to aid healthcare professionals in implementing this guideline in daily practice .
this clinical algorithm ( beslisboom hartrevalidatie 2004 ) consists of a flowchart of questions on physical , psychological and social issues , using both measurement instruments ( for example , the shuttle walk test , macnew heart disease health - related quality - of - life questionnaire [ 9 , 10 ] ) and clinical judgment .
the algorithm uses this information to set individual treatment goals and to advise the clinician on the contents of the cr programme , which may consist of four different group - based treatment modalities ( exercise training , education therapy , lifestyle change therapy , and relaxation / stress management ) , and/or individual treatment .
concurrent with the clinical algorithm , a computerised decision support system was developed , serving to facilitate the use of the clinical algorithm .
this system ( cardiac rehabilitation decision support system , cardss ) actively guides its users through the clinical algorithm , prompting for necessary information and calculating scores of questionnaires .
a recent trial in a cohort of 2,787 patients from 21 centres showed that cardss increases the compliance with guideline - recommended therapeutic decisions .
however , this study also revealed shortcomings of the 2004 clinical algorithm , as illustrated by large variations in several baseline variables between hospitals , even after correction for differences in patient populations .
inter - practice variations were particularly large if assessments were based on clinical judgements rather than on clinical assessment instruments .
another shortcoming of the clinical algorithm was the deficiency of several items that have been shown to be relevant for cr ( e.g. assessment of anxiety , depression and cardiac risk profile ) . to overcome the mentioned shortcomings and deficiencies of the 2004 clinical algorithm and to further improve implementation of the dutch cr guideline , the algorithm was recently updated .
this article describes the process and the changes that were made and discusses its role in the development of cr in the netherlands .
as already mentioned , this was done by assessing inter - practice variations in the data recorded during the needs assessment procedure .
in addition , cognitive , organisational and environmental barriers to guideline implementation were assessed by conducting interviews with healthcare professionals using cardss .
a multidisciplinary expert advisory panel was established , consisting of healthcare professionals who were involved in the development of the 2004 dutch cardiac rehabilitation guidelines .
members of this panel were instructed to select deficiencies and shortcomings needing revision from a list that was composed during the first phase .
in addition , they were requested to provide specific recommendations for revision of these items based on available scientific evidence .
the utility of clinical assessment instruments was evaluated by criteria developed by the scientific advisory committee ( sac ) of the medical outcomes trust .
finally , the expert advisory group was asked to propose new items that should be considered for inclusion in the clinical algorithm . in the third phase ,
a guideline development group , consisting of members of all dutch professional associations involved in cr , decided which revisions proposed by the expert advisory group were finally included in the clinical algorithm 2010 . in this phase , decisions were not only based on scientific evidence but also on applicability in daily clinical practice .
table 1 shows all shortcomings / deficiencies of the 2004 clinical algorithm that were selected during the revision process and the changes that were made in the 2010 version .
three items were changed because of new insights provided by more recent cr guidelines , and six items were revised because of significant between - centre variation of the baseline assessment data in the 2004 algorithm .
for example , after correction for differences in patient populations , the mean percentage of patients with a psychological problem ranged from 17% to 37% when assessed by clinical interview , as compared with ranging from 54% to 59% when assessed by the macnew questionnaire .
this indicates that assessment by clinical interview not only results in a larger between - centre variation but also in an underestimation and , consequently , to undertreatment of psychological problems .
the latter problem also exists when looking at estimation of exercise capacity : the assessment of patients exercise capacities by clinical interview led to a lower percentage of patients being judged as having an insufficiency than when bicycle ergometry or incremental shuttle walk test was used ( 73% vs. 84% , p < 0.01 ) . in all revised items , subjective assessment methods ( clinical experience or unstructured clinical interview )
in addition , based on new scientific insights , the multidisciplinary expert advisory panel added four new key items to the 2010 algorithm : screening for anxiety / depression , stress , cardiovascular risk profile and alcohol consumption .
table 1recommendations in the clinical algorithm 2004 and the revised recommendations in the clinical algorithm cardiac rehabilitation 2010 for all items in the five main domains of the needs assessment proceduredomainitemclinical algorithm 2004clinical algorithm 2010physical functioningobjective exercise capacitymaximal symptom - limited exercise test or clinical judgementmaximal symptom - limited exercise testsubjective exercise capacitymacnew quality - of - life questionnaire [ 9 , 10 ] or clinical judgementmacnew quality - of - life questionnaire [ 9 , 10]psychological functioningemotional functioningmacnew quality - of - life questionnaire [ 9 , 10 ] or clinical judgementmacnew quality - of - life questionnaire [ 9 , 10]anxiety / depressionabsenthospital anxiety and depression scale , patient health questionnaire stressabsentclinical interview ( interheart study ) social functioningsocial functioningmacnew quality - of - life questionnaire [ 9 , 10 ] or clinical judgementmacnew quality - of - life questionnaire [ 9 , 10]attitude of partnerclinical interview , unstructuredclinical interview , structured ( 3 questions)work resumptionclinical interview , unstructuredclinical interview , structured ( 2 to 7 questions , two - stage screening)cardiovascular risk profilecardiovascular risk profileabsentassessment of cardiovascular risk factorsrisk behavioursmoking statusclinical interview , structuredclinical interview , structured and specific treatment advicephysical activityclinical interview , unstructuredmonitor physical activity and health dietary habitsclinical interview , unstructuredindividual screening by dietician on indicationalcohol consumptionabsentfive shot questionnaire [ 29 , 30 ] recommendations in the clinical algorithm 2004 and the revised recommendations in the clinical algorithm cardiac rehabilitation 2010 for all items in the five main domains of the needs assessment procedure
the benefit of physical activity for cardiac patients was already noted more than 200 years ago .
however , it was not until the 1960s that cr programmes were gradually implemented in clinical practice . in 1965 , the netherlands heart foundation and netherlands society of cardiology formed a committee on cr . at that time , the main focus of cr was on physical reconditioning and work resumption . in the early 1990s , a broader and more versatile view on cr was gradually developed . based on these new insights , the cr committee released the first dutch cr guideline in 1995 .
in addition to exercise training , the cr programme also comprised education therapy and psychoeducative prevention therapy ( pep module ) . in 2004 ,
this guideline was replaced by the current dutch cr guideline , which gives more attention to risk prevention through behavioural changes ( e.g. smoking cessation , increasing physical activity , improving dietary habits ) as well as to psychosocial recovery . in addition , several new diagnosis groups were included ( patients with chronic heart failure , congenital heart disease , patients who underwent heart transplantation or icd implantation and elderly patients ) .
the number of possible treatment modalities was extended to four group - based interventions ( exercise training , education therapy , lifestyle change therapy and relaxation / stress management ) and , in selected cases , additional individual treatment ( e.g. by psychologist or dietician ) . in 2008 ,
the netherlands society of cardiology instituted a project group to revise the dutch guideline with respect to psychosocial and work - related aspects of cr ( paahr project ) .
it is expected that the revised guideline will be released later this year . a general problem with guidelines for cardiovascular disease , and in particular when guidelines are complex and multidisciplinary such as in cardiac rehabilitation , is that physician adherence is often poor [ 18 , 19 ] .
this lack of adherence may be related to insufficient knowledge , the physicians attitude ( e.g. a lack of agreement with the guideline , a lack of outcome expectancy and/or a lack of motivation ) or to the presence of external barriers ( e.g. lack of resources , insufficient reimbursement or organisational constraints ) . whereas it is difficult to overcome external barriers ,
however , previous studies showed only moderate effects of educational outreach by experts or trained facilitators . yet
, this effect can be increased by providing feedback and reminders to professionals . as such , clinical decision aids
have been shown to be effective in improving guideline adherence , mainly by increasing knowledge , reducing the tendency to stick to previous practice and by reducing guideline complexity [ 22 , 23 ] .
therefore , given its complex and multidisciplinary nature , cr is particularly suitable for the use of clinical decision aids such as a clinical algorithm .
in addition to a cardiologist , the cr team consists of a coordinator ( e.g. nurse , nurse practitioner or physician assistant ) , a physical therapist , a dietician , a social worker and in selected cases a medical psychologist , a sports physician and/or a rehabilitation physician . as individual patients may need different combinations of treatments from different healthcare professionals from the cr team , a clinical algorithm can be a useful and efficient tool to delegate tasks and responsibilities . in order to be effective for improving guideline adherence
, a clinical algorithm should include well - defined , non - ambiguous procedures for assessing the rehabilitation needs for individual patients .
therefore , the main focus of the present study was to include more objective measurement instruments as compared with the 2004 clinical algorithm .
as current cr guidelines do not provide specific measurement instruments , a multidisciplinary expert advisory panel was established to elect these instruments , using specified ( sac ) criteria for its evaluation .
although this approach has been implemented successfully previously , the effect of the updating procedure on inter - practice variation of variables obtained during the needs assessment procedure , as well as on guideline adherence , is yet to be investigated .
in addition to improving guideline adherence , the updated clinical algorithm may also serve to improve the quality of cr in the netherlands ; by registering data through a computerised decision support system incorporating the clinical algorithm , knowledge on barriers to the implementation of cr can be expanded , and implementation strategies can be improved . furthermore , these data can provide benchmark information for improving quality in individual cr centres .
notwithstanding the fact that using a clinical algorithm in general is an effective strategy to improve guideline adherence , it may not always be successful .
this is particularly the case when external barriers play an important role , such as organisational or procedural changes that are beyond the tasks and responsibilities of the healthcare professionals .
also , it should be acknowledged that the application of a clinical algorithm is only one among several strategies that can be used to improve adherence to cr guidelines .
it may be even more effective when incorporating other strategies , such as educational outreach visits to healthcare professionals .
additional interventions may also be aimed at the patient , e.g. by providing all eligible patients ( web - based ) information at discharge to improve insight into disease severity , risk behaviour and follow - up plans .
although the 2004 dutch clinical algorithm for assessing patient needs in cardiac rehabilitation was shown to be effective for improving compliance with guideline - recommended therapeutic decisions , several shortcomings and deficiencies were revealed .
therefore , a thorough revision was performed according to the latest scientific insights , using more objective assessment instruments . the new clinical algorithm ( beslisboom poliklinische indicatiestelling hartrevalidatie 2010 )
provides an efficient decision procedure to delegate tasks and responsibilities to healthcare professionals active in the field of cr . | backgroundin 2004 , the netherlands society of cardiology released the current guideline on cardiac rehabilitation .
given its complexity and the involvement of various healthcare disciplines , it was supplemented with a clinical algorithm , serving to facilitate its implementation in daily practice . although the algorithm was shown to be effective for improving guideline adherence , several shortcomings and deficiencies were revealed .
based on these findings , the clinical algorithm has now been updated .
this article describes the process and the changes that were made.methodsthe revision consisted of three phases .
first , the reliability of the measurement instruments included in the 2004 clinical algorithm was investigated by evaluating between - centre variations of the baseline assessment data .
second , based on the available evidence , a multidisciplinary expert advisory panel selected items needing revision and provided specific recommendations .
third , a guideline development group decided which revisions were finally included , also taking practical considerations into account.resultsa total of nine items were revised : three because of new scientific insights and six because of the need for more objective measurement instruments . in all revised items ,
subjective assessment methods were replaced by more objective assessment tools ( e.g. symptom - limited exercise instead of clinical judgement ) .
in addition , four new key items were added : screening for anxiety / depression , stress , cardiovascular risk profile and alcohol consumption.conclusionbased on previously determined shortcomings , the clinical algorithm for cardiac rehabilitation was thoroughly revised mainly by incorporating more objective assessment methods and by adding several new key areas . |
type 2 diabetes mellitus ( t2 dm ) is a clinical syndrome characterized by elevated blood glucose caused by a combination of insulin resistance and progressive failure of insulin secretion by the -cells in pancreatic islets of langerhans [ 1 , 2 ] .
the cellular mechanisms underlying -cell failure in t2 dm are not well understood , but several recent studies suggest that pancreatic stellate cells ( pscs ) might play an important role in this process [ 37 ] .
thus , pscs have a well - established role in the pathological fibrotic responses observed in inflammatory pancreatic disorders such as chronic pancreatitis and pancreatic cancer [ 810 ] , and islet fibrosis has been reported in the late stage of t2 dm in humans and in animal models [ 2 , 3 , 1115 ] .
pscs are activated to proliferate and generate fibrotic extracellular matrix ( ecm ) by a range of environmental stimuli which are also associated with t2 dm , including hyperglycemia [ 3 , 16 ] , renin - angiotensin system ( ras ) activation , oxidative stress [ 4 , 13 ] , platelet - derived growth factors , and inflammatory cytokines .
studies in animal models of t2 dm have shown that treatments which improve glucose tolerance including angiotensin - converting enzyme inhibitors ( acei ) , antioxidants , conophylline , and inhibition of nadph oxidase , also suppress psc activation , reduce islet fibrosis , and improve -cell mass and function [ 3 , 5 , 7 , 20 ] .
more recently , pscs were shown to induce apoptosis in a -cell line in vitro , although functional correlates were not reported in this study .
together , these observations are consistent with an important role for pscs in the progressive -cell dysfunction associated with the development of t2 dm . in this study
we have combined in vivo and in vitro models to explore the direct effects of pscs on -cell function and to investigate the importance of the diabetic microenvironment in psc/-cell interactions .
wistar and gk rats were housed three per cage and given access to food and water ad libitum . after a one - week acclimation period
the pscs were cultured in dulbecco 's modified eagle 's medium / ham 's f12 ( dmem / f12 , gibco , ca , usa ) containing 10% fetal bovine serum ( fbs , gibco , ca , usa ) .
three experimental groups were established , each of at least five experimental animals : ( 1 ) untreated gk ( gu ) and wistar ( wu ) rats ; ( 2 ) sham - operated gk ( gs ) and wistar ( ws ) rats ; and ( 3 ) psc - transplanted gk ( gt ) and wistar ( wt ) rats .
1 10 pscs suspended in 100 l pbs were directly injected into the pancreas of transplanted groups .
blood glucose and insulin levels were measured before and 30 , 60 , 90 , and 120 min after glucose administration .
insulin secretion was measured using a rat insulin elisa kit ( millipore , ma , usa ) .
the area under the curve for glucose ( aucg ) and insulin ( auci ) was calculated using a trapezoidal estimation from the values .
hba1c was measured using the dca 2000 + hba1c reagent kit ( bayer , berlin , germany ) . to measure the -cell mass , apoptosis , and islet fibrosis , insulin immunofluorescent staining , tunel ( roche , upper bavaria , germany ) , and
masson 's trichrome stain ( sigma , victoria , australia ) were performed on sections of pancreas , respectively .
the morphometric analysis was done manually using image j software to measure the insulin - positive area , the fibrosis - positive area , and the islet and the total pancreatic area at 200 magnification .
the relative -cell mass was expressed as the insulin - positive area / total pancreas area .
the islet fibrosis was calculated as the fibrosis - positive area / islet area . to measure -cell apoptosis ,
tunel - positive / insulin - positive cells were calculated . to prepare pscs - cm ,
cells grown to near confluence were cultured for 48 h in dmem / f12 serum - free medium supplemented with 0.2% bsa ( 5 10 cells/25 ml medium ) .
the medium was collected , centrifuged to remove any nonadherent cells , and stored at 20c until use .
the rat - derived , insulin - secreting ins-1 cell line was treated with pscs - cm diluted 50% ( v / v ) with standard rpmi-1640 ( cm group ) . to mimic the hyperglycemic microenvironment in the gk rat , other groups of ins-1 cells were treated by exposure to high glucose alone ( 25 mm , hg group ) or to a combination of high glucose and psc - conditioned medium ( cm + hg group ) .
cells were treated for 24 or 48 h , after which insulin secretion , cell viability , apoptosis , and er - stress were assessed .
the cell counting kit-8 ( cck-8 , sigma , ca , usa ) was used for quantitation of viable cell numbers in cytotoxicity assays .
after 24 or 48 h treatment , the viable cells were evaluated by absorbance measurements at 450 nm ( a450 nm ) using an automicroplate reader .
apoptosis was assessed by flow cytometry using the annexin v - fitc / propidium iodide ( annexin v / pi ) apoptosis detection kit ( promega , wi , usa ) according to the manufacturer 's protocol .
after treatment for 24 h , approximately 1 10 ins-1 cells were used for each analysis , and experiment was repeated on three separate occasions .
the expression by ins-1 cells of chop , an established marker for er - stress , was assessed at protein and mrna levels using immunoblotting and real - time quantitative rt - pcr , respectively . as a positive control ,
ins-1 cells were treated with thapsigargin ( 1 mol / l , 24 h ) , an established er stress - inducing agent .
measurements of potassium - stimulated insulin secretion ( ksis ) by ins-1 cells were performed as described .
insulin secretion and insulin content in cell lysates were measured using an insulin radioimmunoassay kit ( beijing technology company , beijing , china ) .
statistical significance was determined by unpaired student 's t - test or one - way variance ( anova ) , as appropriate , and differences between treatments were considered statistically significant at p < 0.05 .
all of the statistical analyses were performed using the statistical product and services solutions ( spss ) package .
to determine whether the pscs have direct effects on -cell in vivo , we assessed the effects of intrapancreatic transplantation of pscs in wistar and gk rats .
pscs treatment had no effect on glucose tolerance in wistar rats , as shown in figure 1(a ) .
thus , there was no significant difference in glucose clearance after oral glucose loading between the three wistar treatment groups ( wu , ws , and wt ) .
figure 1(b ) shows that all of the gk groups have impaired glucose tolerance ( figures 1(a ) and 1(b ) ) .
moreover , compared with gu and gs , gt group had significantly elevated blood glucose at 30 min in the gt group ( p < 0.05 , figure 1(b ) ) , leading to a significantly increased aucg ( 2953.2 296.1 versus 2474.4 374 and 2525.4 273.3 mmol / lmin , p < 0.05 ) . in accordance with the glucose tolerance curves , psc - treatment had no effects on glucose - stimulated insulin secretion in wistar rats ( figure 1(c ) ) .
the reduced levels of insulin secretion observed in control gk rats ( gu , gs ; figure 1(d ) ) were further suppressed by intra - pancreatic transplantation of pscs , with the gt groups showing significantly decreased plasma insulin at 0 and 30 minutes after oral glucose , resulting in significantly reduced auci in the gt group compared to the gu and gs groups ( 8259.2 2345.8 versus 21364.9 8173.4 and 19245.7 4191.9 pg / mlmin , p
measurements of hba1c confirmed the chronic hyperglycemic / diabetic status of the gk rats , with significantly higher hba1c levels in all gk treatments groups compared to normal wistar rats .
intrapancreatic transplantation of pscs had no effect on hba1c levels in wistar rats ( wt ) , but caused significant elevations in gk rats ( 9.8 1.4% versus 8.8 1.0% and 8.5 1.0% , p < 0.05 ) . to determine whether the in vivo effects of pscs to impair glucose tolerance in gk rats were caused by direct effects on -cells , we used immunofluorescence methods to assess -cell mass and apoptosis in the pancreas in situ .
psc transplantation in gk rats ( gt ) caused a large reduction in the -cell mass ( ~40% , p < 0.05 , figure 2 ) , and a significant increase in -cell apoptosis ( ~2-fold , p < 0.05 ) when compared to untreated or sham - operated controls ( gu , gs ) .
in contrast , psc transplantation had no detectable effects on -cell mass or apoptosis in wistar rats , in accordance with the lack of effects on glucose tolerance and hba1c in these normoglycemic control rats .
total collagen deposition evaluated with masson 's trichrome stain showed extensive staining in the wall of blood vessels and pancreatic ducts , as well as in fibrotic islets , as shown in figure 3 .
the degree of islet fibrosis in gk rats was higher than that in normal wistar rats , and the extent of islet fibrosis in gk rats was significantly increased ( ~1.3-fold , p < 0.05 ) after intrapancreatic transplantation of pscs ( gt ) when compared to untreated ( gu ) or sham - operated ( gs ) gk rat islets .
treatment of ins-1 cells with pscs - cm for 24 or 48 h in vitro had marked effects on ins-1 cell viability and function .
figure 4(a ) shows the decline in ins-1 cells viability determined using the cck-8 cell viability assay , in which a450 nm is proportional to viable cell number .
incubation with pscs - cm at 25% , 50% , and 75% ( v / v ) caused a significant and concentration - dependent reduction in cell number compared to control ins-1 cells incubated in standard tissue culture medium .
the deleterious effects of pscs - cm were significant by 24 h and were further enhanced after 48 h treatment ( figure 4(a ) ) . to investigate in vitro interactions between pscs and high concentrations of extracellular glucose
, ins-1 cells were incubated for 24 h with 50% pscs - cm in the presence or absence of 25 mm glucose ( hg ) .
figure 4(b ) shows that both pscs - cm and hg were alone sufficient to reduce ins-1 cell viability and that their effects were additive , with the combination of pscs - cm and hg exerting a significantly more detrimental effect than either treatment alone ( figure 4(b ) , p < 0.01 ) .
the pscs - cm and/or hg - dependent reductions in ins-1 cell viability were associated with increased apoptosis , as measured using the annexin v - fitc / pi apoptosis detection kit , with annexin v / pi cells being considered as in the early stages of apoptosis . in accordance with their effects on cell viability , both pscs - cm and hg alone caused marked and significant increases in ins-1 cell apoptosis , as shown in figure 5 . as for ins-1 cell viability , the effects of both treatments on apoptosis were additive , with the combination of pscs - cm and hg exerting a significantly more detrimental effect than either treatment alone ( 6.1 0.7- , 7.9 0.3- , and 16.3 0.5-fold of control ) .
measurement of chop expression suggests that the effects of pscs - cm and hg on ins-1 cell viability and apoptosis may be mediated through er stress , at least in part , as shown in figure 6 .
chop mrna expression was upregulated in ins-1 cells exposed for 24 h to pscs - cm or hg alone and , again , their effects were additive ( figure 6(a ) ) .
the increases in chop mrna were associated with increased levels of chop protein expression , as shown in figure 6(b ) .
as expected , treatment with thapsigargin induced a marked increase in both chop mrna and protein , indicated by the significantly upregulated expression of chop and validating chop expression as a marker for er stress .
the deleterious effects of pscs on ins-1 cell viability were also associated with reduced insulin secretory function .
thus , ksis by ins-1 cells was significantly reduced by treatment ( 24 h ) with both pscs - cm ( 71.3 20.3% of control , p
< 0.05 ) and hg ( 65.8 24.6% of control , p < 0.01 ) alone , with the combination of pscs - cm and hg exerting a significantly more inhibitory effect on insulin secretion than either treatment alone ( 37.3 16.9% of control , p < 0.01 ) .
the involvement of pscs in pancreatic fibrosis in response to chronic inflammation or pancreatic cancer is well established [ 810 , 2630 ] , but there have been many fewer studies into their potential role in islet fibrosis and progressive -cell failure in t2 dm .
for the first time , pscs transplantation and pscs - cm were used to detect the direct effects of pscs on -cells .
previous studies have linked activation of endogenous pscs with the development of fibrosis / t2 dm [ 3 , 6 , 7 , 20 ] , and in the current study we have used in vivo and in vitro models to demonstrate a direct deleterious effect of pscs on -cell function , leading to -cell loss and impaired insulin secretion and glucose tolerance .
these observations are consistent with a pivotal role for pscs in the pathogenesis of t2 dm . our in vivo experiments using intrapancreatic transplantation of pscs demonstrate that pscs did not influence -cell mass , insulin secretion , glucose handling , or hba1c in normoglycemic wistar rats , but had marked effects to exacerbate all of these ( patho)physiological parameters in gk rats , which are an established model of spontaneous - onset t2 dm [ 2 , 31 ] . before birth , gk rats present decreased -cell mass and low plasma insulin as compared with wistar control rats , proceeding to hyperglycemia and insulin resistance around weaning ( 3 - 4 weeks of age ) .
the development of t2 dm in the gk rat is associated with chronic hyperglycemia [ 31 , 32 ] , activation of the renin - angiotensin system ( ras ) , and increased levels of oxidative stress [ 3335 ] and inflammatory cytokines [ 2 , 31 , 32 ] , all of which are known to have marked effects to activate pscs and enhance their proliferation and generation of ecm .
thus , our observations suggest that under normal conditions pscs exert no effect on islet function , but in the hyperglycemic , inflammatory , and diabetic microenvironment they become activated and , as in the exocrine pancreas [ 810 , 2630 ] , initiate intraislet fibrosis leading to deleterious effects on -cell survival and function . in accordance with this ,
a recent study demonstrated pscs localized around islets in young ( 6 weeks ) gk rat pancreas , but not in age - matched wistar rats , implicating pscs at an early stage of the development of the diabetic phenotype .
our in vitro studies confirm a direct interaction between pscs and -cells and implicate paracrine mediators as one mechanism for that interaction .
thus psc - conditioned , cell - free medium had marked and consistent effects to reduce ins-1 cell viability and function .
these observations are consistent with a recent study using the rin5 -cell line . in that study
, rin5 cells were indirectly cocultured with pscs , confirming a direct effect of psc - derived soluble mediators on -cell viability and function . in the current study we extended the in vitro observations to demonstrate that a high concentration of glucose aggravates the detrimental effects of pancreatic stellate cells on -cell viability and function , consistent with the reduced glucose - induced insulin secretion in the gk rat recipients of intrapancreatic psc transplantations .
chronic exposure to elevated extracellular glucose is known to be a factor in the progressive loss of -cell function in t2 dm , often referred to as glucotoxicity .
our study confirmed the existence of glucotoxic effects on ins-1 cell survival and secretory function and also demonstrated that the deleterious effects of high glucose were additive to those of pscs .
one important mechanism of -cell failure in t2 dm is the development of er stress as a response to an imbalance between the rate of protein synthesis and folding capacity of the endoplasmic reticulum in hypersecreting -cells , eventually resulting in -cell apoptosis [ 37 , 38 ] .
our measurements of increased chop mrna and protein levels in response to pscs - cm and/or high glucose suggest that the detrimental effects of pscs on -cells may be associated with the activation of the er stress response .
overall , our in vitro observations concur with our in vivo studies and suggest that the hyperglycemia and systemic inflammation associated with the onset of t2 dm cause the activation of quiescent pscs .
the activated pscs have deleterious effects on -cell survival resulting in reduced -cell mass and on -cell function resulting in reduced insulin secretion with consequent impaired glucose tolerance . in this model ,
pscs do not initiate the onset of t2 dm but act to amplify the consequences of the hyperglycemic , inflammatory environment , enhancing the development of -cell fibrosis and their ultimate functional failure .
we therefore propose that pscs offer a novel therapeutic target through which to slow or halt the progression of t2 dm . | background and aims .
we here assess the effects of pscs on -cell function and apoptosis in vivo and in vitro .
materials and methods .
pscs were transplanted into wistar and goto - kakizaki ( gk ) rats .
sixteen weeks after transplantation , -cell function , apoptosis , and islet fibrosis were assessed . in vitro
the effects of pscs conditioned medium ( pscs - cm ) and/or high concentration of glucose on ins-1 cell function was assessed by measuring insulin secretion , ins-1 cell survival , apoptosis , and endoplasmic reticulum stress ( er stress ) associated chop expression .
results .
pscs transplantation exacerbated the impaired -cell function in gk rats , but had no significant effects in wistar rats . in vitro ,
pscs - cm caused impaired ins-1 cell viability and insulin secretion and increased apoptosis , which were more pronounced in the presence of high glucose .
conclusion .
our study demonstrates that pscs induce -cell failure in vitro and in vivo . |
a 79-year - old woman with a history of hypertension was transferred in a near - shock state from a regional hospital where she had presented 10 days previously , complaining of one week of nausea , diarrhea , and nocturnal fever . despite various conservative treatments including antibiotics , dyspnea with palpitations
she denied any history of trauma , recent invasive procedures such as dental care , infections , or tuberculosis .
one year previously , she refused to undergo recommended surgical treatment for symptomatic severe aortic stenosis at st .
her vital signs at arrival were a blood pressure of 70/30 mmhg , an irregular heart rate of 127 beats / min , a respiratory rate of 26 cycles / min , and a body temperature of 36.9c .
laboratory testing revealed a white blood cell count of 3,730/mm , a hemoglobin level of 9.0 g / dl , an aspartate transaminase / alanine transaminase ratio of 168/89 iu / l , and a c - reactive protein level of 23.7 mg / dl , while other parameters were within their normal limits .
severe cardiomegaly and bilateral pulmonary edema were apparent in chest radiography , and electrocardiography showed atrial fibrillation with rapid ventricular response .
portable transthoracic echocardiography ( tte ) revealed a massive pericardial effusion , which prompted emergency pericardiocentesis with the drainage of large amounts of serous pericardial fluid .
the findings of tte and trans - esophageal echocardiography ( tee ) after pericardiocentesis were suspicious for a 1.2-cm hypermobile vegetation on the anterior mitral valve leaflet with calcification through the posterior mitral annulus and severe aortic valve stenosis .
brain magnetic resonance imaging revealed focal acute embolic infarction of the left basal ganglia , without focal neurological signs .
chest computed tomography showed extensive mitral valve calcification ( mac ) through the posterior mitral annulus , pulmonary edema , and pleural effusion around both lungs ( fig .
emergency surgery was performed with a diagnosis of mitral infective endocarditis with extensive posterior mac and aortic valve stenosis .
after aortic cross - clamping , exposure of the mitral valve was achieved through a longitudinal left atriotomy via the interatrial groove , revealing multiple friable vegetations attached to the anterolateral commissure and leaflets , as well as extensive mac through the posterior annulus .
the anterior mitral valve leaflet was nearly completely necrotized with inflammation , but the annulus and subvalvular apparatus were preserved with only degenerative changes .
an abscess pocket containing dirty yellowish pus was found on the posterior annulus between p2 and p3 , extending into the endocardium of the left ventricular posterior wall , without an intracavitary connection to the left ventricle ( fig .
after complete debridement and resection of the infected tissue leaving the mac intact , multiple 2 - 0 polyester pledgeted mattress sutures were placed on the left atrial wall and the remaining posterior leaflet for plication .
we passed the valve sutures from the atrial wall to the posterior leaflets , while avoiding passing through the mac and obliterating the abscess pocket site .
the sutures were sequentially passed through bovine pericardium , and then through the sewing ring of a prosthetic valve ( 27-mm carpentier - edwards perimount magna valve ; edwards lifesciences inc . , irvine , ca , usa ) ( fig .
the bovine pericardium was sutured to the left atrial wall with 4 - 0 polypropylene running stitches to cover the abscess pocket site and mac . after repairing the left atriotomy ,
the aortic valve was also replaced with a prosthetic valve ( 21-mm carpentier - edwards perimount magna valve ; edwards lifesciences inc . ) .
preoperative blood culture and tissue culture were negative due to the use of preoperative antibiotics , and the patient was treated with broad - spectrum antibiotics ( ampicillin - sulbactam , 8 g / day ; gentamicin , 3 mg / kg / day ) for six weeks .
follow - up tte on postoperative day 19 showed well positioned and functioning prosthetic valves without paravalvular leakage or dehiscence ( fig .
although mitral valve repair is the preferred surgical approach if circumstances allow , the complete debridement of infectious necrotic lesions on the mitral valve may often hinder valve repair , inevitably requiring replacement .
in particular , the existence of extensive mac renders mitral valve replacement a risky procedure because aggressive annular de - calcification is associated with an increased risk of fatal complications , including intractable hemorrhage from atrioventricular disruption or ventricular rupture , as well as acute myocardial infarction secondary to circumflex artery injury .
partial annular decalcification can lead to a postoperative paravalvular leak and/or dehiscence and fracture of the remaining calcified annulus .
several surgical techniques have been reported for addressing the presence of an unsuitable mitral annulus during mitral valve replacement , and can be divided into two categories : those for mac and those for the management of conditions involving destruction of the annulus , such as infective endocarditis or redo valve replacement .
one approach involves leaving the mac intact and building a new annulus , securing the prosthesis to the leaflet tissue with intra - atrial implantation of a mitral prosthesis .
some surgeons have also used the cavitron ultrasonic surgical aspirator ( cavitron surgical systems inc . , stanford , ct , usa ) to decalcify mac . despite their variety , the proposed surgical techniques must be modified in practice for cases combining an extensive endocarditis lesion and mac , as in the case presented here . in this case , we planned to avoid decalcification of the mac and to reconstruct a stronger annulus with the hope of minimizing the risk of grave postoperative complications .
we used bovine pericardium to reinforce the new anchoring site after plicating both the mitral posterior leaflet and the left atrial wall to secure the mitral prosthesis without decalcifying the mac .
the bovine pericardium not only reinforced the suture on the new annulus , but also acted as a barrier to prevent perivalvular leakage , and may have strengthened the vulnerable left atrial wall by covering the fragile site after debridement of the abscess pocket . in conclusion , the presence of an unsuitable mitral annulus remains a challenge for current surgical techniques .
we report a surgical modification used to successfully treat a patient with extensive endocarditis complicated by subendocardial abscess and posterior mac by reconstructing the new annulus with plication between the left atrial wall and posterior mitral leaflet , reinforcing the bovine pericardium without decalcifying the mac .
further evaluation and extended follow - up are warranted to assess the effectiveness of this surgical technique . | the concomitant presence of posterior mitral annular calcification and infectious mitral valve lesions poses a technical challenge with considerable perioperative risk when using previously proposed techniques for mitral valve surgery .
herein , we report a case of the use of a modified surgical technique to successfully treat a patient with mitral infective endocarditis complicated by a subendocardial abscess and extensive posterior mitral annular calcification . |
kawasaki disease ( kd ) is a form of systemic vasculitis , the etiology of which is unknown .
diagnosis is based on clinical signs raising suspicion of the disease , including fever of over 5 days in duration and at least four of the following five symptoms : bilateral bulbar conjunctival infection , changes in the oral mucosa , changes in the peripheral extremities , polymorphous rash , and cervical adenopathy more than 1.5 cm in diameter .
complete kd ( ckd ) is diagnosed if all diagnostic criteria are fulfilled , whereas incomplete kd ( ikd ) , which occurs in 1520% of kd patients , is diagnosed when not all clinical features are present provided that several suspicious features are evident , laboratory findings suggest the presence of kd , and alternative diagnoses have been excluded . because only some ( thus not all ) clinical symptoms typical of ckd are apparent , ikd diagnosis can be delayed [ 5 , 6 ] , and such delay is a risk factor for development of coronary artery lesions [ 7 , 8 ] .
although ikd is considered to be an incomplete form of ckd , because ikd and ckd patient demographic and laboratory findings are similar [ 9 , 10 ] , several differences in clinical and laboratory presentations have been described .
relatively more children with icd are found at the extremes of the childhood age spectrum ( 1 year of age , or 59 years old ) , and low levels of both serum alanine aminotransferase ( alt ) and gamma - glutamyltransferase and low proportions of hyponatremia and pyuria have been reported in children with ikd [ 11 , 12 ] .
procalcitonin is a peptide precursor of calcitonin , the level of which increases in response to proinflammatory stimuli , especially those of bacterial origin .
procalcitonin levels > 5 ng / ml are closely associated with the presence of severe infection , usually bacteremia .
. found that patients with kd had serum procalcitonin levels significantly higher than normal and bacterial infections occurred more frequently in kd patients compared to those with autoimmune diseases or viral infections , or healthy children .
the cited authors also found that four patients who developed coronary aneurysms had elevated procalcitonin levels .
in contrast , chakrabartty and apong reported that all 38 kd patients studied had low procalcitonin levels ( < 0.5 ng / ml ) but elevated c - reactive protein ( crp ) concentrations and erythrocyte sedimentation rates ( esrs ) . to date , no report examining possible differences in procalcitonin levels between patients with ckd and ikd has appeared .
we therefore measured the serum concentrations of procalcitonin in such patients and compared these levels with those of patients with other febrile illnesses .
we enrolled child patients admitted to chonnam national university hospital from november 2009 to november 2011 . informed written consent
ikd was diagnosed if fewer than three diagnostic criteria were met ( although the presence of fever was required ) ; the laboratory findings were typical of kd patients ; and no alternative diagnosis was apparent .
these patients had experienced more than 3 days of fever , ranged in age from 3 months to 7 years , and had elevated crp ( > 2.0 mg / dl ) levels .
we performed two - dimensional echocardiography on four occasions on each kd patient : on the day of diagnosis ( prior to treatment ; d0 ) and 2 ( d2 ) , 14 ( d14 ) , and 56 days ( d56 ) after intravenous immunoglobulin ( ivig ) infusion .
we checked all scans for the presence of pericardial effusion , coronary artery lesions , and mitral regurgitation , and we assessed left ventricular function including the ejection fraction ( ef ) .
the size of each coronary artery was converted to a z - score to compare dilatation among individuals .
a coronary artery lesion was regarded as present if the z - scores were 2.5 for both the proximal right coronary artery and the left anterior descending artery .
peripheral venous blood was drawn to measure serum procalcitonin levels in ckd and ikd patients and controls .
we also obtained complete blood cell counts and esrs and measured the levels of crp , total protein , albumin , sodium , blood urea nitrogen ( bun ) , creatinine , aspartate aminotransferase ( ast ) , alt , creatine kinase ( ck ) , ck - mb , myoglobin , troponin - i , and n - terminal pro - brain natriuretic peptide ( nt - probnp ) .
we measured serum procalcitonin levels on four occasions in each kd patient : at d0 , d2 , d14 , and d56 .
control data were collected only during the acute stage of disease ( d0 ) . we compared the d0 values of the ckd , ikd , and control groups and those from ckd and ikd patients gathered at later times .
serum nt - probnp was analyzed using an electrochemiluminescence immunoassay ( elecsys probnp sandwich immunoassay ; roche diagnostics , basel , switzerland ) .
serum procalcitonin levels were quantitated by immunoassay ( elecsys brahms pct procalcitonin , roche ) .
the lower and maximum limits of detection were 0.02 and 100 ng / ml , respectively .
the chi - squared test was used to assess the statistical significance of differences in the values of independent variables , and analysis of variance ( anova ) , followed by application of tukey 's post hoc test , was employed to compare data among the three groups .
two - way repeated - measures anova followed by use of tukey 's post hoc test was employed for intragroup analysis .
the software package spss ( version 20.0 ; spss , chicago , il ) was used for all data analysis .
the present study was approved by the institutional review board of chonnam national university hospital ( protocol no .
seventy - seven ckd patients were enrolled ; the male - to - female ratio was 2.08 .
mean patient age was 2.5 1.9 years ( range , 5 months to 6.8 years ) and the mean body weight 13.5 3.8 kg .
twenty - four ikd patients were enrolled , and the male - to - female ratio was 1.7 .
mean patient age was 2.1 2.4 years ( range , 3 months to 10 years ) and the mean body weight 16.5 11.0 kg .
the proportion of patients at the age extremes ( < 1 year or 5 years ) in the ikd group ( n = 14 , 58.3% ) was higher than in the ckd group ( n = 27 , 35.0% ) ( p = 0.043 ) . all kd patients ( those with either ckd or ikd ) were treated with 2 g / kg intravenous immunoglobulin ( ivig ) given as a single infusion . all patients were prescribed 3050 mg / kg oral aspirin during the acute inflammatory stage , and the dose was tapered to 35 mg / kg after the acute stage had passed .
twelve patients with ckd and one with ikd who failed to respond to initial ivig therapy received a second dose of ivig .
two ckd patients who did not respond to this second dose of ivig were given infliximab .
forty - one patients were enrolled as controls ; the male - to - female ratio was 2.15 .
the mean age was 2.8 1.9 years ( range , 11 months to 8.0 years ) and the mean body weight 16.5 11.1 kg .
the clinical diagnoses of controls were as follows : pneumonia ( n = 21 ) , bacteremia ( n = 5 ) , acute tonsillitis ( n = 4 ) , cervical lymphadenitis ( n = 2 ) , acute bronchiolitis ( n = 2 ) , acute gastroenteritis ( n = 2 ) , acute epiglottitis ( n = 2 ) , bacterial keratoconjunctivitis ( n = 1 ) , acute otitis media ( n = 1 ) , and acute cellulitis ( n = 1 ) .
only two control patients yielded positive bacterial blood cultures . at admission , of the 77 patients with ckd , 74 ( 96% ) had bilateral bulbar conjunctival infections , 71 ( 92% ) a polymorphous rash , 73 ( 94% ) changes in the oral mucosa , 61 ( 79% ) cervical adenopathy with lymph nodes greater than 1.5 cm in diameter , and 50 ( 64% ) changes in the peripheral extremities . among 24 patients with ikd , 21 ( 87% ) had bilateral bulbar conjunctival infections , 15 ( 62% ) rash , 18 ( 75% ) changes in the oral mucosa , 7 ( 29% ) cervical adenopathy , and 7 ( 29% ) changes in the peripheral extremities .
all clinical features except bilateral bulbar conjunctival infection were significantly more prevalent in ckd than in ikd patients ( table 2 ) .
the efs ( derived using the cubed method ) in the ckd group were 56.9 28.6% ( d0 ) , 57.3 30.0% ( d2 ) , 61.5 28.6% ( d14 ) , and 51.7 34.6% ( d56 ) .
the efs in the ikd group were 64.6 23.5% ( d0 ) , 62.4 27.7% ( d2 ) , 64.4 24.1% ( d14 ) , and 65.1 59.4% ( d56 ) .
the number of patients with dilatation of the coronary artery was significantly greater in ckd than in ikd patients ( four [ 5.2% ] versus nil ) .
peripheral blood from all 101 kd patients was drawn on d0 , d2 , d14 , and d56 and from the 41 controls on d0 and d2 .
on d0 , the wbc ( p = 0.004 ) and platelet count ( p = 0.001 ) , the esr ( p < 0.001 ) , and the nt probnp level ( p = 0.021 ) were significantly higher in kd patients than controls .
crp ( p = 0.004 ) and troponin i ( p = 0.006 ) levels were significantly higher in the ckd group than controls .
the hematocrit was significantly lower in the ikd group than controls ( p = 0.028 ) .
the potassium concentration was significantly higher in the ikd group than in either the ckd or control groups ( p = 0.006 ) .
the laboratory data from all three groups at diagnosis ( d0 ) are shown in table 3 .
on d2 , the total protein level was significantly higher in the ckd group than in ikd patients or controls ( p = 0.001 ) . to explore the significance of this finding
, we sought to correlate total protein levels with the total ivig dose , body weight , and body surface area .
on d14 , both platelet ( p = 0.002 ) and albumin levels ( p = 0.001 ) were significantly higher in ckd than in ikd patients .
no significant difference in any of the wbc , the esr , or crp and nt probnp levels was evident between ckd and ikd patients at any later time .
ckd and ikd patients experienced significant reductions in the wbc and crp level at d2 , d14 , and d56 , compared to the d0 findings .
the nt - probnp levels of ckd patients at d2 and d14 were significantly lower than those at d0 ( figure 1 ) .
of the 101 kd patients and 41 controls , we measured serum procalcitonin levels of 85 patients ( ckd : 64 , ikd : 21 ) and 34 controls at d0 . the mean d0 serum procalcitonin levels in ckd patients ( 0.71 1.36 ng / ml ) and controls ( 0.67 1.06 ng / ml ) were significantly higher than those of ikd patients ( 0.26 0.26 ng / ml ) ( p = 0.014 and p = 0.041 , resp . ) ( figure 2 ) .
we measured d2 serum procalcitonin levels in 82 patients ( ckd : 65 , ikd : 17 ) and 15 controls ( after resolution of fever ) ; d14 levels in 85 patients ( ckd : 66 , ikd : 19 ) ; and d56 levels in 22 patients ( ckd : 18 , ikd : 4 ) . the mean control procalcitonin level ( 0.30 1.33 ng / ml ) at d2 was significantly higher than that of the ikd group ( 0.14 1.10 ng / ml ) ( p = 0.035 ) .
however , no significant difference in mean serum procalcitonin level was evident between ckd and ikd patients at any later time .
ckd patients showed significant reductions in procalcitonin levels at d14 ( p < 0.001 ) and d56 ( p
< 0.001 ) compared to d0 . likewise , ikd patients experienced significant reductions at d2 ( p = 0.028 ) and d14 ( p = 0.004 ) ( figure 2 ) .
no significant difference was evident between the four ckd patients with coronary artery dilatation ( 0.8 1.0 mm ) and those without dilatation ( 0.7 1.4 mm ) . also , no significant correlation was evident between procalcitonin level and the presence of a coronary artery lesion at any later time .
however , a lack of statistical power may have precluded identification of such a correlation ; very few patients had coronary artery dilatation .
in the present study , we have shown the utility of measuring serum procalcitonin levels in patients with ckd , ikd , and other febrile illnesses , at different stages of disease .
kd patients had significantly higher wbc and platelet counts and esr than did those with other febrile illnesses . also , during the acute stage of disease , ckd patients and those with other febrile illnesses had significantly higher levels of serum procalcitonin than did ikd patients .
procalcitonin has been suggested to be an acute - phase reactant , because synthesis thereof is thought to be induced by il-6 or tnf- , and procalcitonin levels increase after development of inflammation [ 13 , 17 ] .
although crp is a sensitive marker of inflammation , crp levels rise and then become normalized , only at later stages of disease .
several studies have reported high procalcitonin levels in the absence of infection in patients with behcet 's disease , kd , and active wegener 's granulomatosis [ 14 , 1921 ] .
okada et al . suggested that procalcitonin level could serve as a novel marker for clinical diagnosis of kd .
the serum concentration of procalcitonin was significantly higher in 25 acute kd patients ( 2.3 3.0 ng / ml ) and 17 with bacterial infections ( 2.2 2.9 ng / ml ) than in patients with autoimmune diseases ( n = 10 , 0.4 0.4 ng / ml ) or viral infections ( n = 17 , 0.4 0.3 ng / ml ) , or healthy children ( n = 18 , 0.2 0.1 ) .
the cited authors concluded that the serum procalcitonin level may be of clinical utility when determining the severity of kd and might assist in the differential diagnosis of patients with inflammatory diseases .
it was also found that four patients who developed coronary aneurysms exhibited higher procalcitonin levels ( 7.2 3.8 ng / ml ) .
procalcitonin levels are not elevated in patients with inflammatory ( including autoimmune ) diseases , but the levels of other acute phase reactants including crp , il-6 and/or neopterin may indeed be higher than normal [ 22 , 23 ] .
in contrast , chakrabartty and apong reported that kd patients expressed low levels of procalcitonin .
the cited authors sought a marker assisting in differentiation of kd from sepsis so that kd patients would not be unnecessarily prescribed antibiotics .
procalcitonin levels were low ( < 0.5 ng / ml ) in all of 38 admitted kd patients , but all had high esrs and crp levels . in our present study , we considered that procalcitonin was an acute - phase reactant and compared the levels thereof in patients with both types of kd and controls with other febrile illnesses . during the acute stage of disease ,
procalcitonin levels were significantly higher in patients with ckd and other febrile illnesses than in ikd patients , decreased in both types of kd patients to d14 after ivig treatment , and remained steady thereafter .
no statistically significant correlation between the presence of coronary artery disease and procalcitonin level was evident although , as explained above , our analysis may have lacked adequate statistical power .
the observed differences in procalcitonin levels may be explained by the fact that ckd patients exhibit more advanced inflammatory reactions than do ikd patients .
, we found that serum procalcitonin levels were relatively low in both ckd ( 0.71 1.36 ng / ml ) and ikd ( 0.26 0.26 ng / ml ) patients compared with the level ( 2.3 3.0 ng / ml ) reported by okada et al . .
one possible explanation is that the cited authors worked with only 25 patients ; we studied 101 . also , a relatively small proportion ( 4% ) of our patients had coronary artery lesions , compared to 25% of patients in the cited study .
chakrabartty and apong reported low procalcitonin levels ( < 0.5 ng / ml ) in their 38 kd patients .
most kd patients have an elevated crp level or esr , and an elevated nt - probnp level at the acute stage of kd appears to be a reliable indicator of the presence of either ckd or ikd [ 25 , 26 ] . a recent large - scale study by manlhiot et al
. showed that ckd ( n = 738 ) and ikd ( n = 217 ) patients yielded similar laboratory findings in terms of wbc , esr and albumin , ast , and crp levels but not alt levels .
the level of alt in ckd patients ( median : 27 u / l , range : 4328
u / l ) was higher than in patients with ikd ( median : 22 u / l , range : 2154 u / l ; p = 0.02 ) .
perrin et al . reported that the serum levels of both alt and gamma - glutamyltransferase were significantly higher in 39 patients with ckd ( 67.1 91.1 u
/ l , resp . ) than in 20 patients with ikd ( 37.6 21.8 u / l , 31.2 31.8 u / l , resp . ) .
however , none of crp or esr and blood cell count ( including wbc ) differed significantly between ckd and ikd patients at admission . in our present study , wbc and platelet count and
the esr were significantly higher in kd patients compared to those with other febrile illnesses , at the time of diagnosis .
also , in the subacute stage , wbc and platelet counts , the esr , and the crp level were significantly higher in kd patients than controls .
the total protein level was significantly higher in the ckd than in the ikd or control groups and was not correlated with any of ivig dose , body weight , or body surface area .
however , none of the wbc , the esr , or the levels of crp and nt - probnp differed significantly between ckd and ikd patients .
none of the levels of total protein , albumin , ast , alt , sodium , or chloride differed significantly among the two kd groups and controls . only serum potassium level ( in the ikd group )
one limitation of the present study is that some follow - up data are missing probably because parents refused to permit blood to be drawn as recovering children appeared to be healthy . in conclusion , this is the first report of serial procalcitonin measurements in patients with both types of kd and controls .
the mean d0 serum procalcitonin levels of ckd and controls were significantly higher than those of ikd patients during the acute stage of disease .
in contrast , the levels of other inflammatory markers including the wbc , the esr , and crp level did not significantly differ between patients with ckd and ikd .
further exploration of differences in procalcitonin levels between ckd and ikd patients is needed to better understand the incomplete presentation of ikd . | incomplete kawasaki disease ( ikd ) is considered to be a less complete form of kawasaki disease ( ckd ) , and several differences in the laboratory presentations of ikd and ckd have been noted .
we investigated serum procalcitonin levels in patients with ikd , ckd , and other febrile diseases ( a control group ) .
seventy - seven patients with ckd , 24 with ikd , and 41 controls admitted to our hospital from november 2009 to november 2011 were enrolled in the present study .
we obtained four measurements of serum procalcitonin levels and those of other inflammatory markers from each patient .
samples were taken for analysis on the day of diagnosis ( thus before treatment commenced ; d0 ) and 2 ( d2 ) , 14 ( d14 ) , and 56 days ( d56 ) after intravenous immunoglobulin infusion .
we obtained control group data at d0 .
the mean d0 serum procalcitonin levels of ckd patients ( 0.71 1.36 ng / ml ) and controls ( 0.67 1.06 ng / ml ) were significantly higher than those of ikd patients ( 0.26 0.26 ng / ml ) ( p = 0.014 and p = 0.041 , resp . ) .
no significant difference in mean procalcitonin level was evident among groups at any subsequent time . in conclusion ,
the serum procalcitonin level of patients with acute - stage ckd was significantly higher than that of ikd patients . |
central retinal vein occlusion ( crvo ) is a common cause of vision impairment which can occur at any given age .
most of the cases are unilateral ; bilateral cases are rare , usually having an underlying systemic illness such as a hyperviscosity syndrome or an inflammatory condition.1 here , we present the case of a patient , who came to us with a bilateral crvo and was later diagnosed to have chronic myeloid leukemia ( cml ) and was successfully treated with oral imatinib mesylate for cml and repeated intravitreal bevacizumab injections for macular edema of crvo , without any complications .
a 50-year - old asian indian male presented with complaints of sudden painless diminution of vision in the right eye followed by left eye over a span of 2 days .
there was no history of any other ocular complaints , trauma , or any ocular intervention .
there was no history of bleeding tendencies , fainting episodes , and malnutrition or drug intake . on examination ,
the best corrected visual acuity was 0.8 logmar ( od ) and 1.0 logmar ( os ) .
anterior segment examination revealed a clear cornea with grade one nuclear sclerosis in both eyes .
the intraocular pressure was measured to be 14 mmhg ( od ) and 17 mmhg ( os ) by goldmann applanation tonometry .
posterior segment examination revealed a clear media with normal sized optic disc and a cup : disc ratio of 0.3 in both eyes .
the vessels were dilated and tortuous with multiple flame - shaped and dot - blot hemorrhages in all four quadrants of both eyes .
there was the presence of roth 's spots in midperiphery in both eyes , yellowish white subretinal infiltrates , and perivenular sheathing suggestive of leukemia [ figure 1a and b ] .
fundus fluorescein angiography revealed delayed arteriovenous transit time and areas of capillary nonperfusion and blocked fluorescence due to retinal hemorrhages .
optical coherence tomography ( oct ) showed spongy macular edema in both eyes with a foveal thickness of 536 3 ( od ) and 530 19 ( os ) [ figure 1a and b ] .
systemic detailed workup of the patient was done to confirm leukemia as the cause of bilateral crvo .
a differential leukocyte count revealed polymorphs 40% , lymphocytes 11% , monocytes 2% , eosinophils 1% , basophils 5% , blasts l3% , myelocytes 16% , metamyelocytes 12% , and platelets of 2.55 lac/l .
( a ) fundus photograph ( od ) showing superficial and deep retinal hemorrhages in all four quadrants with roth 's spots ( encircled ) in midperiphery , white retinal infiltrates ( fine black arrow ) , and perivascular sheathing ( thick arrow ) .
( b ) fundus photo and optical coherence tomography picture of the left eye ( os ) showing similar features bone marrow aspiration gave a picture of a hypercellular marrow with 3% blasts and other cells in the maturation stages of myeloid series [ figure 2a ] .
trephine biopsy also showed a hypercellular marrow and preponderance of myeloid series showing cells in varying stages of maturation [ figure 2b ] .
the final diagnosis of cml in the chronic phase was made , and the patient was started on oral imatinib mesylate 400 mg once a day .
( a ) bone marrow aspirate ( using may - grunwald - giemsa stain , in oil immersion ) showing hypercellularity with 3% blasts and cells of myeloid series ( yellow arrowhead pointing towards a blast cell ) .
( b ) hematoxylin- and eosin - stained trephine biopsy ( 20 ) showing hypercellularity , cells in varying stages of maturation and preponderance of myeloid series of cells the yellowish retinal infiltrates gradually increased over the next 1 month [ figure 3 ] .
fundus photo showing increase in the intensity and size of retinal infiltrates , 1-month following initiation of systemic therapy the patient became systemically stable after 3 months of starting systemic therapy .
however , neovascularization of the disc was noted , and the patient was subjected to panretinal photocoagulation in both eyes [ figure 2b ] .
after the systemic status was controlled , intravitreal bevacizumab 0.125 mg in 0.05 ml was injected in the right eye followed by the left eye .
an oct at 2 months recorded the central macular thickness ( cmt ) of 201 55 ( od ) and 214 44 ( os ) and the patient regained vision of 0.7 and 0.3 logmar in od and os , respectively .
he was kept under regular monthly follow - up , and bevacizumab was given on pro re nata basis .
a total of four injections , each of bevacizumab were given over a period of 1 year .
the macular edema stabilized after six injections of intravitreal bevacizumab in od and seven injections in os .
at the end of 4 years follow - up , the patient is doing well systemically .
he is in a period of remission on imatinib mesylate , the total leukocyte count is 5200 mm and logmar visual acuity of 0.6 and 0.3 in od and os , respectively , was documented at the last follow - up .
eye is a window to systemic diseases ; a careful ocular examination could clinch the diagnosis and prompt referral to a hemato - oncologist could be lifesaving .
published the largest case series of three cases with waldenstrom 's macroglobulinemia as a cause of bilateral crvo.2 a similar case with bilateral simultaneous crvo in waldenstrom 's macroglobulinemia was also reported by chanana et al.3 in these cases , the ocular symptoms improved with improvement in the systemic condition .
however , in this case , intravitreal anti - vascular endothelial growth factor ( vegf ) agent was required for stabilization of the ocular condition and resolution of macular edema despite systemic control .
similar reports of bilateral crvo have also been documented in association with other hypercoagulable states such as hyperhomocysteinemia,4 multiple myeloma,5 and acute myeloid leukemia.6 the above case report points toward an ocular presentation of cml wherein ophthalmic evaluation pointed toward the underlying disease .
ocular involvement is noted in about one - half of the patients of leukemia at the time of diagnosis7 although it is frequently asymptomatic .
vision loss as the presenting feature of cml is rare and has been reported attributable to bilateral crvo,8 leukemic retinopathy,9 and proliferative retinopathy.10 in a prospective study of 120 patients with newly diagnosed leukemia of all cell types , schachat et al .
diagnosed a crvo in five patients - all of whom had myeloid leukemia with extremely high white blood cell counts or platelet counts - and attributed the venous occlusion to blood hyperviscosity.11 gordon et al.12 emphasized the importance of distinguishing between infectious and neoplastic retinal infiltrates in patients with a history of leukemia .
they found that neoplastic ( or leukemic ) retinal infiltrates occurred in patients who had newly diagnosed leukemia and those patients in blast crisis .
the present case showed an initial increase in leukemic infiltrates as the patient was started on systemic therapy ; however , with systemic remission , these disappeared over the course of 3 months .
the macular edema due to crvo remained persistent at this stage and responded to intravitreal bevacizumab injections .
bcr - abl protein in cml also increases vegf levels , and imatinib has anti - vegf properties.13 notably , after systemic remission with imatinib , the patient showed favorable response to intravitreal anti - vegf and panretinal photocoagulation .
the patient 's coagulation profile was normal , and there was no history of bleeding tendencies .
thus , we conclude that systemic anti - vegf agents may not be sufficient to overcome the ocular manifestations of the growth factor and intravitreal injections of anti - vegf agents would be needed to counteract the high levels of vegf responsible for ocular morbidity , without any adverse effects .
| central retinal vein occlusion ( crvo ) is a common pathology of the retinal vasculature .
patients with crvo usually present with a drop in visual acuity .
the condition bears no specific therapy ; treatment is aimed at the management of potentially blinding complications , of which there are many . with majority of cases being unilateral , bilateral crvo
is usually associated with an underlying systemic illness such as a hyperviscosity syndrome . here ,
we present a case of a patient , who presented with a bilateral drop in vision diagnosed as bilateral crvo on ophthalmic evaluation .
systemic workup revealed the presence of an underlying undiagnosed chronic myeloid leukemia .
an initial presentation to the ophthalmologist is a rare occurrence in leukemic patients .
this case report highlights the role of the ophthalmologist in diagnosing a potentially life - threatening hematological illness . |
cardiac remodelling is central to the pathophysiology of heart failure ( hf ) and is an established prognostic factor in patients with hf .
left ventricular ( lv ) enlargement has been shown to be associated with an increased risk for adverse cardiac events , while reduced lv ejection fraction ( lvef ) is a powerful predictor of cardiovascular outcomes and all - cause mortality . the therapeutic effects of beta - blockade , angiotensin - converting enzyme ( ace ) inhibition , and cardiac resynchronization therapy have been linked to their beneficial effects on cardiac remodelling . a recent analysis compared 30 clinical event trials and 88 remodelling trials with the same drug or device therapy and reported that the odds ratio associated with therapy for long - term mortality correlate with the shorter term effect of the therapy on lvef , end - systolic volume , and end - diastolic volume .
it is therefore relevant to evaluate the impact of novel hf therapies on cardiac remodelling .
the heart rate lowering effect of ivabradine , a specific inhibitor of the if current in the sinoatrial node , has been found to be beneficial in patients with hf .
the results of the systolic heart failure treatment with the if inhibitor ivabradine trial ( shift ) showed that treatment with ivabradine superimposed on background therapy for hf was associated with an 18% reduction in risk for the primary composite endpoint of cardiovascular death or hospitalization for worsening hf ( p < 0.0001 ) . in this article , we present the results of the prespecified echocardiographic substudy of the shift trial , aiming to evaluate the effects of ivabradine vs. placebo on lv remodelling and function in hf .
briefly , this randomized , double - blind , placebo - controlled , parallel - group , international multicentre clinical trial evaluated 6505 male or female patients with moderate - to - severe chronic hf and documented lv systolic dysfunction ( lvef 35% ) .
eligible patients were in sinus rhythm and had resting heart rate of 70 beats per minute ( bpm ) on 12-lead electrocardiography ; they had been clinically stable for 4 weeks , and had been admitted to hospital for worsening hf within the previous 12 months .
other inclusion and exclusion criteria are detailed elsewhere . after a 2-week run - in period
randomization was stratified according to beta - blocker intake at randomization and country . at subsequent visits at 14 days , 28 days , and
every 4 months thereafter ( or at any other time if necessary ) , the dosage of blinded study medication could be adjusted upward ( 7.5 mg bid ) or downward ( 2.5 mg bid ) depending on resting heart rate and tolerability .
the shift echocardiography substudy was performed in 89 centres in 21 countries participating in the main shift study . to avoid selection bias ,
all the patients in each participating centre were invited to enter the substudy , and those included signed a specific informed consent form in addition to the documents related to the main study .
the substudy was carried out in accordance with the declaration of helsinki , 1964 , and its text revisions .
the primary endpoint of the shift echocardiography substudy was the change in the lv end - systolic volume index ( lvesvi ) from baseline to 8 months .
secondary endpoints were changes in lv end - diastolic volume index ( lvedvi ) , non - indexed lv end - systolic and end - diastolic volumes ( lvesv and lvedv ) , and lvef during the same interval . tertiary endpoints included left atrial end - systolic volume index , grade of diastolic dysfunction ( assessed by doppler imaging ) , grade of mitral regurgitation , right ventricular ( rv ) myocardial performance index , and rv s wave peak velocity ( assessed by doppler imaging ) .
the 8-month duration of the substudy was selected because it was considered long enough to allow detection of a benefit on lv remodelling on the basis of the observed effects of ace inhibitors and beta - blockers while minimizing loss of patients to follow - up because of death in this hf population .
echocardiography was performed at baseline ( in the 2 weeks between selection and inclusion ) and within 1 month of the 8-month visit .
the two recordings were to be made by the same technician using the same technique and the same equipment .
transthoracic echocardiography was performed with a phased - array imaging system equipped with a transducer with second harmonics capability .
images were obtained in the parasternal long- and short - axis and apical views , with the subject lying in the left lateral position .
all participating sites received a detailed imaging manual before initiation of the substudy , needed to be certified by the core laboratory , and were regularly monitored for and informed of quality of imaging recordings throughout the study .
the echocardiograms were read centrally according to predefined procedures in the core laboratory at the montreal heart institute ( canada ) by technicians supervised by two cardiologists , all of them blinded to treatment and examination sequence ( baseline vs. follow - up ) .
both recordings from the same patient were assessed during two separate imaging analysis sessions by the same technician and validated by the same cardiologist , without knowledge of the results of the other imaging exam ( i.e. results of the first recording analysis were not known to the core laboratory team when the second recording from the same patient was analysed ) .
all measurements were made according to the recommendations of the american society of echocardiography ( ase ) and the european association of echocardiography .
each assessment was analysed for at least three consecutive cycles ( or at least five in case of atrial fibrillation ) , avoiding post - ectopic beats .
two patients in the ivabradine group had atrial fibrillation at the time of the 8-month echocardiogram ; four patients ( two ivabradine , two placebo ) sustained atrial fibrillation during the substudy ( m0 to m8 ) . primary and secondary criteria were assessed on two - dimensional apical four- and two - chamber views , using biplane views when they were analysable at both time points ; if biplane apical views were not available at baseline and follow - up , the analysis was conducted using the apical four - chamber view .
patients who only had analysable images from the apical two - chamber view were excluded from the substudy .
each recording was graded for the quality of lv endocardial border resolution : grade 1 , acceptable four- and two - chamber views ; grade 2 , acceptable four- or two - chamber view ; grade 3 , poor quality four- and two - chamber views .
grade 3 was assigned to recordings with insufficient resolution of lv endocardial border in 4 or more of the 17 ventricular segments ( using the ase 17-segment model ) .
inter - reader and intra - reader variability was evaluated at the echocardiography core laboratory using 60 recordings analysed twice by each technician .
intra - class correlation coefficients ( iccs ) were found to be satisfactory for all readers ( ranges for lvesvi : intra - reader icc , 0.960.99 ; inter - reader icc , 0.950.98 ) . left ventricular diastolic function was classified as normal , impaired lv relaxation , pseudonormal , or restrictive based on standard criteria .
the sample size was estimated to demonstrate the superiority of ivabradine over placebo on the change from baseline to 8 months in lvesvi ( primary substudy endpoint ) , using a two - sided student 's t - test for independent samples at a 5% type i error rate . considering a standard deviation of 25 ml / m
, it was estimated that 414 patients would be necessary to detect at least 8 ml / m difference between treatment groups with 90% power .
the sample size was adjusted to 520 patients since it was anticipated that the change from baseline in lvesvi would not be assessable for approximately 20% of patients , on the basis of the estimated rate of mortality and the projected proportion of non - assessable echocardiographic recordings .
baseline characteristics were presented by treatment group on all patients randomized in the main study and participating in the echocardiography substudy , with means sd for continuous variables , and numbers and percentages for categorical variables .
changes in primary and secondary endpoints were compared between treatment groups on patients included in the substudy who had taken at least one dose of study drug and with an evaluation of the primary endpoint considered reliable , using a covariance analysis adjusted for beta - blocker intake at randomization , country , and baseline value .
the treatment effect was estimated using adjusted least square means from this model , with associated two - sided 95% confidence intervals ( ci ) and p - values ( significance level set at 5% ) . a sensitivity analysis with imputation for missing values of lvesvi ( imputing conservatively
no change in the ivabradine arm and a mean change from baseline of 10 ml / m in the placebo arm ) indicated that the results are not unduly influenced by missing data .
changes in lvesvi and lvef from baseline to 8 months were divided into three classes ( improvement , no change , and worsening ) according to clinical thresholds ( 15% for lvesvi and 5% for lvef , respectively ) .
the percentages of patients with an improvement were compared between treatment groups using a test .
the relationship between baseline echocardiography criteria and the shift primary composite endpoint ( defined as first event among hospitalization for worsening hf or cardiovascular death ) as well as its components was investigated in patients included in the substudy and allocated to the placebo group .
cox proportional hazards models adjusted for the echocardiography endpoint as a dichotomous variable ( below and above the median value ) and for other relevant factors at baseline [ age , new york heart association ( nyha ) class , heart rate , ischaemic aetiology , systolic blood pressure , estimated glomerular filtration rate , and beta - blocker intake ] were used to estimate hazard ratio comparing classes of baseline echocardiography endpoints , with associated 95% cis and p - values .
time - to - event curves were presented by class of baseline lvesvi and estimated using the kaplan meier method . the number and percentage of shift primary composite endpoints subsequent to 8 months was calculated in each group divided by tertile of relative change in lvesvi from baseline to 8 months , for ivabradine and placebo groups separately .
the correlation between changes in lvef and heart rate was tested using the linear regression model including the two treatment groups .
the analyses on primary and secondary endpoints of the substudy were all prespecified ( lvesvi , lvedvi , lvesv , lvedv , and lvef ) , as was the case for tertiary echocardiographic endpoints ( e.g. mitral regurgitation ) .
the exploratory analyses linking echocardiographic parameters to outcomes or heart rate were performed on a post hoc basis .
the independent centre for statistics keyrus biopharma ( nantes , france ) verified all analyses .
the cardiologists in charge of the core laboratory and the shift executive committee were responsible for the study design , the interpretation of the results , the development and writing of the report , and the decision to submit for publication and had full access to all data . members of the medical and scientific departments of the sponsor supported the work of the executive committee , but did not make any scientific or research decisions independent of this committee .
briefly , this randomized , double - blind , placebo - controlled , parallel - group , international multicentre clinical trial evaluated 6505 male or female patients with moderate - to - severe chronic hf and documented lv systolic dysfunction ( lvef 35% ) .
eligible patients were in sinus rhythm and had resting heart rate of 70 beats per minute ( bpm ) on 12-lead electrocardiography ; they had been clinically stable for 4 weeks , and had been admitted to hospital for worsening hf within the previous 12 months .
other inclusion and exclusion criteria are detailed elsewhere . after a 2-week run - in period
randomization was stratified according to beta - blocker intake at randomization and country . at subsequent visits at 14 days , 28 days , and
every 4 months thereafter ( or at any other time if necessary ) , the dosage of blinded study medication could be adjusted upward ( 7.5 mg bid ) or downward ( 2.5 mg bid ) depending on resting heart rate and tolerability .
the shift echocardiography substudy was performed in 89 centres in 21 countries participating in the main shift study . to avoid selection bias ,
all the patients in each participating centre were invited to enter the substudy , and those included signed a specific informed consent form in addition to the documents related to the main study .
the substudy was carried out in accordance with the declaration of helsinki , 1964 , and its text revisions .
the primary endpoint of the shift echocardiography substudy was the change in the lv end - systolic volume index ( lvesvi ) from baseline to 8 months .
secondary endpoints were changes in lv end - diastolic volume index ( lvedvi ) , non - indexed lv end - systolic and end - diastolic volumes ( lvesv and lvedv ) , and lvef during the same interval . tertiary endpoints included left atrial end - systolic volume index , grade of diastolic dysfunction ( assessed by doppler imaging ) , grade of mitral regurgitation , right ventricular ( rv ) myocardial performance index , and rv s wave peak velocity ( assessed by doppler imaging ) .
the 8-month duration of the substudy was selected because it was considered long enough to allow detection of a benefit on lv remodelling on the basis of the observed effects of ace inhibitors and beta - blockers while minimizing loss of patients to follow - up because of death in this hf population .
echocardiography was performed at baseline ( in the 2 weeks between selection and inclusion ) and within 1 month of the 8-month visit .
the two recordings were to be made by the same technician using the same technique and the same equipment .
transthoracic echocardiography was performed with a phased - array imaging system equipped with a transducer with second harmonics capability .
images were obtained in the parasternal long- and short - axis and apical views , with the subject lying in the left lateral position .
all participating sites received a detailed imaging manual before initiation of the substudy , needed to be certified by the core laboratory , and were regularly monitored for and informed of quality of imaging recordings throughout the study .
the echocardiograms were read centrally according to predefined procedures in the core laboratory at the montreal heart institute ( canada ) by technicians supervised by two cardiologists , all of them blinded to treatment and examination sequence ( baseline vs. follow - up ) .
both recordings from the same patient were assessed during two separate imaging analysis sessions by the same technician and validated by the same cardiologist , without knowledge of the results of the other imaging exam ( i.e. results of the first recording analysis were not known to the core laboratory team when the second recording from the same patient was analysed ) .
all measurements were made according to the recommendations of the american society of echocardiography ( ase ) and the european association of echocardiography .
each assessment was analysed for at least three consecutive cycles ( or at least five in case of atrial fibrillation ) , avoiding post - ectopic beats .
two patients in the ivabradine group had atrial fibrillation at the time of the 8-month echocardiogram ; four patients ( two ivabradine , two placebo ) sustained atrial fibrillation during the substudy ( m0 to m8 ) .
primary and secondary criteria were assessed on two - dimensional apical four- and two - chamber views , using biplane views when they were analysable at both time points ; if biplane apical views were not available at baseline and follow - up , the analysis was conducted using the apical four - chamber view .
patients who only had analysable images from the apical two - chamber view were excluded from the substudy . each recording was graded for the quality of lv endocardial border resolution : grade 1 , acceptable four- and two - chamber views ; grade 2 , acceptable four- or two - chamber view ; grade 3 , poor quality four- and two - chamber views .
grade 3 was assigned to recordings with insufficient resolution of lv endocardial border in 4 or more of the 17 ventricular segments ( using the ase 17-segment model ) .
inter - reader and intra - reader variability was evaluated at the echocardiography core laboratory using 60 recordings analysed twice by each technician .
intra - class correlation coefficients ( iccs ) were found to be satisfactory for all readers ( ranges for lvesvi : intra - reader icc , 0.960.99 ; inter - reader icc , 0.950.98 ) . left ventricular diastolic function was classified as normal , impaired lv relaxation , pseudonormal , or restrictive based on standard criteria .
the sample size was estimated to demonstrate the superiority of ivabradine over placebo on the change from baseline to 8 months in lvesvi ( primary substudy endpoint ) , using a two - sided student 's t - test for independent samples at a 5% type i error rate . considering a standard deviation of 25 ml / m
, it was estimated that 414 patients would be necessary to detect at least 8 ml / m difference between treatment groups with 90% power .
the sample size was adjusted to 520 patients since it was anticipated that the change from baseline in lvesvi would not be assessable for approximately 20% of patients , on the basis of the estimated rate of mortality and the projected proportion of non - assessable echocardiographic recordings .
baseline characteristics were presented by treatment group on all patients randomized in the main study and participating in the echocardiography substudy , with means sd for continuous variables , and numbers and percentages for categorical variables .
changes in primary and secondary endpoints were compared between treatment groups on patients included in the substudy who had taken at least one dose of study drug and with an evaluation of the primary endpoint considered reliable , using a covariance analysis adjusted for beta - blocker intake at randomization , country , and baseline value .
the treatment effect was estimated using adjusted least square means from this model , with associated two - sided 95% confidence intervals ( ci ) and p - values ( significance level set at 5% ) . a sensitivity analysis with imputation for missing values of lvesvi ( imputing conservatively
no change in the ivabradine arm and a mean change from baseline of 10 ml / m in the placebo arm ) indicated that the results are not unduly influenced by missing data .
changes in lvesvi and lvef from baseline to 8 months were divided into three classes ( improvement , no change , and worsening ) according to clinical thresholds ( 15% for lvesvi and 5% for lvef , respectively ) .
the percentages of patients with an improvement were compared between treatment groups using a test .
the relationship between baseline echocardiography criteria and the shift primary composite endpoint ( defined as first event among hospitalization for worsening hf or cardiovascular death ) as well as its components was investigated in patients included in the substudy and allocated to the placebo group .
cox proportional hazards models adjusted for the echocardiography endpoint as a dichotomous variable ( below and above the median value ) and for other relevant factors at baseline [ age , new york heart association ( nyha ) class , heart rate , ischaemic aetiology , systolic blood pressure , estimated glomerular filtration rate , and beta - blocker intake ] were used to estimate hazard ratio comparing classes of baseline echocardiography endpoints , with associated 95% cis and p - values .
time - to - event curves were presented by class of baseline lvesvi and estimated using the kaplan meier method . the number and percentage of shift primary composite endpoints subsequent to 8 months was calculated in each group divided by tertile of relative change in lvesvi from baseline to 8 months , for ivabradine and placebo groups separately .
the correlation between changes in lvef and heart rate was tested using the linear regression model including the two treatment groups .
the analyses on primary and secondary endpoints of the substudy were all prespecified ( lvesvi , lvedvi , lvesv , lvedv , and lvef ) , as was the case for tertiary echocardiographic endpoints ( e.g. mitral regurgitation ) .
the exploratory analyses linking echocardiographic parameters to outcomes or heart rate were performed on a post hoc basis .
the independent centre for statistics keyrus biopharma ( nantes , france ) verified all analyses .
the cardiologists in charge of the core laboratory and the shift executive committee were responsible for the study design , the interpretation of the results , the development and writing of the report , and the decision to submit for publication and had full access to all data . members of the medical and scientific departments of the sponsor supported the work of the executive committee , but did not make any scientific or research decisions independent of this committee .
a total of 613 patients were screened for the substudy and 611 were included ( 304 ivabradine , 307 placebo , figure 1 ) .
a total of 200 patients were excluded leaving 411 patients ( 67% of the included set , 208 ivabradine and 203 placebo ) for analysis .
the main reasons for exclusion were poor quality of two- or four - chamber views ( 104 patients with insufficient resolution of lv endocardial border , precluding reliable measurement of lv volumes ) , no baseline and/or 8-month recording received at the core laboratory ( 34 patients ) , and no matching apical biplane or apical four - chamber views ( nine patients ) .
other reasons for exclusion were death ( 36 patients ) and consent withdrawal ( 17 patients ) .
the characteristics of the substudy population at baseline are presented in table 1 , and compared with those of the main study population .
mean resting heart rate at baseline was 78.6 9.1 bpm and 66% had hf of ischaemic aetiology .
most of the patients were receiving a beta - blocker ( 92% ) and a renin
the main beta - blockers used were carvedilol , bisoprolol , and metoprolol succinate ( table 2 ) , with 29% of the population at guidelines - based target dose of beta - blocker and 56% receiving 50% of the target dose .
the characteristics of the substudy population were similar to those of the population in the main study , except for a lower rate of history of hypertension in the substudy ( 59 vs. 66% ) and a higher rate of use of aldosterone antagonists ( 72 vs. 60% ) .
there were no clinically relevant differences between the treatment groups in the substudy , between the shift patients included in the substudy and shift patients not included in the substudy ( see online supplementary material ) , or between patients randomized in the study and the full analysis set for the substudy .
table 1baseline characteristics of the substudy population ( included set ) vs. the population of the main shift study ( randomized set)shift echocardiography substudymain study ( n = 6505)ivabradine ( n = 304)placebo ( n = 307)all ( n = 611)demographic parameters age ( years)60.4 10.959.1 11.159.7 11.060.4 11.4 sex ( male)244 ( 80%)252 ( 82%)496 ( 81%)4970 ( 76% ) caucasian276 ( 91%)281 ( 92%)557 ( 91%)5771 ( 89% ) current smoker48 ( 16%)48 ( 16%)96 ( 16%)1118 ( 17% ) bmi ( kg / m)27.7 4.527.7 4.927.7
9.179.9 9.6 heart rate 77 bpm137 ( 45%)129 ( 42%)266 ( 44%)3357 ( 52% ) sitting sbp ( mm hg)120.5 15.9119.1 15.4119.8 15.6121.7 16.0 sitting dbp ( mm hg)75.2 9.375.4 9.075.3 9.175.7 9.5 egfr ( mdrd formula ) ( ml / min/1.73 m)73.7 20.675.8 25.374.7 23.174.7 23.0nyha class class ii146 ( 48%)140 ( 46%)286 ( 47%)3169 ( 49% ) class iii156 ( 51%)164 ( 53%)320 ( 52%)3223 ( 50% ) class iv2 ( 0.7%)3 ( 1.0%)5 ( 0.8%)111 ( 1.7%)medical history duration of heart failure ( years)3.5 4.03.5 4.53.5
4.33.5 4.2primary cause of heart failure ischaemic205 ( 67%)200 ( 65%)405 ( 66%)4418 ( 68% ) non - ischaemic99 ( 33%)107 ( 35%)206 ( 34%)2087 ( 32% ) myocardial infarction180 ( 59%)178 ( 58%)358 ( 59%)3666 ( 56% ) hypertension179 ( 59%)175 ( 57%)354 ( 58%)4314 ( 66% ) diabetes96 ( 32%)98 ( 32%)194 ( 32%)1979 ( 30% ) previous stroke25 ( 8%)28 ( 9%)53 ( 9%)522 ( 8% ) history atrial fibrillation and/or atrial flutter18 ( 6%)20 ( 7%)38 ( 6%)522 ( 8% ) left bundle branch block45 ( 15%)40 ( 13%)85 ( 14%)865 ( 13%)treatment at randomization -blocker281 ( 92%)281 ( 92%)562 ( 92%)5820 ( 90% ) ace inhibitor243 ( 80%)255 ( 83%)498 ( 82%)5116 ( 79% ) arb51 ( 17%)36 ( 12%)87 ( 14%)927 ( 14% ) diuretic ( excluding antialdosterone)264 ( 87%)266 ( 87%)530 ( 87%)5414 ( 83% ) aldosterone antagonist224 ( 74%)218 ( 71%)442 ( 72%)3922 ( 60% ) digitalis83 ( 27%)99 ( 32%)182 ( 30%)1416 ( 22%)devices at least one device10 ( 3%)13 ( 4%)23 ( 4%)244 ( 4% ) icd9 ( 3%)11 ( 4%)20 ( 3%)207 ( 3%)data are numbers of patients ( % ) or means sd.bmi , body mass index ; bpm , beats per minute ; sbp , systolic blood pressure ; dbp , diastolic blood pressure ; egfr , estimated glomerular filtration rate ; nyha , new york heart association ; ace , angiotensin - converting enzyme ; arb , angiotensin ii receptor blocker ; icd , implantable cardioverter defibrillator ; mdrd , modification of diet in renal disease study equation .
table 2types and doses of beta - blockers at baseline in the substudy population ( included set ) vs. the population of the main shift study ( randomized set)shift echocardiography substudymain study ( n = 6505)ivabradine ( n = 304)placebo ( n = 307)all ( n = 611)-blockers281 ( 92%)281 ( 91.5%)562 ( 92%)5820 ( 90% ) carvedilol160 ( 57%)149 ( 53%)309 ( 55%)2604 ( 45% ) bisoprolol46 ( 16%)60 ( 21%)106 ( 19%)1486 ( 26% ) metoprolol succinate48 ( 17%)45 ( 16%)93 ( 17%)815 ( 14% ) metoprolol tartrate16 ( 6%)19 ( 7%)35 ( 6%)618 ( 11% ) nebivolol9 ( 3%)9 ( 3%)18 ( 3%)198 ( 3% ) other2 ( 0.7%)2 ( 0.4%)107 ( 1.8%)mean daily dosage of -blocker ( mg ) carvedilol23.5 16.023.5 17.123.5
3.25.9 3.26.2 3.3 metoprolol succinate131.3 80.2131.9 73.3131.6 76.589.9 60.0 metoprolol tartrate72.7 46.2115.8 50.896.1 52.869.1 47.4 nebivolol6.4 2.84.3 2.65.4 2.85.9
2.9patients at target dose of -blocker76 ( 27%)87 ( 31%)163 ( 29%)1488 ( 26%)patients at 50% target dose of -blocker159 ( 57%)154 ( 55%)313 ( 56%)3181 ( 56%)data are numbers of patients ( % ) or means sd . baseline characteristics of the substudy population ( included set ) vs. the population of the main shift study ( randomized set ) data are numbers of patients ( % ) or means sd .
bmi , body mass index ; bpm , beats per minute ; sbp , systolic blood pressure ; dbp , diastolic blood pressure ; egfr , estimated glomerular filtration rate ; nyha , new york heart association ; ace , angiotensin - converting enzyme ; arb , angiotensin ii receptor blocker ; icd , implantable cardioverter defibrillator ; mdrd , modification of diet in renal disease study equation . types and
doses of beta - blockers at baseline in the substudy population ( included set ) vs. the population of the main shift study ( randomized set ) data are numbers of patients ( % ) or means sd .
median treatment duration during the substudy was 8.1 months ( iqr , 7.88.3 ) and median follow - up after the 8-month echocardiogram was 16.1 months ( iqr , 1319.9 ) . mean dosage of ivabradine during the substudy was 6.0 1.6 mg bid .
heart rate was reduced by 14.7 11.4 bpm in the ivabradine group to 63.5 9.5 bpm at 8 months vs. by 5.8 10.8 bpm to 72.2 12.4 bpm with placebo .
mean baseline lvesvi was similar in the ivabradine and placebo groups ( 65.2 29.1 and 63.6 30.1 ml / m , respectively ) .
treatment with ivabradine for 8 months was associated with a significant reduction in lvesvi vs. placebo [ 7.0 16.3 vs. 0.9 17.1 ml / m , estimate ( se ) 5.8 ( 1.6 ) , 95% ci 8.8 to 2.7 , p < 0.001 , table 3 ] .
more patients in the ivabradine group had a 15% or more decrease in lvesvi than in the placebo group ( 38 vs. 25% , respectively , p= 0.005 ) ( figure 2 ) .
the ivabradine - associated improvement in lvesvi was consistent in all the prespecified subgroups : ischaemic vs. non - ischaemic hf ( estimate of treatment - placebo difference 5.51 vs. 7.61 ) , with or without half target beta - blocker dose at randomization ( 4.39 vs. 7.89 ) , and baseline lvef above and below 32% ( 6.08 vs. 5.77 ) .
table 3echocardiographic parameters at baseline , 8 months , and their changes in the substudyvariableivabradineplacebotreatment effectnbaseline8 monthschangenbaseline8 monthschangee ( se ) , 95% cip - valueprimary endpoint lvesvi ( ml / m)20865.2 29.158.2 28.37.0 16.320363.6 30.162.8 28.70.9 17.15.8 ( 1.6 ) , 8.8 to 2.7<0.001secondary endpoints lvesv ( ml)208123.8 55.6110.8 54.613.0 31.6203122.2 59.8120.9 56.41.3 32.811.2 ( 3.0 ) , 17.1 to 5.4<0.001 lvedvi ( ml / m)20493.9 32.885.9 30.97.9 18.919990.8 33.189.0 31.61.8 19.05.5 ( 1.8 ) , 8.9 to 2.00.002 lvedv ( ml)204178.4 63.4163.7 60.614.7 36.4199174.7 67.6171.7 63.82.9 36.810.9 ( 3.4 ) , 17.6 to 4.20.001 lvef ( % ) 20432.3 9.134.7 10.22.4 7.719931.6 9.331.5 10.00.1 8.02.7 ( 0.8 ) , 1.3 to 4.2<0.001data are numbers of patients ( % ) or means sd .
parametric approach with adjustment for country , beta - blocker intake at randomization , and baseline value .
e ( se ) , estimate ( standard error of treatment effect ) ; ci , confidence interval.lvesv , left ventricular end - systolic volume ; lvesvi , left ventricular end - systolic volume index ; lvedv , left ventricular end - diastolic volume ; lvedvi , left ventricular end - diastolic volume index ; lvef , left ventricular ejection fraction .
figure 2(a ) relative change in left ventricular end - systolic volume index ( lvesvi ) and ( b ) absolute change in left ventricular ejection fraction ( lvef ) from baseline to 8 months .
the grey and white bars represent percentages of patients reaching echocardiographic criteria for the ivabradine and placebo groups , respectively .
echocardiographic parameters at baseline , 8 months , and their changes in the substudy data are numbers of patients ( % ) or means sd .
parametric approach with adjustment for country , beta - blocker intake at randomization , and baseline value .
e ( se ) , estimate ( standard error of treatment effect ) ; ci , confidence interval .
lvesv , left ventricular end - systolic volume ; lvesvi , left ventricular end - systolic volume index ; lvedv , left ventricular end - diastolic volume ; lvedvi , left ventricular end - diastolic volume index ; lvef , left ventricular ejection fraction .
( a ) relative change in left ventricular end - systolic volume index ( lvesvi ) and ( b ) absolute change in left ventricular ejection fraction ( lvef ) from baseline to 8 months .
the grey and white bars represent percentages of patients reaching echocardiographic criteria for the ivabradine and placebo groups , respectively .
the mean values of the secondary efficacy criteria were similar at baseline in the ivabradine and placebo groups ( table 3 ) .
significant treatment effects were found at 8 months for ivabradine vs. placebo on lvedvi ( p= 0.002 ) , lvesv ( p < 0.001 ) , lvedv ( p= 0.001 ) , and lvef ( p
0.001 ) ( table 3 ) . left ventricular ejection fraction was increased by 2.4 7.7% in the ivabradine group but was unchanged in the placebo group ( 0.1 8.0% ) .
more than a third ( 36% ) of the patients in the ivabradine group had 5% increase in lvef vs. less than a quarter ( 23% ) of the placebo group ( p= 0.003 ) ( figure 2 ) .
there were no significant changes in left - atrial end - systolic volume index or rv myocardial performance index in either group , while rv s wave peak velocity increased over 8 months with ivabradine and decreased with placebo ( data not shown ) .
there were 351 patients who had an evaluation of mitral regurgitation at baseline and 8 months .
of these , mitral regurgitation improved by at least one grade in 18 patients ( 18/176 , 10% ) in the ivabradine group vs. 14 patients ( 14/175 , 8% ) in the placebo group ( p= 0.47 ) .
more patients improved by at least one grade of lv diastolic function from baseline to 8 months with ivabradine [ 26 patients out of 120 fully documented patients ( 22% ) ] than with placebo [ 11 patients out of 106 ( 10% ) , p= 0.02 ] . to explore the impact of baseline values of echocardiographic parameters reflecting lv systolic function
, we divided the placebo group by median baseline lvesvi ( 59 ml / m ) and found that the incidence of the primary composite endpoint of hospitalization for worsening hf or cardiovascular mortality was significantly greater in the patients with higher values ( hr 1.62 , 95% ci 1.032.56 , p= 0.04 , figure 3 ) .
the increase in risk appeared to be driven by both hospitalizations for worsening hf ( hr 1.80 , 95% ci 1.063.07 , p= 0.03 ) and cardiovascular deaths ( hr 1.56 , 95% ci 0.783.10 , p= 0.21 , table 4 ) .
similar changes in risk were observed when the placebo group was split by median baseline lvedvi ( 85 ml / m ) , or for median baseline lvef ( 31% ) , though the differences were not statistically significant for lvef ( table 4 ) .
table 4relationship between echocardiography parameters at baseline and the shift primary composite endpoint and its components in the placebo group of the substudy ( included set echo)number of eventshr ( 95% ci)p - valuelvesvi ( n = 263 ) shift primary composite endpoint 59 ml / m561.62 ( 1.032.56)0.04 < 59
ml / m35 hospitalization for worsening heart failure 59 ml / m441.80 ( 1.063.07)0.03 < 59 ml / m25 cardiovascular death 59 ml / m281.56 ( 0.783.10)0.21 < 59 ml / m15lvedvi ( n = 257 ) shift primary composite endpoint 85 ml / m561.75 ( 1.112.75)0.02 < 85 ml / m34 hospitalization for worsening heart failure 85 ml / m441.96 ( 1.153.34)0.01 < 85 ml / m24 cardiovascular death 85 ml / m281.65 ( 0.843.23)0.14 < 85 ml / m15lvef ( n = 257 ) shift primary composite endpoint < 31%501.21 ( 0.781.88)0.40
31%391.28 ( 0.772.13)0.34 31%29 cardiovascular death < 31%261.29 ( 0.672.51)0.45 31%17lvesvi , left ventricular end - systolic volume index ; lvedvi , left ventricular end - diastolic volume index ; lvef , left ventricular ejection fraction ; hr , hazard ratio ; ci , confidence interval.there were 43 cardiovascular deaths in the placebo group over the entire duration of the main shift study ( table 4 ) , compared with 36 deaths in the two study arms combined during the 8 months of follow - up of the echocardiography substudy ( figure 1 ) .
figure 3kaplan meier cumulative event curves for the shift primary composite endpoint of cardiovascular death or hospitalization for worsening heart failure in the placebo group split by median left ventricular end - systolic volume index ( lvesvi ) 59 vs. < 59
relationship between echocardiography parameters at baseline and the shift primary composite endpoint and its components in the placebo group of the substudy ( included set echo ) lvesvi , left ventricular end - systolic volume index ; lvedvi , left ventricular end - diastolic volume index ; lvef , left ventricular ejection fraction ; hr , hazard ratio ; ci , confidence interval .
there were 43 cardiovascular deaths in the placebo group over the entire duration of the main shift study ( table 4 ) , compared with 36 deaths in the two study arms combined during the 8 months of follow - up of the echocardiography substudy ( figure 1 ) .
meier cumulative event curves for the shift primary composite endpoint of cardiovascular death or hospitalization for worsening heart failure in the placebo group split by median left ventricular end - systolic volume index ( lvesvi ) 59 vs. < 59 ml / m . in the shift echocardiography substudy population , there were 102 primary composite endpoint events ( 81 hospitalizations for worsening hf and 21 cardiovascular deaths ) , 63 of which occurred after 8 months .
this relatively low number of events provided little power to detect an effect of treatment on outcomes according to echocardiographic parameters . to obtain some measure of the effect
, we divided the ivabradine group and the placebo group by tertiles of change in lvesvi and determined the incidence of primary composite endpoint after 8 months for each third . despite few events in this exploratory analysis , patients in the lowest third ,
i.e. those with the greatest relative reductions in lvesvi , had numerically lower event rates than those with smaller relative reductions or relative increases ( data not shown ) .
the relationships between changes in heart rate and both changes in lv volumes and lvef were explored .
there was an inverse relation between the change in heart rate and the change in lvef ( r = 0.17 , p= 0.0006 ) ; expressed differently , larger decreases in heart rate were associated with greater increases in lvef .
in contrast , there was no significant relationship between changes in heart rate and changes in lv volumes for all patients .
however , when split by baseline median heart rate , those patients who were above the median of 77 bpm had larger lv volumes ( lvesvi : 68.1 29.3 vs. 61.8 30.3 ml / m , p= 0.019 ) and lower lvef ( 29.4 9.0 vs. 33.1 8.8% , p < 0.001 ) at baseline .
our results indicate a reversal of cardiac remodelling with ivabradine , with significant reductions in lv volumes and an increase in lvef over 8 months of treatment .
these changes were consistent in the prespecified subgroups , irrespective of baseline lvef , beta - blocker intake , and aetiology of hf .
moreover , these results occurred despite treatment with beta - blockers and raas antagonists , each used in more than 90% of patients .
our results have important clinical implications since cardiac remodelling is a central feature of the progression of hf .
we explored the association between the risk of clinical cardiovascular adverse outcomes and echocardiographic parameters in a series of complementary analyses .
left ventricular end - systolic volume index 59 ml / m at baseline was associated with a 62% increase in relative risk for the shift primary composite endpoint of cardiovascular death or hospitalization for worsening hf vs. patients with lvesvi < 59 ml / m . using the median to divide these data
may have diluted the relation to the shift primary composite endpoint observed at higher lv volumes .
in addition , the patients with the largest treatment - related reductions in lvesvi at follow - up had a numerically lower rate of the shift primary composite endpoint than patients with smaller reductions in lvesvi .
this exploratory analysis suggests that there is a link between treatment - related improvement in lv function and prognosis in hf patients , and is consistent with the observation that short - term effects of drugs or devices are associated with long - term effects on mortality .
the study provides further support for the role of heart rate reduction with ivabradine to improve cardiac remodelling .
compared with those with lower heart rates , patients with a heart rate 77 bpm at baseline had larger lvesvi and lower lvef at baseline .
furthermore , there was a statistically significant albeit weak inverse relationship between the change in heart rate and the change in lvef , which means that larger decreases in heart rate during follow - up were associated with greater increases in lvef .
a positive effect of ivabradine on lv remodelling was also demonstrated in the echocardiographic substudy of the beautiful ( morbidity - mortality evaluation of the if inhibitor ivabradine in patients with coronary disease and left ventricular dysfunction ) trial , in a different population of patients with stable coronary artery disease and lv systolic dysfunction .
results in experimental studies are consistent with the effects of ivabradine on cardiac remodelling that we observed . in a rat model of chronic mild hf ,
these changes were linked to modifications in the extracellular matrix and in cardiac myocyte function .
ivabradine also has had beneficial effects on the global cardiac remodelling process involved in hf , including optimization of energy consumption , reverse electrophysiological and structural cardiac remodelling .
similar effects with ivabradine have been found by other workers in a rat model of chronic severe hf , including reductions in fibrosis , local raas stimulation , and sympathetic drive , as well as improvement in endothelial function .
experiments in a canine model also indicated a positive impact on sarcoplasmic reticulum calcium overload .
together , these experimental results support a beneficial role of ivabradine on lv remodelling and function that may contribute to the reduction in cardiac morbidity and mortality found in patients with hf . the reduction in morbidity and mortality with raas antagonists and beta - blockade in hf
our analysis was not designed to clarify the time - course of treatment effects and could not evaluate the acute effect of ivabradine . in the absence of acute measurements , the degree to which volumetric changes are influenced by heart rate itself can not be determined .
our finding of improved lv function with ivabradine shows that further improvement in reverse cardiac remodelling and slowing of disease progression can be achieved , in addition to that obtained with background therapy for hf .
patients needed to be treated with a beta - blocker to be eligible for this study , and the shift trial sought to exclude patients who were not receiving guidelines - driven beta - blocker dosing . given the beta - blocker dosage observed in shift ( which was similar to other recently published data ) and the effect on lv ejection fraction of higher doses , further investigation would be required to shed light on the comparison between ivabradine and more aggressive beta - blockade ( when tolerated ) .
our data were recorded in patients with heart rates 70 bpm and in sinus rhythm and predominantly in men , which may limit the generalization of our results to all patients with hf .
another limitation of our study is that about a third of patients were excluded from the analysis , usually for reasons related to the quality or collection of the echocardiographic recordings .
the reduction in morbidity and mortality with raas antagonists and beta - blockade in hf is associated with improvement in lv function .
our analysis was not designed to clarify the time - course of treatment effects and could not evaluate the acute effect of ivabradine . in the absence of acute measurements , the degree to which volumetric changes are influenced by heart rate itself can not be determined .
our finding of improved lv function with ivabradine shows that further improvement in reverse cardiac remodelling and slowing of disease progression can be achieved , in addition to that obtained with background therapy for hf .
patients needed to be treated with a beta - blocker to be eligible for this study , and the shift trial sought to exclude patients who were not receiving guidelines - driven beta - blocker dosing . given the beta - blocker dosage observed in shift ( which was similar to other recently published data ) and the effect on lv ejection fraction of higher doses , further investigation would be required to shed light on the comparison between ivabradine and more aggressive beta - blockade ( when tolerated ) .
our data were recorded in patients with heart rates 70 bpm and in sinus rhythm and predominantly in men , which may limit the generalization of our results to all patients with hf .
another limitation of our study is that about a third of patients were excluded from the analysis , usually for reasons related to the quality or collection of the echocardiographic recordings .
heart rate reduction with ivabradine reverses lv remodelling in patients with hf and lv systolic dysfunction .
treatment with ivabradine is associated with marked reductions in lv volumes and a significant improvement in lvef , therefore suggesting that it is modifying disease progression in patients with hf .
this work was supported by servier , france . funding to pay the open access publication charges for this article was provided by servier .
conflict of interest : all authors have received fees , research grants , or both from servier .
| aimsthe shift echocardiographic substudy evaluated the effects of ivabradine on left ventricular ( lv ) remodelling in heart failure ( hf).methods and resultseligible patients had chronic hf and systolic dysfunction [ lv ejection fraction ( lvef ) 35% ] , were in sinus rhythm , and had resting heart rate 70 bpm .
patients were randomly allocated to ivabradine or placebo , superimposed on background therapy for hf .
complete echocardiographic data at baseline and 8 months were available for 411 patients ( ivabradine 208 , placebo 203 ) .
treatment with ivabradine reduced lvesvi ( primary substudy endpoint ) vs. placebo [ 7.0 16.3 vs. 0.9 17.1 ml / m2 ; difference ( se ) , 5.8 ( 1.6 ) , 95% ci 8.8 to 2.7 , p < 0.001 ] .
the reduction in lvesvi was independent of beta - blocker use , hf aetiology , and baseline lvef .
ivabradine also improved lv end - diastolic volume index ( 7.9 18.9 vs. 1.8 19.0 ml / m2 , p= 0.002 ) and lvef ( + 2.4 7.7 vs. 0.1 8.0% , p < 0.001 ) .
the incidence of the shift primary composite outcome ( cardiovascular mortality or hospitalization for worsening hf ) was higher in patients with lvesvi above the median ( 59 ml / m2 ) at baseline ( hr 1.62 , 95% ci 1.032.56 , p= 0.04 ) .
patients with the largest relative reductions in lvesvi had the lowest event rates.conclusionivabradine reverses cardiac remodelling in patients with hf and lv systolic dysfunction . |
glaucoma is characterized by progressive optic nerve damage resulting in irreversible loss of vision and is a leading cause of blindness worldwide.1 the only available treatment to delay the progression of glaucoma involves lowering the intraocular pressure ( iop ) .
iop reduction is most commonly accomplished by applying topical medications that reduce the rate of aqueous humor formation or reduce resistance to aqueous humor outflow.2 invasive procedures such as trabeculectomy or glaucoma drainage device implantation are typically performed when medical therapy fails . despite improvements in glaucoma patient outcomes , compliance to prescribed topical treatments remains an issue , and many surgical procedures are accompanied by significant morbidity . as a result , new micro - invasive techniques and implants capable of providing significant iop reduction have been developed .
micro - invasive glaucoma surgery with the glaukos istent and istent inject ( istent is fda approved in the us , ce marked in the european union , and has medical device approval in canada .
istent inject is an investigational device in the us , and is ce marked in the european union ; glaukos corporation , laguna hills , ca , usa ) is intended to improve physiologic outflow of aqueous humor from the anterior chamber through the conventional outflow pathway.3 previous evaluations by an anterior segment perfusion model,4,5 flow theory,6 and clinical studies,713 have shown that one or more istents placed in schlemm s canal through the trabecular meshwork resulted in lower iop and reduced medication burden .
the istent ( figure 1a ) was designed to be implanted in schlemm s canal via an ab - interno procedure through a self - sealing clear corneal incision .
the self - trephining tip of the stent pierces the trabecular meshwork , and the curved stent body with retention arches maintains the position of the stent within schlemm s canal .
the open half - pipe design of the stent body assures that the canal remains patent and collector channels are not obstructed .
the tubular portion of the stent , the snorkel , conducts the flow through the trabecular meshwork into schlemm s canal .
the istent inject ( figure 1b ) was designed to be implanted over a trocar that pierces the trabecular meshwork .
the head of the stent facilitates implantation and maintains the position of the stent across the trabecular meshwork .
four side ports are present to direct flow circumferentially into the lumen of schlemm s canal . while istent and istent inject
have been evaluated in the anterior segment perfusion model,4,5 the stents have not been previously evaluated in whole eye ex vivo models . unlike the whole globe model , the anterior chamber model lacks the lens , iris , and choroid which can affect the tension on the scleral spur , and such tension has been shown to affect outflow facility.1418 while the differences in the models were not anticipated to have a large effect on the facility change effected by manipulation of the trabecular meshwork , to verify this assumption the istent was evaluated in the whole globe model , and the results were compared with bahler et al.4 engineering approximations ( hagen poiseuille ) of the resistance through the stents suggest that the resistance is extremely low . the engineering approximations were verified using computational fluid dynamics ( cfd ) , and total outflow resistances with and without stents were calculated .
human globes from pseudophakic donors with no history of glaucoma were obtained from various eye banks around the country .
approval was obtained from the colorado multiple institutional review board for the use of human material , and the tenets of the declaration of helsinki were followed , prior to initiation of this study .
informed consent was obtained from donors or relatives for use in research by the various eye banks .
the perfusion system ( figure 2 ) incorporated a standard programmable syringe pump ( pump 11 plus ; harvard apparatus , holliston , ma , usa ) .
pressure was monitored via an in - line real - time pressure transducer ( research grade pressure transducer ; harvard apparatus ) connected to a single channel chart recorder ( pharmacia rec-481 ; pharmacia / pfizer , new york , ny , usa ) .
polyethylene tubing with a 1.14 mm inner diameter ( pe-160 ; warner instruments , hamden , ct , usa ) was used for all connections .
the human globe , in each case , was first prepared by injecting dulbecco s modified eagle s medium ( dmem ; life technologies , carlsbad , ca , usa ) through the optic nerve with a 26-gauge needle until the globe had returned to a spherical shape .
the perfusion system is illustrated in figure 2 . a fluid line ( terminating in another 26-gauge needle )
was inserted diagonally through the anterior chamber of the eye , passing through the cornea and pupil and ending with the tip beneath the iris ( not shown ) .
the globe was surrounded by damp gauze , and the perfusion pump ( filled with dmem ) was set to an initial inflow rate of 7 l / minute .
iop was allowed to increase until it reached 30 mmhg to ensure the globe was completely inflated . the infusion rate
was then reduced to 2.5 l / minute , and a steady - state iop was maintained for at least 60 minutes prior to insertion of the devices .
stent placement was not targeted to any specific features of the trabecular meshwork or schlemm s canal , although they were implanted on the nasal side to mimic the clinical procedure .
when more than one stent was inserted , the second stent was placed three clock hours from the first .
a water tight closure of the corneal wound was then achieved with a 10 - 0 nylon suture .
the flow rate remained constant , and iop was measured until steady state was achieved for at least 1 hour post - insertion of the device(s ) in all cases in order to determine that the true steady state was achieved . at low flow rates , the stent resistance , rs , can be estimated using the hagen
the assumptions of the equation are that the flow is laminar , viscous , and incompressible , and the flow is through a constant circular cross - section where the length of the tube is substantially longer than its diameter . according to hagen poiseuille : where l is the length of the tube , r is the radius of the tube , and = 7.2 10 pa sec is the aqueous humor viscosity at 37c.19 the equivalent circuit models used for calculating resistance through the stents are shown in figure 3 .
the istent is a single resistance element calculated via equation 1 , with a length of 250 m and a radius of 60 m : rs 4.42 10 mmhg/(l / minute ) .
the istent inject presents resistance from the central lumen in series with the parallel resistances from the side ports . in this approximation
the central lumen dimensions were approximately l = 265 m and r = 40 m , and the side lumen dimensions were l = 46 m and r = 25 m : rs 3.72 10 mmhg/(l / minute ) .
the application of hagen poiseuille suggests that the resistance of the istent is 12% of the resistance of the istent inject .
inlet and outlet effects ( entrance head loss and exit head loss ) would be expected to raise the effective resistance somewhat from what is calculated by hagen
poiseuille for both stents . however , as shown below , the flow resistance of both stents is negligible compared with the total outflow resistance of the eye .
cfd models of the two devices were created and solved using the commercial code cfd research corporation ( esi group , paris , france ) . due to the simple geometry of fluid passageway through the istent , an axisymmetric ( two - dimensional ) mesh could be used .
for the istent inject , a three - dimensional mesh was created containing 280,328 three - dimensional hexahedral elements . in preprocessing ,
the mesh was configured such that only two of the four side outlets maintained flow ; this assumed that when placed in schlemm s canal , the other outlets would be blocked by tissue contact . while additional outlets may be unblocked in situ , modeling it in this way represents a worst - case scenario .
aqueous humor is modeled as an incompressible newtonian fluid with constant dynamic viscosity of 7 10 pa sec and density 1.00 g / cm . in this initial application of cfd
, we included inlet and outlet regions corresponding to the anterior chamber and schlemm s canal , respectively .
the no - slip condition was applied at the stent and non - inlet / exit surfaces of the anterior chamber inlet section and schlemm s canal exit section . neglecting any uveoscleral flow in the ex vivo whole human eye perfusion model , the outflow resistance
is simply calculated per the following equation : where rn is the total outflow resistance , iopn is the iop when n stents are implanted in the eye , and qa is the total flow into the eye .
the results from the ex vivo perfusion studies are summarized in table 1 along with bahler et als4 data from the anterior chamber model .
a single istent resulted in a lowering of iop from baseline in all three eyes tested .
the addition of a second istent to an existing device resulted in additional iop lowering that was statistically significant .
the nominal rate of aqueous humor production is 2.5 l / minute.20 a worst - case scenario for examining the performance of the stent , ie , flow rate of 5 l / minute , twice the nominal , was considered .
poiseuille approximation , shown in equation 1 , suggest that a pressure differential of only 0.021 mmhg is sufficient to drive 5 l / minute through the istent , and only 0.18 mmhg is sufficient to drive 5 l / minute of flow through the istent inject under the worst - case assumption made above of only two side ports unobstructed .
poiseuille approximations were verified via cfd by applying static pressure differentials to the stents over these ranges .
the steady - state flow of the stent was evaluated with regard to the boundary conditions .
these results are shown in figure 4 and were compared to the expected flow based on the resistance calculated from the hagen
the linearity in figure 4 indicates that at these flow rates , the resistance was pressure independent , and the actual resistance of the stents was somewhat higher than that approximated by hagen
the cfd results indicate that the flow resistance of the istent is 6.410 mmhg/(l / minute ) and 5.610 mmhg/(l / minute ) for the istent inject .
cfd analysis performed with and without the inlet and outlet regions indicated that much of the increase was due to inlet and outlet flow effects : the pressure loss due to inlet and outlet effects with the istent was 26.9% , and 18.5% for the istent inject .
regardless , the cfd models estimated that a single istent or istent inject was wholly sufficient to conduct the entire 2.5 l / minute of aqueous humor production at negligible pressure differentials of only 0.016 and 0.14 mmhg , respectively .
figure 5 shows a pressure color map through the istent body generated by the model at a pressure differential of 0.016 mmhg , resulting in a flow rate of 2.5 l / minute .
cooler colors indicate lower pressures , while warmer colors indicate higher pressures ; the pressure scale , shown above the stent body , indicates color correspondence to pressure in mmhg .
the pressure within the stent transitioned smoothly from the inlet pressure to the outlet pressure , with minimal inlet effects .
maximum axial velocity at the centerline was 0.76 cm / second , and the maximum reynolds number was 1.34 .
a detailed inspection of the flow through the snorkel confirmed that the velocity profile was indeed parabolic ( not shown ) .
figure 7 shows the pressure differential inside the istent inject , and figure 8 illustrates the flow capability of the stent , at a pressure differential of 0.14 mmhg , resulting in a flow rate of 2.5 l / minute .
as with the istent , the pressure in the istent inject transitioned smoothly from the inlet pressure to the outlet pressure , with minimum inlet effects .
maximum axial velocity along the centerline was 1.76 cm / second , yielding a reynolds number of 2.01 , and 2.15 cm / second at the exit ports , yielding a reynolds number of 1.54 .
again , these numbers indicate that the laminar flow assumption is correct , although some minor additional pressure losses may be associated with secondary ( ie , non - axial ) velocities seen at the outlet junction on the right - hand side of figure 8 . inserting the ex vivo data ( table 1 ) into equation 2 , the total outflow resistances calculated with zero , one , and two stents were r0 = 7.9 mmhg/(l / minute ) , r1 = 5.5 mmhg/(l / minute ) , and r2 = 4.4 mmhg/(l / minute ) , respectively .
the current whole globe ex vivo studies verify previous findings that the istent is an effective iop lowering device , and that efficacy is enhanced when combining two devices compared with a single device .
the addition of a second stent lowered the pressure by another 2.91.1 mmhg to a total pressure reduction of 8.92.2 mmhg .
these findings agree with perfusion in the anterior segment model,4 theoretical analysis,6 and clinical studies.713 use of an ex vivo model is advantageous because it is fast , inexpensive , and it allows new devices to be tested using the appropriate anatomy prior to clinical studies , especially when the only comparable animal models are primates .
the data in table 1 suggest that either the anterior segment model or the whole globe model is an appropriate ex vivo model for evaluating trabecular meshwork stents .
cfd analysis revealed that flow through the istent and istent inject is free of significant resistance , only 0.0062 and 0.057 mmhg/(l / minute ) , respectively , at physiological flow rates .
potential effects of circumferential resistance through schlemm s canal and distal resistance through the collector channels21 are not completely understood and were not incorporated into the cfd model . instead , experimental data from the ex vivo studies were utilized to evaluate total outflow resistance . applying equation 2 ,
the total outflow resistance was calculated to be r0 = 7.9 mmhg/(l / minute ) , r1 = 5.5 mmhg/(l / minute ) , and r2 = 4.4 mmhg/(l / minute ) , for zero , one , and two stents , respectively .
thus , a single istent reduced the total outflow resistance by 30% , and two istents reduced the total outflow resistance by 44% .
these results agree with previous ex vivo studies.4,22 the cfd resistance values are somewhat higher than the resistance predicted by simple circuit models and illustrate the capability of cfd to consider inlet and outlet effects that are often neglected by simpler models . however , the resistance values are so low that a single istent is capable of carrying the entire aqueous humor outflow from the anterior chamber into schlemm s canal at a pressure difference of only 0.016 mmhg or less .
the resistance of the istent is two or three orders of magnitude less than the total outflow resistance . in practice
, the aqueous humor outflow utilizes a combination of the istent , the trabecular meshwork , schlemm s canal , and the distal outflow pathways .
the low resistance of the istent serves to lower the overall outflow resistance and iop .
consistent with the findings of bahler et al,4 adding a second istent further decreased iop , probably by accessing additional regions of the distal outflow pathway .
a general limitation of computational and laboratory models is that their results may not exactly represent in vivo outcomes .
it is encouraging , therefore , that extensive clinical experience with istent demonstrates prolonged reduction of iop and medication burden , combined with an excellent safety profile.713
in the whole globe ex vivo model , a single istent reduced iop by 6.0 mmhg from baseline , and combination of two istent implants lowered iop by 8.9 mmhg from baseline . despite the micro - size of the istent and istent inject trabecular bypass implants , analytical approximations supported by cfd modeling indicated that each stent presented a negligible flow resistance that resulted in significant iop reduction .
cfd modeling also indicated that flow through the istent and istent inject lumens was smooth and laminar .
these and prior clinical and laboratory studies show that istent and istent inject therapy is potentially titratable , allowing physicians to reach lower target iops with the implantation of additional stents . | micro - invasive glaucoma surgery with the glaukos istent or istent inject ( glaukos corporation , laguna hills , ca , usa ) is intended to create a bypass through the trabecular meshwork to schlemm s canal to improve aqueous outflow through the natural physiologic pathway .
while the istent devices have been evaluated in ex vivo anterior segment models , they have not previously been evaluated in whole eye perfusion models nor characterized by computational fluid dynamics . intraocular pressure
( iop ) reduction with the istent was evaluated in an ex vivo whole human eye perfusion model .
numerical modeling , including computational fluid dynamics , was used to evaluate the flow through the stents over physiologically relevant boundary conditions . in the ex vivo model ,
a single istent reduced iop by 6.0 mmhg from baseline , and addition of a second istent further lowered iop by 2.9 mmhg , for a total iop reduction of 8.9 mmhg .
computational modeling showed that simulated flow through the istent or istent inject is smooth and laminar at physiological flow rates .
each stent was computed to have a negligible flow resistance consistent with an expected significant decrease in iop .
the present perfusion results agree with prior clinical and laboratory studies to show that both istent and istent inject therapies are potentially titratable , providing clinicians with the opportunity to achieve lower target iops by implanting additional stents . |
the process of aligning images so that the corresponding features can be related is called image registration .
image registration methods have been discussed and
classified in books [ 14 ] and surveys [ 510 ] .
a way to reduce the ad hoc nature of registration methods is to
require them to satisfy certain properties .
researchers have realized the
importance of symmetry and transitivity of registration methods [ 1120 ] . in ,
ashburner et al . proposed an approximately symmetric
image registration method that uses symmetric priors . in
, christensen and johnson
proposed a registration algorithm that approximately satisfies the symmetry
property .
( christensen and johnson used the term inverse consistency for
what we refer to as symmetry .
) their idea is to estimate the forward and
reverse transformation simultaneously by minimizing an objective function
composed of terms that measure the similarity between the two images and the
consistency of the forward and reverse transformations .
this approach requires
maintaining two transformations , computing their inverses and it has a tradeoff
among the terms in the objective function . in ,
rogelj and kovai proposed a
registration method that uses symmetrically designed forces that deform the two
images .
the method is approximately symmetric , it requires maintaining forward
and backward transformation , but it does not use their inverses . in , krinjar and tagare proposed
an exactly symmetric registration method that is based on a symmetrically
designed objective function , but it requires the computation of the inverse
transformation . in , lorenzen et al .
proposed an exactly symmetric
registration method that is based on a symmetrically designed fluid model .
the
method uses two transformations whose compositions define the forward and
backward transformations in such a way that they are inverses of each other .
beg and khan in
and avants et al . in used an exactly symmetric registration method that
maintains two functions , which when composed appropriately give forward and backward transformations that are exact inverses of each other . in
, cachier and rey analyzed
the reasons behind the asymmetry in registration , proposed symmetrized
similarity and smoothing energies , but their implementation of the method was
not exactly symmetric . in , tagare et al .
proposed an exactly symmetric
registration method that does not require to maintain both forward and reverse
transformations and compute their inverses .
instead , the objective function ,
which can be based on intensity differences ( e.g. , mean squared difference ,
normalized cross - correlation ) or distributions ( e.g. , mutual information ,
normalized mutual information ) is modified such that the method is symmetric
and only the forward transformation is needed .
consequently , the objective
function has only one term , which avoids the problem of tradeoff among multiple
terms .
their implementation is symmetric up to the machine precision . in , christensen and johnson
realized the importance of transitivity of image registration but did not provide a way to satisfy it .
the above methods are either approximately or exactly
symmetric but none of them is transitive . in this paper , we propose a method to
modify any image registration algorithm such that it is provably symmetric and
transitive on an image sequence . symmetry and transitivity are especially
important in motion tracking applications ; they insure that a physical point is
tracked in the same way regardless of the order in which the images are
registered .
if there are topological changes present in the image sequence , the
two properties can hold only over corresponding regions .
motion within the body is present at
the system level , organ level , tissue level , cellular level , subcellular level ,
and molecular level .
in addition to the normal motion , pathology - induced motion
or changes can occur ( e.g. , osteoporosis , multiple sclerosis , and tumor
growth ) . in both normal and pathology - induced motion or changes
, it is often
useful to compute the motion , that is , to register image sequences .
such
information can improve our understanding of the normal function and diseases
as well as help develop better treatments .
the presented approach is
illustrated on sequences of cardiac mr images , which if accurately registered
can provide clinically useful myocardial displacement and strain information .
however , the same or similar approach can be used for the registration of any
other image sequence .
let denote a set of
real numbers and an n - dimensional
metric space .
an n - dimensional
intensity image is a function i : . intensity images will be referred to as just images .
without loss of generality
the set of all images is denoted as . an n - dimensional
geometric transformation is a function t : . geometric transformations will be referred to as
just transformations .
the set of all transformations is denoted as . let tid denote the
identity transformation , that is , tid(r ) = r , r . let denote the
composition of transformations .
an image registration operator is a function : . ideally , any image registration operator should have
the following three properties i , j , k .
identity .
an image registration operator ,
the result of the registration should not depend on the order of images , that
is , when an image registration operator is applied to two images , the obtained
transformation should be the inverse of the transformation obtained when the
order of images is reversed .
the generated transformation from the second to the third
image composed with the generated transformation from the first to the second
image should be equal to the generated transformation from the first to the
third image .
an image registration operator , when applied to two identical images should
generate the identity transformation .
the result of the registration should not depend on the order of images , that
is , when an image registration operator is applied to two images , the obtained
transformation should be the inverse of the transformation obtained when the
order of images is reversed .
the generated transformation from the second to the third
image composed with the generated transformation from the first to the second
image should be equal to the generated transformation from the first to the
third image .
the proposed
approach is simple ; select a reference image and then perform the registration
of any two images from the sequence of images through the reference .
the
reference can be an image from the sequence of images or an image similar to
the images in the sequence .
let the reference be denoted as r and let represent an
image registration operator that does not necessarily have any of the
properties from section 2.2 . a new image registration operator is defined
as(4)(i , j)=[(j , r)]1(i , r),where i and j are any two
images from the sequence .
it is assumed that the transformations generated by from images in
the sequence to the reference image are invertable , that is , that [ (j , r ) ] always exists .
proofidentity holds since(5)(i , i)=[(i , r)]1(i , r ) by ( 4)=tid .
symmetry holds
since(6)(j , i)(i , j)=[(i , r)]1(j , r)[(j , r)]1 (i , r ) by ( 4)=[(i , r)]1(i , r)=tid by ( 4 ) .
transitivity holds
since(7)(j , k)(i , j)=[(k , r)]1(j , r)[(j , r)]1 (i , r ) by ( 4)=[(k , r)]1(i , r)=(i , k ) by ( 4 ) .
identity holds since(5)(i , i)=[(i , r)]1(i , r ) by ( 4)=tid .
symmetry holds
since(6)(j , i)(i , j)=[(i , r)]1(j , r)[(j , r)]1 (i , r ) by ( 4)=[(i , r)]1(i , r)=tid by ( 4 ) .
transitivity holds
since(7)(j , k)(i , j)=[(k , r)]1(j , r)[(j , r)]1 (i , r ) by ( 4)=[(k , r)]1(i , r)=(i , k ) by ( 4 ) .
it should be noted that the only requirement for the
above result to hold is that [ (j , r ) ] exists .
this
means that the obtained transformations and their inverses do not need to be
differentiable .
however , a number of registration methods involve
regularization terms that use transformation derivatives , in which case the
registration operator needs to generate diffeomorphic transformations . if the jacobian of the transformation (j , r ) is positive
then the inverse transformation [ (j , r ) ] exists .
if the
jacobian of the transformation
(j , r ) is zero or
negative , the inverse transformation does not exist and the
reference - based registration operator given by ( 4 ) can not be used or it can be
used only over the part of the domain where the jacobian is positive .
many
registration methods control the jacobian either directly [ 2226 ] or indirectly [ 12 , 2730 ] by using a smoothness term that penalizes extreme
warps to prevent singularities ( zero jacobian ) or folding of the space
( negative jacobian ) , in which case the inverse transformation exists and the
reference - based registration can be used .
while the
result of the previous section holds for any registration operator that
generates invertable transformations , here we illustrate the approach on two
sequences of cardiac magnetic resonance images using two nonrigid image
registration algorithms .
we acquired a
3d anatomical cine mri scan together with a 3d tagged cine mri scan of a
healthy volunteer and then repeated the acquisitions four months later .
the
volunteer was a 27-year - old male subject with no history of heart disease .
the
purpose of the tagged scan was to validate the myocardial deformation recovered
from the anatomical scan .
the scans were acquired using steady - state
free - precession short axis cine imaging ( flip angle = 65 , tr = 3.4 ms , te = 1.7 ms ) covering the entire heart
on a 1.5 t clinical mri scanner ( intera , philips medical systems , best , the
netherlands ) .
all the scans had contiguous short - axis slices with similar field
of view and phases covering the entire cardiac cycle and their parameters are
given in table 1 .
the tags were applied immediately after the detection of the
r - wave from the ekg signal and the first frame was acquired at a delay of 15
milliseconds after the r - wave .
two orthogonal sets of parallel planar tags with
about 9 mm plane separation were oriented orthogonal to the image planes . for both scans , for each acquired slice the scanner
recorded the rigid body transformation from the scanner coordinate system to
the slice .
this allowed us to map all the slices to a common coordinate system ,
that is , to spatially align the anatomical and tagged scans .
similarly , the
scanner recorded the start time for each phase ( frame ) relative to the peak of
the r wave , which allowed us to temporally align the anatomical and tagged
scans .
since the heart rate , that is , the duration of the cardiac cycle , was
not the same for the anatomical and tagged scans , we used relative time ( as a
percentage of the cardiac cycle ) for the temporal alignment . to recover the
myocardial deformation , we use thin plate splines ( tps ) to represent the
transformation between any two frames and then maximize the normalized mutual
information to determine
the transformation parameters ( tps node positions ) .
we use normalized mutual
information since it was shown to outperform several other images similarity
measures .
since
myocardium is nearly incompressible and its volume does not change by more than
4% over the cardiac cycle , we constrain the optimization of the tps node
positions such that the jacobian of the transformation never deviates from 1
( which corresponds to exact incompressibility ) by more than 4% .
( the purpose of this section is to illustrate the
approach of section 2.3 ( construction of symmetric and
transitive registration algorithms from nonsymmetric and nontransitive
registration algorithms ) , and instead of the method given in we could have used any
other registration method . for this reason we did not present here all the
details of the used registration method and the interested reader
this registration
algorithm we denote as 1 , while 2 represents its
unconstrained version .
given that near incompressibility is a physical property
of the myocardium , 1 is expected to
be more accurate than 2 . each of the two operators was used to recover
myocardial deformation from the two cardiac mr image sequences in two ways :
sequential and reference - based . in the sequential approach ,
the deformation was
recovered from the first to the second frame , then from the second to the third
frame , and so on . in the reference - based approach ,
figure 2 shows
a short - axis section through the 3d image and 3d lv wall model surface for the
sequential and referenced - based recoveries using the two registration
operators . to quantitatively evaluate the deformation recovery
accuracy we compared the cardiac deformation recovered from the anatomical cine
mri against the corresponding information from the tagged cine mri .
we
developed a tool for interactive positioning of virtual tag planes in tagged mri
scans .
the tag planes are modeled as splines that the user can position and
deform by moving control points .
this allows the user to fit the virtual tag
planes to the tagged image as well as to compute tag plane intersections .
once
the cardiac deformation is recovered from the anatomical cine mri using the
proposed method , it is applied to the virtual tag planes at ed and then
compared to the interactively positioned tag planes in other frames . in each
image slice , we compute the distances between the corresponding intersections
of orthogonal virtual tag lines ( in - slice cross - sections of the virtual tag
planes ) generated interactively and by the model .
this allows for in - plane
( short - axis ) deformation recovery validation .
the out - of - plane ( long - axis ) deformation
is not evaluated with this procedure since the tag planes , being perpendicular
to the short - axis image slices , do not contain information about the
out - of - plane motion .
virtual tag lines for the sequential and referenced - based
recoveries for the two operators are shown in figure 3 .
table 2 contains the
distances between corresponding intersections of virtual and real tag lines at
end systole , which is the most deformed state relative to end diastole .
let 1 and 2 represent the
symmetric and transitive modifications of 1 and 2 , respectively .
operators 1 and 2 are defined by
( 4 ) ( the end diastole frame is used for r ) , which
involves transformation inversion . since we use tps for transformation representation
and the inverse of a tps transformation can not be obtained analytically , we
invert the transformation numerically using the newton - raphson method for
solving nonlinear systems of equations .
the numerical error of the computation of the
inverse transform is denoted as . if the jacobian of the transformation is positive
then the inverse transformation exists and can be
specified to be an arbitrary small positive number , that is , the inverse
transformation can be computed with an arbitrary small error . while can be set to
an arbitrary small positive value , in practical applications little is gained
if is set to a
value smaller than two orders of magnitude smaller than the pixel size .
the
reason for that is that the registration error is on the order of the pixel
size , and by setting to one
hundredth the pixel size the error of the computation of the inverse
transformation is already negligible compared to the registration error , and
further reducing does not
improve the registration accuracy . for a given image registration operator , we define the following three errors .
identity error of image i is(8)eiden = maxr(i , i)(r)r.
symmetry
error of images i and j is(9)esym = maxr(j , i)((i , j)(r))r.
transitivity
error of images i , j , and k is(10)etran = maxr(j , k)((i , j)(r))(i , k)(r).
identity error of image i is(8)eiden = maxr(i , i)(r)r.
symmetry
error of images i and j is(9)esym = maxr(j , i)((i , j)(r))r.
transitivity
error of images i , j , and k is(10)etran = maxr(j , k)((i , j)(r))(i ,
k)(r). identity , symmetry and transitivity errors for 1 , 2 , 1 , and 2 are given in
tables 3 , 4 , and 5 , respectively .
the errors for 1 and 2 in the three
tables were computed using = 0.001 mm .
the
dependence of eiden , esym , and etran on is depicted in
figure 4 for 1 .
theorem 1 says
that reference - based modification of any registration operator satisfies
identity , symmetry , and transitivity on an image sequence .
while the theorem
always holds and the modified registration operator satisfies the three
properties exactly , section 3 demonstrates a practical application of the
reference - based registration using a relatively accurate registration algorithm
( 1 ) and its less accurate version ( 2 ) .
the method was applied to two cardiac cine mri
scans , which are periodic image sequences , but the same conclusions hold in the
case of linear image sequences .
as expected , the constrained registration ( 1 ) outperformed its unconstrained version ( 2 ) , which can be seen in figures 2 and 3 and in table
2 for both sequential and reference - based approaches .
the two figures and the
table also show that in this case the reference - based registration was more
accurate than the sequential registration .
the advantage of the sequential
registration is that the difference between any two consecutive frames is
relatively small , while the reference - based registration deals with larger
deformations ( e.g. , from end diastole to end systole ) .
however , the
disadvantage of the sequential registration is that there is accumulation of
error from frame to frame , which seems to overweigh the advantage of small
frame - to - frame difference .
the reference - based constrained registration
( column ( c ) in figures 2 and 3 ) was more accurate than the other three algorithms
( table 2 ) .
the difference in accuracy of the four algorithms is most
pronounced at end systole , and it can be seen as the different level of
agreement between the model contours and the underlying image in figure 2 and
between the virtual tag lines and the underlying image in figure 3 .
since 1 and 2 use tid as the initial
transformation in searching for the transformation that maximizes the
normalized mutual information , both operators satisfy ( 1 ) exactly and
consequently have eiden = 0 for any image ,
as it can be seen in table 3 .
operators 1 and 2 involve
transformation inversion as defined in ( 4 ) , which is done numerically with an
accuracy of . this is why the identity errors for 1 and 2 in table 3
are approximately equal to . the symmetry errors ( table 4 ) for 1 and 2 are
approximately 1 mm , while for 1 and 2 they are
approximately 2. the reason for this is that the evaluation of ( 9 )
involves a composition of the operators , each contributing approximately to esym due to the
numerical inversion of transformation in ( 4 ) .
similarly , the transitivity errors ( table 5 ) for 1 and 2 are
approximately 2 mm , while for 1 and 2 they are
approximately 2. the reason for this is that the evaluation of ( 10 )
involves a composition of the operators , each contributing approximately to etran due to the
numerical inversion of transformation in ( 4 ) .
it should be noted that 1 and 2 have similar
values for eiden , esym , and etran .
the reason for this is that the three errors depend
only on the accuracy of the computation of the inverse transformation , and not
on the registration accuracy ( 1 is more accurate than 2 ) .
in fact , if
the inverse transformation could be computed exactly , the three errors would be
zero regardless of the registration operator .
the three errors scale with , as shown in figure 4 , and they can be made as small
as the machine precision .
figure 4 also shows that eiden , esym 2 , and etran 2. thus , the reference - based registration slightly
worsens the identity error and it significantly improves the symmetry and
transitivity errors over the sequential registration .
for the two tested image sequences , the symmetric and
transitive registration methods 1 and 2 had smaller
registration errors than their nonsymmetric and nontransitive counterparts 1 and 2 , respectively ( table 2 ) . while all four registration
methods had either zero or nearly zero identity errors ( table 3 ) , 1 and 2 had very small
( nearly zero ) symmetry and transitivity errors and 1 and 2 had these
errors on the order of the pixel size or even larger ( tables 4 and 5 ) .
thus ,
in this limited study , very small ( nearly zero ) symmetry and transitivity
errors were accompanied by reduced registration errors .
however , to determine
if this is always the case one would need to prove it mathematically or at
least repeat the experiment on a large number of image sequences . to simplify the notation
, it was assumed that all the
images had the same domain , but the same conclusions hold when the domains are
different .
furthermore , the method involves the transformation inversion , which
is done only once ( after both images are registered to the reference ) , as
opposed to the methods proposed in [ 12 , 1416 ] that require computing the
inverse transformation in each iteration of the optimization .
we used the normalized mutual information as the image
similarity measure for the registration algorithms 1 and 2 .
we repeated all the experiments by using the mutual
information , mean square difference , and normalized cross - correlation as
alternative image similarity measures and the obtained results were nearly
identical to those reported in section 3 and for this reason they are not
included in the paper .
these repeated experiments confirm that the conclusions
of the paper are not specific to the normalized mutual information .
the
reference - based registration of image sequences is provably symmetric and
transitive .
furthermore , a limited study showed that the reference - based
registration was more accurate than the sequential registration , although this
can not be guaranteed to always hold . | this paper presents a method for constructing symmetric and transitive algorithms for registration of image sequences from
image registration algorithms that do not have these two properties .
the method is applicable to both rigid and nonrigid registration
and it can be used with linear or periodic image sequences . the symmetry and transitivity properties are satisfied exactly ( up to
the machine precision ) , that is , they always hold regardless of the image type , quality , and the registration algorithm as long as
the computed transformations are invertable .
these two properties are especially important in motion tracking applications since
physically incorrect deformations might be obtained if the registration algorithm is not symmetric and transitive .
the method was tested on two sequences of cardiac magnetic resonance images using two different nonrigid image registration
algorithms .
it was demonstrated that the transitivity and symmetry errors of the symmetric and transitive modification of the
algorithms could be made arbitrary small when the computed transformations are invertable , whereas the corresponding errors
for the nonmodified algorithms were on the order of the pixel size .
furthermore , the symmetric and transitive modification of the
algorithms had higher registration accuracy than the nonmodified algorithms for both image sequences . |
adrenal failure is a potentially fatal disorder , and the prevalence of primary and secondary adrenal failure is 93140 and 150280 per million , respectively .
because symptoms of adrenal failure such as anorexia , nausea , vomiting , weight loss and fatigue are rather non - specific , they may well be mistaken as those occurring from other disorders in patients suffering from severe co - morbidities .
we encountered a rare case of a chronic haemodialysis patient with type 2 diabetes , alcoholic cirrhosis and pancreatitis , presenting as hypoglycaemic coma due to severe adrenal failure caused by acth deficiency .
a 62-year - old man was admitted in emergency in the early morning because he could not be awakened .
he had started haemodialysis for diabetic nephropathy 3 years previously and had been receiving regular haemodialysis three times a week .
he also suffered from hypertension , alcoholic cirrhosis and chronic pancreatitis . during the last 15 months , with progressive appetite loss and fatigue ,
seven weeks previously , he had experienced hypoglycaemia and was treated with intravenous ( iv ) glucose .
insulin dosing was 4 u novorapid twice a day at that time , which had been stopped since then .
upon arrival , the level of consciousness was glasgow coma scale 14 ( e4v4m6 ) without focal neurological deficit .
he appeared malnourished ( body mass index 16 kg / m ) without skin pigmentation .
his body temperature was 33.1c ( on a warm june day ) , blood pressure ( bp ) 146/78 mm hg , pulse rate 66/min , respiration rate 14/min , arterial ph 7.383 and o2 saturation 100% ( with room air ) .
the general laboratory data ( table 1 ) were compatible with the known morbidities of chronic renal failure , liver cirrhosis and chronic pancreatitis , except for hypoglycaemia and a lowered hba1c for a patient with diabetes .
ten grams of glucose was administered iv and , 30 min later , capillary blood glucose ( bg ) was 5.7 mmol / l , and he was fully conscious .
ast , aspartate aminotransferase ; alt , alanine aminotransferase ; ldh , lactate dehydrogenase ; gtp , glutamyl transpeptidase .
the patient was receiving 7500 u epogen per week.cortisol and acth were determined before breakfast on day 2 ; other endocrine data were obtained at 1000 h on day 7 when capillary blood glucose was 6.2
mmol / l ; cortisol was undetectable by radioimmunoassay ( immunotech ) with the detection limit being 5.5 nmol / l .
cortisol was undetectable with a very low acth level ( table 1 ) , which established a diagnosis of adrenal failure due to acth deficiency .
the magnetic resonance imaging revealed no hypothalamic or pituitary abnormality , and serum antipituitary antibody was negative .
9 mm hg ( mean sd , n = 17 ) during days 18 . upon diagnosing adrenal failure , 25 mg of oral hydrocortisone ( 10 mg , 10 mg and 5 mg after each meal , respectively ) was started on day 9 , which promptly restored appetite and well - being .
bp was 151 18/83 14 mm hg ( n = 15 ) during days 916 with 5 mg of amlodipine , which was modestly higher than the values recorded before the treatment with hydrocortisone ( p = 0.005 for systolic bp and p < 0.001 for diastolic bp , mann
hypoglycaemia did not recur , and insulin was resumed on day 11 , because bg ( mean sd from the premeal and the midnight values ) for the preceding 2 days was 11.8 2.2 mmol / l ( n = 7 ) with 1800 kcal diet .
in this patient , adrenal failure was due to acth deficiency , not due to addison s disease , hence there was no skin pigmentation .
loss of appetite , general fatigue , malnutrition and previous hypoglycaemic events had been considered to be due alcoholic binge
as far as we are aware , adrenal crisis or hypoglycaemic coma due to adrenal failure has not been reported in chronic haemodialysis patients .
toth and lee analysed 36 patients with uraemic hypoglycaemia in the literature and identified 19 haemodialysis patients .
the number of dialysis patients is estimated to be 1.5 million and > 90% of them are receiving haemodialysis .
the prevalence of primary and secondary adrenal failure is 93140 and 150280 per million , respectively .
, cortisol production must have been virtually absent because plasma cortisol was undetectable despite the cortisol metabolism is depressed in haemodialysis patients . however , the patient presented with only hypoglycaemic coma and mild hypothermia , but not with circulatory collapse or electrolyte abnormalities .
we consider haemodialysis had protected him from adrenal crisis because the circulating volume and electrolytes were maintained by haemodialysis . because the glucose concentration of the dialysis solution was 8.3 mmol / l , hypoglycaemia , if any , might have been relieved to some extent during each haemodialysis session .
adrenal crisis develops not only in patients with addison s disease but also in patients with acth deficiency , and hyponatraemia is not rare in the latter group .
this is in part due to the fact that cortisol concentration in plasma is 1000-fold higher than that of aldosterone , and cortisol elicits its effects not only through the glucocorticoid receptor but also through the mineralocorticoid receptor . because cortisol is a tonic inhibitor of antidiuretic hormone ( adh ) , adh excess is often present in adrenal insufficiency , which also contributes to hyponatraemia in adrenal failure .
haemodialysis most likely protects the patients with either primary or secondary adrenal failure against volume depletion and hyponatraemia . in summary ,
adrenal failure should be suspected in chronic haemodialysis patients with hypoglycaemia even if other salient features of adrenal failure are absent . | a 62-year - old man , receiving chronic haemodialysis and suffering from alcoholic liver cirrhosis and chronic pancreatitis , presented with hypoglycaemic coma .
plasma cortisol was undetectable
( < 5.5 nmol / l ) with suppressed adrenocorticotropic hormone ( acth ) , which established a diagnosis of adrenal failure due to acth deficiency .
twenty - five milligrams of oral hydrocortisone eradicated hypoglycaemia .
presentation of adrenal failure in this patient was atypical because he was hypertensive , serum electrolytes including sodium were normal and anaemia was unremarkable , which were all due to end - stage renal disease and its treatment with haemodialysis . as far as we are aware , this is the first case report of hypoglycaemic coma due to adrenal failure in a chronic haemodialysis patient . |
chewing gum is a habit practiced regularly by a relatively high proportion of individuals in many countries including india , and its use has increased within the last decade . in 1999 , available data suggested that some 374 billion pieces of chewing gum were sold worldwide , representing approximately u.s .
besides the obvious pleasant taste imparted by flavors and sweetening agents in chewing gum , the practice has the potential to benefit oral and dental health .
the introduction of sugar - free or sorbitol - containing chewing gums renewed interest in this aspect of diet , and caries reduction with sugarless and sugar phosphate products was reported by finn and gimision .
short - term plaque studies on the other hand demonstrated that sugar - free gums neither altered plaque accumulation nor removed established plaque .
mechanical supragingival plaque control is the most rational and efficient method for the prevention of periodontal diseases .
daily removal of plaque by the patient is also of concern for a beneficial long - term treatment outcome .
the majority of individuals find difficult or impossible to comply with a proper oral hygiene regimen .
moreover , even in those patients who do achieve high levels of oral cleanliness , plaque control deteriorates over time .
the effective use of mechanical devices to reduce dental plaque , in fact , is highly dependent on compliance with oral hygiene instructions given by dental professionals .
reasons for non - compliance are many and may include level of education , domestic circumstances , disposable income , beliefs and attitudes regarding personal and oral care , stressful life events , psychomotor skills , frequency of dental visit , and age . thus , a need exists for adjunctive methods of mechanical plaque removal that are simple , inexpensive , and convenient for use by patients . in the last decade , sugar - free chewing gum has been claimed to provide oral health benefits , including caries reduction and plaque control .
the caries preventive effects of these products have been well documented in many experimental and clinical investigations .
the mechanical cleaning effects of chewing gum have also been studied , but the results on the antiplaque efficacy , particularly at the smooth ( buccal and lingual ) surfaces of the teeth , have been equivocal .
indirect effect of chewing gums to oral health could be through salivary flow stimulation and mechanical tooth cleaning .
supragingival plaque is exposed to saliva and to the natural self - cleansing mechanisms existing in the oral cavity .
the maintenance of an effective plaque control is the cornerstone of any attempt to prevent and control periodontal disease .
the aim of this study was to investigate the effects of sugar - free and sugar - containing gums on both plaque formation and established plaque over the whole surfaces of the teeth .
a concurrent parallel double blind randomized control trial was designed involving 16 subjects ( 11 female and 5 male students ) from a dental college , aged 20 to 25 years , who volunteered to participate in this 4-day plaque regrowth study .
the subjects were selected on the basis of definite inclusion criteria of having 20 healthy natural teeth free from conservative restorations .
exclusion criteria included with no signs of periodontitis , temporomandibular joint disorder , malocclusion , or xerostomia and no medical or pharmacotherapy history that could affect the result of the study . the subjects who could not fulfill the set inclusion criteria
the total sample size of 16 was deliberately taken with the help of similar studies conducted around the world considering a total of 11 samples by hanham and addy , 12 samples by pizzo et al .
, 10 samples by addy et al . , and 12 samples by pizzo et al .
along with this , a pilot study was conducted on six samples ( each n = 3 ) ; based on pilot study , sample size was calculated .
the study was powered ( 85% ) to detect a difference in means of 2 units on the primary outcome variable using a 2-sided significance level t - test ; therefore , a total of 16 subjects were recruited for the present study .
the subjects who have given the consent and satisfied the criteria of the study were given a through prophylaxis , polishing , and flossing in order to obtain zero baseline plaque , and randomly allocated into group a ( sugar containing ) and group b ( sugar - free ) of eight each .
participating subjects were supplied with chewing gums which were similar in appearance and sufficient in quantity for the complete study period .
the gums were provided in unmarked boxes , each coded with the subject 's identity and the study period , along with verbal and written instructions on their use .
the products tested are shown in table 1 , together with the active ingredients and the manufacturers .
they were asked to chew one piece of chewing gum for 30 minutes 3 times a day after meal and to suspend all oral hygiene practices like brushing , mouth rinsing during the study period .
chewing gums , active ingredients , and manufacturers the subjects were also requested to maintain their customary dietary habits , but during the first hour after a chewing gum they avoided eating and drinking . on the 5 day , subjects were scored for plaque with the help of silness and loe index . the scoring criteria : ( silness and loe 1964 ) .
0 no plaque1 a film of plaque adhering to the free gingival margin and adjacent area of the tooth .
the plaque may be seen in situ only after application of disclosing solution or by using the probe on the tooth surface.2 moderate accumulation of soft deposits within the gingival pocket , or the tooth and gingival margin which can be seen with the naked eye.3 abundance of soft matter within the gingival pocket and/or on the tooth and gingival margin .
a film of plaque adhering to the free gingival margin and adjacent area of the tooth .
the plaque may be seen in situ only after application of disclosing solution or by using the probe on the tooth surface .
moderate accumulation of soft deposits within the gingival pocket , or the tooth and gingival margin which can be seen with the naked eye .
abundance of soft matter within the gingival pocket and/or on the tooth and gingival margin .
the scores of the sugar and sugar - free chewing gum group were analyzed by using
all the data collected were tested for normality before entering for analysis using spss 11.0 version software .
the study design was approved by the local ethical committee and conformed to the requirements of the declaration of helsinki as adopted by the 18 world medical assembly in 1964 and subsequently revised .
there has been higher mean scores for upper and lower arch by sugar - containing gums ( 1.39 and 1.05 ) when compared with sugar - free gums ( 0.98 and 0.96 ) . and
, similar results were found when the total mouth pi scores were compared between the two gums , i.e. , 1.22 and 0.97 , respectively .
result difference was statistically significant when tested among the sugar - containing and sugar - free gums of upper arch .
mean pi scores in relation to upper and lower quadrant in sugar containing and sugar - free gums ( mann - whitney u test ) in table 3 , the mean pi scores among the anterior and posterior arches when tested for the sugar - containing and sugar - free gums revealed higher mean pi scores for sugar - containing gums , i.e. , 1.10 and 1.32 , among anterior and posterior arches , respectively .
comparison of mean pi scores in anterior and posterior quadrant in relation to sugar containing and sugar - free gums ( mann - whitney u test ) in table 4 , the mean pi scores for upper and lower left quadrant were found to be lower in comparison to the upper and lower right quadrant . and , higher mean pi scores were reported in sugar - containing gums of all the four quadrants , i.e. , 1.37 , 1.42 , 1.12 , and 0.98 , respectively .
significant difference has been shown for upper left quadrant between the sugar - free and sugar - containing gums .
comparison of mean pi score in all four quadrants in relation to sugar containing and sugar - free gums ( mann - whitney u test ) in table 5 , the mean pi scores were found to be lower in upper lingual teeth ( i.e. , 0.65 ) in comparison with buccal and lingual surfaces of upper and lower arches .
comparison of mean pi scores buccal and lingual surfaces in relation to sugar containing and sugar - free gums ( mann - whitney u test )
the chewing gum is a common habit in many countries and belief exists amongst the general public that as with fibrous foods , chewing gum has cleansing effect on the teeth and gingiva .
some support for mechanical cleansing by chewing gum can be found in earlier reports . despite the lack of evidence for long - term benefits of chewing gum on dental health , some short - term advantages resulting from the mechanical removal of debris may be derived . with such objectives to test the efficacy of chewing gum containing sugar or sugar free in a 4-day double blind study
, the results showed that there is minimal difference between the sugar - free and sugar - containing gums .
this indicates that sugar - free and sugar - containing gums do not have much effect on plaque accumulation .
there was no significant reduction in plaque accumulation with sugar - containing chewing gums in the present study .
such results have been supported by other authors such as bratthall and ainamo et al .
the reduction in plaque reported may be due to the mechanical forces that little amount of plaque is removed henceforth .
gum chewing elicits an increase in saliva flow rate which increase the buffering capacity of saliva and concurrently leads to an enhanced clearance of fermentable carbohydrates from the mouth .
the low scores recorded lingually on the lower teeth and palatally on the upper teeth in the present study would suggest that natural cleansing mechanisms of foods do alter plaque coverage of tooth surfaces and similar results were found in the another study . nevertheless , since no detrimental effects of chewing gum on the incidence of caries
have been reported in the present study and since sugar - free chewing gum may actually reduce the incidence of caries , the chewing of gum can not at this time be considered a hazard to dental health . however , it is still tempting to concur with the suggestions of ainamo et al . that people who chew gum should be encouraged to use the now readily available sugar - free gums .
such preparations may also act as suitable replacements for other confectionary of known cariogenic potential .
based on the available evidence , the chewing of sugar - free gum after meals has been recommended as a way to prevent caries , even if no mechanical oral hygiene can be performed .
similar studies done on plaque control other than chewing gums employing fibrous food such as carrot have been undertaken by lindhe and wicen and reece and swallow .
it is concluded from these studies that chewing gum is capable of removing plaque deposits from the more exposed aspects of tooth surfaces and will reduce the presence of salivary debris immediately after eating food . | background : chewing gum is a habit practiced regularly by a relatively high proportion of individuals in many countries including india , and its use has increased within the last decade .
the aim of this study was to determine the effects of sugar - free and sugar chewing gums on plaque deposition.materials and methods : the study is a double blind clinical trial involving 16 healthy volunteers ( divided into 2 groups ) in a 4-day plaque regrowth model . on day one , subjects received professional prophylaxis , suspended oral hygiene measures , and commenced chewing their allocated product .
gum chewing was one piece chewed for 30 minutes 3 times a day . on day 5 , subjects were scored for plaque with the help of silness and loe index.results:results show sugar - free group have a lesser mean plaque score of 0.98 as compare to sugar group ( 1.23 ) , though this difference was not statistically significant.conclusion:the sugar - free gums can be used as an adjunct to mechanical oral hygiene measures . |
the central paradigm of protein science suggests that protein functions are directly controlled by protein structures . with the increasing number of solved protein structures
a number of semi or fully automated structure comparison methods have been developed based on methodologies like alignment of secondary structure elements ( 13 ) , environmental profiles ( 4 ) and distance measure matrices ( 5 ) .
most of these methods use regular secondary structure information in their algorithms . by analyzing local protein structures ,
many groups have found recurring short structural motifs also called structural alphabet ( sa ) spanning structural space ( 68 ) .
these short motifs represent local structure variations in protein space upon which backbone model of most proteins can be built .
they have been shown to be informative to analyze protein structures ( 9 ) and have been used in structure prediction ( 10 ) , backbone reconstruction ( 11,12 ) and loop modeling ( 13 ) .
we present a web - based service called protein block expert ( pbe ) for protein structure comparison and analysis using a sa of 16 pentapeptide structural motifs known as protein blocks ' ( pbs ) ( 14,15 ) .
a protein structure can be encoded into sequence of pbs by sliding an overlapping window of five residues .
hence , simplified 1d representation of protein structure can be used just like amino acid sequence analysis to find similarity , dissimilarity and relationship among proteins in terms of structure .
its concept is similar to sa - search ( 18 ) web server , but differs greatly as it uses a specialized sa substitution matrix derived on the basis of aligned homologous proteins present in a large database of phylogeny and alignment of homologous protein structures ( pali ) ( 19,20 ) .
applications and validation of such a matrix have been demonstrated ( m. tyagi , v. s. gowri , n. srinivasan , a. g. de brevern , b. offmann , manuscript submitted ) .
pbe is not only a service to find structural similarities between proteins or a mining tool for recognizing the fold of a protein structure , it also provides an interface to a database to study proteins in terms of pbs at the levels of superfamily and family .
pbe provides the following features to the user :
a tool to encode protein structure into pbs sequence.structure comparison between a pair of proteins using pb description using both local and global alignment algorithms.mining a databank , which is derived from scop , for proteins with similar fold.access to a database of preprocessed pb sequences and pairwise alignments at family and superfamily levels . a tool to encode protein structure into pbs sequence .
structure comparison between a pair of proteins using pb description using both local and global alignment algorithms .
mining a databank , which is derived from scop , for proteins with similar fold .
access to a database of preprocessed pb sequences and pairwise alignments at family and superfamily levels .
the set of pbs consists of 16 structural motifs of each five residue long ( 14,15 ) .
each of the pbs is represented by a vector of eight , dihedral angles associated with five consecutive c atoms and the 16 pbs are denoted by the letters a , b , , p. encoding of protein 3d structure into sequence of pbs as implemented in our server is a two - step process .
first , protein backbone is encoded into sequence of ( , ) angles calculated from backbone atomic positions .
second , an overlapping window of five c atoms , i.e. vector of eight ( , ) angles is moved along the backbone .
pbs for each window is assigned on the basis of smallest dissimilarity measure called root mean square deviation on angular values or r.m.s.d.a .
( 21 ) between observed ( , ) values in the window and the standard dihedral angles for various pbs .
it accepts a structure as an input and lists the sequence of pbs in the structure .
analysis of sequence of pbs using classical amino acid sequence alignment algorithms allows us to explore possibility of finding structural similarities between two proteins using reduced complexity of protein structure .
it allows user to compare two proteins using simple dynamic programming ( dp ) algorithm by aligning two pb sequences using our pb substitution matrix .
the substitution table used in our study was derived by re - encoding in terms of pbs the structurally aligned homologous proteins present in the pali database ( 19,20 ) .
the detailed description of calculation , discussion on pb substitution matrix and proposed applications are reported elsewhere ( m. tyagi , v. s. gowri , n. srinivasan , a. g. de brevern , b. offmann , manuscript submitted ) . indeed , local structural similarities between two uploaded protein structures are found using pb sequence alignment .
this approach has already been successfully benchmarked and compared to standard flexible alignment methods like dali ( 5 ) and rigid body superposition methods like stamp ( 25 ) where > 75% of structurally equivalent residues in our pb alignment method overlapped with those identified with these standard methods . moreover , careful inspection of aligned coordinates from pb - alignc after aligning pbs indeed shows identical and in some cases , lower r.m.s.d .
values than dali ( m. tyagi , v. s. gowri , n. srinivasan , a. g. de brevern , b. offmann , manuscript submitted ) .
these results are expected to improve by using more robust dp algorithm combined with optimized gap penalty .
interestingly , in the same study we have shown how pb alignment method is able to pick up subtle similarities at local level between two proteins .
hence pb alignment is providing both local and global flavors of dp algorithms . in pbe - alignc
, the user is required to upload two protein structures in pdb format . after transforming the 3d structures into 1d pb sequences and the latter
if the uploaded protein structures have more than one chain , option to select any one of the possible pair for alignment is presented .
the output displays the aligned pb sequences along with the information like length of proteins , alignment length , best fit superposition r.m.s.d .
pb alignment is transformed into amino acid alignment to define equivalent regions required by profit and further iterations are done to obtain best fit r.m.s.d .
the server provides the possibility to download the initial pb and corresponding amino acid alignments in fasta format as well as the superimposed coordinates between the two structures . as pbe requires only backbone atoms to generate pb sequence and
is independent of residues , the user can upload anonymous protein structures by changing all residues to any one kind and giving only coordinates of backbone atoms in the pdb format . hence newly solved structures can be easily analyzed without making them public .
pbe - alignm uses a database of protein domains derived from scop database ( 27 ) .
protein structures were extracted from scop 1.65 via the astral ( 28 ) server using a sequence identity cutoff of 95% ( scop95 ) with 9392 domains .
these domains were encoded into pb sequences and are made available for user to query at family and superfamily levels in pbe - sadb database .
further , an extensive all - against - all pairwise pb sequence alignments between all 7195 domains were generated using dp and our pb substitution matrix .
protein domains in scop95 having any chain breaks were not considered for pb sequence alignment process .
pairwise alignments within each of seven major structural classes from scop95 , which amounts 5 405 433 alignments , are featured in pbe - sadb database where option is provided to the user to view / download pairwise pb alignments at the level of family or superfamily . each pb alignment in the generated databank
had raw score given by dp algorithm . to remove the dependence of this value on the length of the two proteins , the score is also provided in the normalized form by dividing the raw score by the length of the alignment including gaps .
this normalized score from global alignment algorithm is used to rank alignments during the following analysis . in the first study we analyzed the efficiency of our method to discriminate between various scop classes or in other words what is the overlap of scores between classes based on pb sequence alignment .
this question is important since 1d representation of protein structure using pb sequence lacks topological information , which can create confusion due to identical linear sequence of regular structures in two proteins having different topologies .
a dataset of 1500 protein domains was selected randomly from scop at 95% keeping the relative proportion of seven major classes same as in original databank .
each time one domain was selected and was queried against the databank to find top 10 ranking pb alignments against the given query and statistics was calculated for true hits at each rank position .
analysis of the distribution of true hits shows that 85.9% of them are at first rank and a hit rate of 98.2% is achieved when first 10 ranking alignments are considered ( data not shown ) .
it should be noted that the value increases from 85.9 to 93% when the same analysis is performed on the 7267 7267 pairwise alignments . a confusion matrix between seven scop classes
matrix is populated simply based on criteria if query protein and first rank protein have same class or not .
alpha plus beta class was most confused class with 76.2% cases finding itself at first rank .
beta class was most well - behaved class with accuracy of 94.4% followed by alpha beta and alpha class .
low accuracy rate of multi - domain and membrane class can be attributed to very low number of proteins of this present in our dataset . in a second study
, we assessed how well a pb alignment can extract protein of similar fold from a databank within given a class . a jackknife approach ( as done in previous analysis , cf .
infra ) was applied to calculate statistics for identifying true fold of a protein as defined by scop at various levels .
it is noteworthy that 81.3% of true hits are from first rank while 89.3% true hits are within top 10 ranking alignments .
further efficiency of mining similar folds within each seven major classes was studied and results are reported in table 2 . for each class ,
hit rate was calculated at three different levels , top10 , top5 and top1 where first 10 , 5 and first ranking alignments were considered , respectively . the ability of our method to extract same scop fold within top10 level vary from a hit rate of 86.1% for alpha class to 93.6% for alpha / beta class .
similarly , at top1 level , the hit rate varies from 70% ( small protein class ) to 88.4% ( alpha / beta class ) .
consistent good level of hit rates across various classes to mine similar fold using pb alignment method gives support to basic ability of the method and quality of the substitution matrix .
these results hence illustrates that the use of pb substitution matrix with simple dp algorithm along with nave scoring function is efficient to extract proteins sharing structural similarities from large dataset .
pbe - alignm provides this facility for mining structural similarities from a databank using a reduced representation of protein structures .
user can upload a protein structure in pdb format and can decide against which databank the structure is to be queried .
pbe gives option to select local or global alignment algorithm , setting up parameters like minimum length of proteins against which query should be aligned .
option is also given to decide if the user would like to align against whole databank or with some specific scop class proteins .
we have created two databases of protein structures and are grouped under the pbe - sadb facility .
first is a database of 9392 protein domains extracted from scop95 that were translated into pb sequences .
second is a database of all possible pairwise pb sequence alignments within each scop class .
an interface gives option for querying any one of these two databases at superfamily or family level by entering appropriate scop code .
list of all family and superfamily codes and their description present in our database is available in the help section .
in addition pb sequences or alignments can also be accessed by specifying a pdb i d of a protein . because pdb was filtered for 95% sequence identity cutoff , the list of the available pdb structures can also be checked in help section .
facility to query and download pb sequences or pb alignments at family or superfamily level is expected to be of great help in studying protein structure conservation .
this can also aid studies on variations in homologous proteins in terms of structural alphabets , which might provide better insight into sequence to structure relationship .
analysis of pb alignments to study conservation or variability of local structures is expected to provide better understanding of relationship between structure and function of homologous proteins .
pairwise pb alignments for each given class were calculated using 32 processor ibm aix52 machine .
web server front end and back end processing are handled using html , cgi and perl scripting along with java ( pbs encoding of protein ) and c ( dp ) programs .
job requests in pbe - alignm are queued and provided a randomly generated job - id that guarantees the inaccessibility of jobs to other users of the servers .
pbe server is maintained on a linux - based single processor machine and is accessible at .
decrease in the complexity of protein space from three dimensions to one dimension with the combination of sequence analysis methods to study protein structure has opened a simple and exciting way of looking at protein structure space .
initial results of mining similar fold structures in databases and finding local structural similarities between proteins has been a promising start though far from exploiting full potential of such methodology .
low confusion rate across various scop classes and high efficiency rate to mine similar fold proteins from large database based on nave scoring scheme indicates that pb alignment method is efficient enough to discriminate between different topologies despite lack of topological information .
this success can be attributed to both , efficiency of pbs to represent local structural properties in more refined form compared to simple sse representation and quality of substitution matrix .
sequence of pbs between regular sses and their alignment or misalignment might be playing important role in discriminating between correct and incorrect hits .
pairwise comparison of proteins using pbe - alignc performed reasonably well when compared to standard methods like dali and this was further validated on a large - scale basis here ( 7195 7195 pairwise alignments ) .
structure alignment using pbs has also shown its efficiency to locate subtle similarities at local level and to very efficiently mine for local structural similarities from large structural databases .
though , pb alignment is expected to be very advantageous in cases of distantly related proteins where residue
finally , because prediction of protein backbone in terms of pb sequences is possible from amino acid sequence ( 14 ) , this work opens up interesting perspectives for large - scale structural annotation of genomic data .
if a given query and extracted alignment have same fold , a hit is counted at that position .
mining scop for similar structures using pb alignment confusion matrix between true ( vertical ) and predicted ( horizontal ) scop classes .
mining scop for similar structures using pb alignment hit rates ( in percentage ) for identifying similar fold within each scop classes . are given rates that take into account top10 , top5 and top1 ranking alignments . | encoding protein 3d structures into 1d string using short structural prototypes or structural alphabets opens a new front for structure comparison and analysis . using the well - documented 16 motifs of protein blocks ( pbs ) as structural alphabet
, we have developed a methodology to compare protein structures that are encoded as sequences of pbs by aligning them using dynamic programming which uses a substitution matrix for pbs .
this methodology is implemented in the applications available in protein block expert ( pbe ) server .
pbe addresses common issues in the field of protein structure analysis such as comparison of proteins structures and identification of protein structures in structural databanks that resemble a given structure .
pbe - t provides facility to transform any pdb file into sequences of pbs .
pbe - alignc performs comparison of two protein structures based on the alignment of their corresponding pb sequences .
pbe - alignm is a facility for mining scop database for similar structures based on the alignment of pbs .
besides , pbe provides an interface to a database ( pbe - sadb ) of preprocessed pb sequences from scop culled at 95% and of all - against - all pairwise pb alignments at family and superfamily levels .
pbe server is freely available at . |
connective tissue nevi of the skin are hamartomas , consisting predominantly from one of the components of the extracellular matrix namely collagen , elastic fibres or proteoglycans .
we hereby describe a case of eruptive collagenoma in a child which started at an early age of three and half years .
we report this case due to its relative paucity of literature in paediatric population coupled with appearance of lesions over an atypical site ( face ) .
a 4 year old girl presented to the dermatology department with history of noticing multiple , grouped , asymptomatic raised skin lesions predominantly in bilateral axillae , upper back and face for 6 m. the lesions appeared on normal skin , without any preceding inflammation or trauma .
there was no history of epilepsy , developmental anomalies , delay in milestones or any pertinent family history .
clinical examination revealed multiple , symmetrically distributed , brownish papules and plaques having a leathery surface ranging in size from 0.2 0.2 cm [ figure 1 ] to 2.5 2.5 cm [ figure 2 ] .
few skin coloured , firm , non tender nodules , with surface showing irregular thickening , giving a peau d orange appearance were noted in the axillae [ figure 3 ] .
well defined brownish plaques having a leathery surface on flexor aspect of left forearm multiple brownish papules and plaques on the face skin coloured nodules with surface showing irregular thickening giving a peau d orange appearance considering a differential diagnosis of connective tissue nevus , histoid hansen 's and neurofibromatosis the patient was investigated .
routine investigations were normal , while histopathology revealed a thickened reticular dermis with haphazardly arranged thickened collagen bundles suggestive of collagen naevus on haematoxylin and eosin stain [ figure 4 ] .
masson 's trichrome highlighted an increase in the collagen tissue in the dermis [ figure 5 ] , while verhoeff - van gieson showed a marked paucity of elastic fibres [ figure 6 ] .
( h and e , 100 ) photomicrograph showing dense thick collagen bundles in the dermis .
( masson 's trichrome stain , 100 ) photomicrograph demonstrating an increased collagen with marked reduction in elastic fibres .
in an attempt to classify collagenomas , uitto et al . characterized them , based on pattern of distribution ( localized or generalized ) and mode of inheritance ( acquired or inherited ) .
while inherited autosomal dominant collagenomas include familial cutaneous collagenomas and shagreen 's patches of tuberous sclerosis , those that are acquired are eruptive collagenomas and isolated collagenomas .
eruptive collagenoma , first reported by cramer in 1966 , presents with sudden symmetrical appearance of several firm skin - coloured papules and nodules of various sizes , usually less than 1 cm in diameter , on the trunk and upper extremities , in the first two decades of life .
the incidence and pathogenesis is unknown with no established family history or associated systemic findings .
histopathologically , the lesions are characterized by an excessive accumulation of randomly arranged dense collagen bundles with either diminished , altered or absent elastic tissue .
eruptive collagenoma should be differentiated from other collagenomas namely familial cutaneous collagenoma , which is an autosomal dominated disorder , first described by henderson et al . , in 1968 , characterized by clinical features similar to eruptive collagenoma .
however , there is a positive family history , third decade presentation and associated systemic involvement .
isolated collagenomas are sporadically acquired collagenomas localized to one body region and not associated with any disease
. they can present as paving stone naevi , plantar collagenoma , zosteriform lesions and papulolinear lesions .
shagreen patch is a collagenoma variant associated with tuberous sclerosis , characterized by single to few asymmetrically distributed skin - coloured plaques of variable size predominantly in the lumbosacral region .
papular elastorrhexis and nevus anelasticus are closely related entities which mimic eruptive collagenomas both clinically and histologically .
papular elastorrhexis is characterised by multiple , 2 mm to 5 mm , flat , firm papules over the trunk and extremities while nevus anelasticus present as perifollicular papules .
both these conditions are associated with a decrease in elastic fibres . due to paucity of reported literature
it is uncertain , whether these entities reflect a part of the same disease process or are separate disease states .
this patient was diagnosed as eruptive collagenoma based on first decade presentation of multiple lesions , histopathological findings and the absence of family history and systemic findings .
though in most cases the onset of presentation has been beyond the first decade , only two cases have been reported with development of lesions within the first decade . the earliest onset of presentation
, in a 2 year old nigerian girl and the other by lee et al . , with onset at 5 years . only 2 case reports by mukhi et al
an extensive review of indian literature has yielded a paucity of case reports of eruptive collagenoma .
although this entity is relatively uncommon , a good clinical acumen and a degree of suspicion help in the clinical diagnosis of such cases , while histopathology confirms the diagnosis .
there is a relative paucity of published case reports in pediatric age group . to the best of our knowledge and after extensive review of literature
, we could not find any case reports in indian pediatric population . besides this , involvement of atypical sites like | eruptive collagenomas are non familial connective tissue nevi of unknown etiology presented with an abrupt onset .
while most cases are reported in young adults , there is a paucity of literature in children .
we report a case of a 4-year - old girl , who presented with multiple asymptomatic , papules , plaques and nodules on the face , trunk and upper extremities with no systemic involvement .
histopathologically , the lesion showed thickened homogenized collagen fibres highlighted by masson 's trichrome stain and paucity in elastic fibres by verhoeff - van gieson stain , confirming the diagnosis of eruptive collagenoma . |
according the latest iteration of the who classification , subcutaneous panniculitis - like t - cell lymphoma(sptcl ) is refer to t - cell derivation cytotoxic tumor .
multiple subcutaneous nodules of varying size or necrotic , mostly located on the trunk , and extremities , may be misdiagnosed as panniculitis because of it 's deceptively benign appearance .
sptcl affects both man and women with a broad age range , but rarely in children younger than 2 years .
some literatures about the treatment of sptcl have been published , but no scheme was involved in relapsed sptcl .
a 25-year - old man presented with a 1-month history of recurrent , multiple , reddish skin lesions and nodules on the anterior chest surface together with low - grade fever , general fatigue and anorexia .
a deep punch biopsy of a nodule from the anterior chest verified the diagnosis of sptcl , immunohistochemistry showed the neoplastic cells were positive for : lca , cd3 , cd8 , granzyme b , and perforin , and negative for cd56 and cd20 .
the symptions improved in 4 days and disappeared in 1 week after prednisone 90 mg daily was given intermittently -which maintained 3 weeks and was tapered off in 6 weeks .
but the same lesions and nodules was founded in the same location with 4-kg weight loss eight months after the termination of prednisone .
the patient was admitted to our hospital with the examination of pale , and febrile(temperature of 39c ) .
local skin examination revealed multiple erythematous , tender , and firm subcutaneous nodules of variable size ( 11.5 cm ) on the anterior chest .
physical examination , chest x ray , ct scan of abdomen , and bone marrow studies , revealed no other site involvement .
laboratory investigations showed pancytopenia , an elevated erythrocyte sedimentation rate ( 66 mm / h ) , normal renal function tests , abnormal hepatic function tests ( alanine aminotransferase 188 u / l , aspartate aminotransferase 236 u / l , alkaline phosphatase 635 u / l , total bilirubin 98 mol / l , conjugated bilirubin 22 mol / l , and high triglycerides 465 mg / dl .
, 48 sec . respectively ; fibrinogen is 74 mg / dl ; and positive fibrinogen degradation products were also noted .
serologic tests for syphilis , hiv , and hepatitis b and c viruses were negative .
a diagnosis of sptcl together hps was made and a choep recipe -cyclophosphamide [ 750 mg / m , intravenously ( i.v . ) , doxorubicin ( 50 mg / m i.v . ) , vincristine ( 2 mg i.v . )
all given on day 1 and prednisone ( 90 mg / day per os ) given on days 15 , etoposide 100 mg / m i.v . on days
fever and skin lesions resolved within 2 weeks after the treatment but relapsed in 1 week later .
then prednisone was removed ( tapered off in 1 month ) and only cyclosporine a 200 mg was taken every day .
after 1 month , this patient was discharged with all the systemic symptoms and skin lesions resolved .
the patient 's condition remained in remission at 12-month follow - up with a normal blood count ; there was no evidence of clinical relapse .
the distinctive clinicopathological features of a t - cell lymphoma involving the subcutaneous tissue were firstly described by gonzalez in 1991 .
it have been identified that it 's drived from cytotoxic t lymphocytes and named sptcl in the revised european american classification of lymphoid neoplasms .
two distinct subtypes of sptcl have been classified by the cellular origins : and t - cell derivation .
now , sptcl is specially refer to the cytotoxic tumor of t - cell derivation . in sptcl , subcutaneous fat is involved that make panniculitis - like appearance .
morphologically , the lymphoma infiltrate involves the lobules of the subcutaneous tissue , resulting in a typical lobular - panniculitis - like pattern .
the lymphoma cells are small to medium in size with moderate pale cytoplasm ; the nuclei are round to irregular and often hyperchromatic .
the neoplastic cells are generally confined to subcutaneous tissue and frequently infiltrate individual fat cells with a rim - like arrangement at the cell border .
tumor cells are most often cd8 cytotoxic lymphocytes expressing one or more cytotoxic granule proteins including tia-1 , granzyme b , and perforin and the tcr .
manifestations of systemic involvement are fever , hepatosplenomegaly , mucosal ulcers and sometimes serosal effusions .
the clinic course may present in an indolent manner but to be aggressive when the hemophagocytic syndrome(hps ) happen .
hps , a presenting feature in 37% of patients with sptcl , is characterized by fever , cytopenia , splenomegaly , abnormal liver function , and the pathologic finding of hemophagocytosis ( phagocytosis of erythrocytes , leukocytes , platelets , and their precursors bymacrophages ) in bone marrow .
the appearance of hps was thought to be related to cytokine and chemokine production by the malignant cells , perhaps in a setting of comprised cytolytic function .
this patient have pancytopenia , liver function abnormalities , hepatosplenomegaly , pleural effusion , hypertriglyceridemia , and coagulopathy , in keeping with the diagnosis of relapsed sptcl complicated by hps .
treatments such as systemic chemotherapy , radiotherapy , stem cell transplantation and limb amputation have been used since this disease described .
first of all , systemic steroids or other immunosuppressive agents should be applied in sptcl without associated hps , whereas radiotherapy should be considered in cases of solitary skin lesions .
multi - agent chemotherapy may be required in cases with progressive disease not responding to immunosuppressive therapy or in cases with hps , but anthracycline - based combination chemotherapy ( chop or chop - like ) were reported to be with unsatisfactory result .
other materials , such as denileukin diftitox , a recombinant fusion protein that combines human interleukin 2 and diphtheria toxin , have been applied on sptcl with challenging outcome .
prednisone have been applied on sptcl with optimistical result although some still be invalid . in this patient , prednisone made a period of 8 months tumor release but be ineffective ultimately .
even hps , a syndrome was reported can be cureed by prednisone , emerging at the eighth month of the application of prednisone , proved the treatment was aborted .
chop together with etoposide , another cytostatic drug which was verified to be effective to hps , so called choep , made no significant effect .
cyclosporine , a calcineurin inhibitor , is a potent immunosuppressant that reduces the production of several growth factors . in spite of the significantly worse prognosis of sptcl complicated by hps , cyclosporine
the mechanism of action of cyclosporin in sptcl is also down - regulation of cytokines . in
this patient , cyclosporine ( 200 mg / d ) made the symptoms improved within 1 week and resolved in 1 month .
there is not standard treatment to sptcl because of the rarity of the disease , especially to relapsed phase .
this report suggest that cyclosporine a may be still an effictive therapy when this disease relapsed . | a 25-year - old man was diagnosised subcutaneous panniculitis - like t - cell lymphoma ( sptcl ) through biopsy of a nodule from the anterior chest . after the treatment with prednisone 90 mg 3 weeks and tapered off in 1 month ,
the disease released , but relapsed together with symptions of hemophagocytic syndrome eight months after the termination of prednisone .
choep recipe was given but with unsatisfactory result until cyclosporine was prescribed .
cyclosporine was removed 6 months later .
there is no evidence of clinical relapse 1 year later .
this case suggest that cyclosporine could be a selectable treatment even in relapsed sptcl . |
the pars plana vitrectomy ( ppv ) is a common procedure for the treatment of vitreoretinal diseases including vitreous hemorrhage , retinal detachment , epiretinal membrane , and macular hole . with the recent introduction of a sutureless vitrectomy technique
, vitreoretinal surgeons are now performing vitrectomies more routinely and with fewer serious complications [ 1 , 2 ] .
it has been well established that the intraocular pressure ( iop ) increases immediately after a vitrectomy [ 36 ] .
the mechanism of this increased iop is unclear , although it responds well to conventional ocular hypotensive medications [ 3 , 4 ] .
although several studies have reported that a vitrectomy may induce glaucoma long after surgery , the association remains unclear [ 3 , 7 , 8 ] . according to yu et al .
, neither the incidence of late - onset open - angle glaucoma ( oag ) nor that of ocular hypertension ( oht ) increases in patients after a vitrectomy .
diurnal iop change and peak iop are important factors in glaucoma development and they are essential for evaluating the precise state of the iop [ 1012 ] .
the studies mentioned above did not investigate the diurnal iop pattern and its variation , and these patterns may provide a crucial link between the vitrectomy and development of glaucoma . in this study , we analyzed the effect of vitrectomy on the diurnal change of iop .
from may to december 2007 , we recruited patients who were admitted for cataract surgery and who had undergone a previous vitrectomy .
this exam included slit biomicroscopy , zeiss four - mirror indentation gonioscopy , dilated fundus indirect biomicroscopy ,
snellen best corrected visual acuity , specular biomicroscopy , and optical biometry ( iol - master , carl zeiss ) .
patient history was evaluated , including other systemic and ocular diseases , current medications , and prior surgeries .
an experienced ophthalmologist used a goldman applanation tonometer to measure the diurnal iop every two hours between 9 a.m. and 11 p.m .. we compared the diurnal iop data in patients who had one vitrectomized eye with a normal fellow eye and with age- , gender- , and axial length - matched controls .
a vitrectomized eye was defined according to the following criteria : ( 1 ) the eye had undergone one successful ppv by a retinal specialist without any complications , ( 2 ) a phakic eye had an open angle without peripheral anterior synechiae ( pas ) , and ( 3 ) the eye was not exposed to antiglaucoma eye drops for at least four months prior to the study . to avoid the influence of tamponade foreign material on iop , we excluded eyes with a history of silicone injection or eyes that had undergone surgery within the last four months .
a patient was defined to have a normal fellow eye by the following criteria : ( 1 ) the eye had an open angle without pas and had never been operated on , ( 2 ) the eye had not been exposed to antiglaucoma eye drops for at least four months prior to the study , and ( 3 ) the eye did not have any pathology or abnormality associated with iop on full ophthalmologic examination .
the inclusion criteria for normal control eyes were the same as those for normal fellow eyes as described above .
patients were excluded from analysis if they had a history of ocular surgery ( not including vitrectomy ) , use of antiglaucoma medication , pas > 90 degrees as measured with gonioscopy , inflammation , or anterior chamber reaction .
we compared the diurnal iop and ocular biometries of vitrectomized eyes to their normal fellow eyes and to control eyes . to minimize selection bias , two separate ophthalmologists measured iop and the data independently and in parallel .
paired t - tests were conducted using spss 18.0 ( chicago , il , usa ) to analyze the difference in iop between vitrectomized and normal eyes .
this study included 21 individuals ( 12 males and 9 females ) with one vitrectomized eye and one normal eye and 21 normal control individuals .
the mean patient age was 57.52 12.80 years ( range : 2178 years ) and 17 ( 81.0% ) of the vitrectomized eyes were right eyes .
the preoperative indications for vitrectomy included vitreous hemorrhage ( 9 eyes , 42.9% ) , retinal detachment ( 5 eyes , 23.8% ) , epiretinal membrane ( 4 eyes , 19.0% ) , macular hole ( 2 eyes , 9.5% ) , and endophthalmitis ( 1 eye , 4.8% ) .
after the vitrectomy , gas was injected into 11 eyes ( c3f8 in 8 eyes and sf6 in 3 eyes ) , and the other eyes were not injected with any tamponade material ( 10 eyes , 47.6% ) .
mean iops were 13.33 2.90 mmhg in the eyes with c3f8 gas tamponade , 13.07 3.35 mmhg in the eyes with sf6 gas tamponade , and 13.61 2.88 mmhg in the eyes without tamponade .
the mean interval time between surgery and iop measurement was 36.12 39.20 months ( range : 4140 months ) ( table 1 ) . there were no strong correlations between the iop of vitrectomized eyes and those of normal fellow eyes . also , the correlation coefficient between the iop of the vitrectomized eye and that of the normal fellow eye was low in the morning when the iop was relatively high .
the mean correlation coefficients of the iops between bilateral eyes are summarized in table 2 .
comparisons of iop and other ocular parameters between vitrectomized eyes and normal fellow eyes are described in table 3 .
there were no significant differences between the diurnal measurements , the average iop , the minimum iop , the maximum iop , or the iop fluctuation ( calculated as [ maximum iop minimum iop ] ) between vitrectomized and normal fellow eyes .
other ocular parameters including endothelial cell count , corneal pachymetry , axial length , anterior chamber depth , and keratometry also were not significantly different between the two groups ( table 3 ) .
the comparisons of iop and ocular parameters between vitrectomized eyes and age- , gender- , and axial length - matched control eyes are described in table 4 .
there were no significant differences in the diurnal measurement , average iop values , minimum iop values , or maximum iop values between the two groups .
however , there was significantly greater iop fluctuation in vitrectomized eyes ( 3.38 3.54 mmhg ) compared to the control eyes ( 1.65 0.95 mmhg , p = 0.031 ) .
no other ocular parameters were significantly different between the two groups ( table 4 ) . following a typical diurnal pattern ,
the iop in all subjects was the highest in the morning and gradually decreased over the course of the day ( figure 1 ) .
in this study , we found that the iop profiles were not significantly different in vitrectomized eyes compared with that of normal eyes except for fluctuations in the diurnal iop .
the iop diurnal fluctuation was larger in vitrectomized eyes than it was in age- , gender- , and axial length - matched controls ' normal eyes .
furthermore , although it was not statistically significant , the iop diurnal fluctuation was larger in patients ' vitrectomized eyes compared to their normal fellow eyes .
it is still controversial whether or not a vitrectomy affects the iop in the development of postoperative glaucoma .
no prior studies have evaluated the diurnal iop . however , there are contradictory results from prior studies with regard to the association between vitrectomies and iop .
yu et al . demonstrated that a vitrectomy did not increase the risk of glaucoma or oht in 441 vitrectomized patients .
they found that oht and oag occurred in 4.31% of vitrectomized eyes ; 2.49% of the control eyes were afflicted with oht and 2.95% of control eyes experienced oag .
although a larger proportion of oht and oag occurred in vitrectomized eyes , there was no statistically significant difference between the two groups .
in contrast , fujikawa et al . reported that the mean iop increased after a vitrectomy in patients with macular holes .
similarly , ki - i et al . reported that although the immediate change in the iop after a phacovitrectomy was minimal , some eyes developed increased iop after several months .
more recently , wu et al . reviewed the iop of 198 patients who underwent a vitrectomy and demonstrated that the incidence of high iop ( 24 mmhg ) or increased iop ( 5 mmhg ) was significantly greater in vitrectomized eyes than in control eyes .
for example , iop can increase as a result of surgical complications including hemorrhage , inflammation , and gas tamponade [ 4 , 1618 ] .
this temporary iop elevation can be easily controlled with antiglaucoma eye drops [ 3 , 4 ] .
in other cases , late - onset glaucoma may develop [ 17 , 19 ] . given this risk
, it is very important to identify any long - term harmful effects that vitrectomy has on the eye .
there are many reports regarding iop and the prevalence of oht or oag after a vitrectomy .
however , none of these studies reported the diurnal iop . therefore , the association between a vitrectomy and diurnal iop had not been previously characterized .
it has been well established that there is a diurnal variation in iop and it may be an important factor in glaucoma development .
iop is not a fixed value and it varies even over short - term periods .
some studies have suggested that the diurnal fluctuation may be increased in glaucoma or oht patients [ 1012 , 20 ] .
the circadian rhythm of ocular perfusion pressure could contribute to the mechanism of changing diurnal fluctuations , though the mechanism is not clearly understood .
regardless , it is important to study diurnal iop patterns in order to understand the variation in iop and characterize pathological iop patterns .
we investigated the diurnal iop to determine whether it is affected by a vitrectomy . in this study , we compared the diurnal iop of vitrectomized eyes with normal fellow eyes .
there were no significant differences between vitrectomized eyes and normal fellow eyes in the diurnal iop , minimum iop , maximum iop , or fluctuations of iop .
one retrospective study of oag patients suggested that daytime peak iop might be important in predicting the progression of glaucoma .
konstas et al . reported that patients with a daytime peak iop of 18 mmhg or lower ( readings at 10 a.m. , 2 p.m. , and 6 p.m. ) generally did not experience worsening of their glaucoma .
we measured iop with a shorter interval in between readings ( 2 hours ) , and there were no significant differences in the peak iop between vitrectomized eyes and normal eyes .
we also compared the diurnal iop of the vitrectomized eye with age- , gender- , and axial length - matched control eyes to eliminate the effects of axial length on the iop .
similarly , there were no significant differences in terms of diurnal iop , minimum iop , or maximum iop values between vitrectomized eyes and control eyes . however , the diurnal iop fluctuations were wider in the vitrectomized eyes than they were in the control eyes .
we did not find a strong correlation in the iop between vitrectomized eyes and normal fellow eyes .
in a previous study , the correlation between right and left eyes in normal subjects almost exceeded 0.8 .
however in our study , there were relatively low correlations between vitrectomized eyes and normal fellow eyes
. even if there are no major complications after the vitrectomy , the iop may change .
chang reported that increased oxidative stress on the trabecular meshwork after a vitrectomy might increase the iop and the risk of glaucoma in the long term .
moreover , fujikawa et al . suggested that inflammatory stress or the physical effects of the vitrectomy could be the cause of increased iop .
although the mechanism is unclear , various effects of a vitrectomy can influence diurnal changes of the iop .
we also used a small sample size and did not control for the preoperative diagnosis .
in addition , because patients who had used antiglaucoma eye drops were excluded , the iop changes after the vitrectomy may have been underestimated .
a well - designed , longitudinal , prospective study is necessary to further evaluate iop in vitrectomized eyes . in conclusion ,
a vitrectomy may change diurnal iop in the long term . however , the iop changes observed herein were within an acceptable range .
considering the importance of the iop diurnal variation , these data help to describe the status of the eye after a vitrectomy . | purpose . to evaluate the diurnal intraocular pressure ( iop ) in eyes after vitrectomy compared to that of healthy eyes .
methods .
twenty - one patients who had undergone vitrectomy and 21 age- and gender - matched normal controls were enrolled during the same period .
we measured the diurnal iop every two hours between 9 a.m. and 11 p.m. in all patients who were admitted for cataract surgery .
patients with a history of eye surgery ( not including vitrectomy ) or use of a medication that is associated with iop were excluded .
the iop and ocular parameters of patients were compared with the same patients ' fellow healthy eyes and with normal eyes of age- and gender - matched controls .
results .
there were no significant differences between vitrectomized eyes and normal fellow eyes with regard to all iop parameters including the maximum , minimum , and iop fluctuation values .
diurnal fluctuation of iop ( or the difference between the maximum and minimum iop ) was larger in vitrectomized eyes than it was in age- and gender - matched control eyes .
conclusions .
vitrectomy did not markedly affect the iop .
although there were no severe complications after vitrectomy , the iop fluctuation was wider in vitrectomized eyes than it was in normal eyes . |
crohn s disease is a chronic relapsing and remitting , inflammatory bowel disease ( ibd ) that has a significant impact on health - related quality of life ( hrqol ) [ 1 , 2 ] .
hrqol has been recognized as an important health outcome and has been defined in the medical setting as a concept encompassing a broad range of physical and psychological characteristics and limitations which describe an individual s ability to function and derive satisfaction from doing so .
crohn s disease frequently begins in early adulthood , causing a heavy disease burden in a relatively young patient population .
consequently , many patients will be affected for most of their adult life . in crohn
s disease , health status is affected as much by psychosocial factors and functional status as by disease activity . for example , the need to wear an ostomy bag may adversely affect self - image , and this has been reported to be a common worry for patients with crohn s disease .
moreover , previous studies have demonstrated a high correlation between disease severity , as measured by the crohn s disease activity index ( cdai ) , and hrqol , as assessed using the inflammatory bowel disease questionnaire ( ibdq ) [ 7 , 8 ] .
a large longitudinal study prospectively assessed the hrqol of patients with crohn s disease over 1 year and determined influencing factors .
the following factors were associated with a negative impact on overall hrqol : female gender , tobacco use , active crohn s disease , involvement of the colon , hospitalization , corticosteroid treatment , and surgery in the past 3 months ( order does not indicate priority ) .
these findings were reflected in a survey of patients with a long - term diagnosis of crohn s disease .
it is therefore important to include improvement in hrqol as a key therapeutic goal in the treatment of patients with crohn s disease .
conventional treatment options for crohn s disease include aminosalicylates , antibiotics , corticosteroids , immunomodulators , and surgery .
however , in some patients , the disease can be refractory or unresponsive to certain pharmacologic treatments .
furthermore , corticosteroids are associated with a risk of dependency and potentially serious side effects ( e.g. , diabetes , osteoporosis , moon face , and acne ) , which are detrimental to a patient s hrqol and may negatively affect self - esteem [ 1113 ] .
such treatments therefore require careful management . additionally , it is rarely possible to achieve long - term relief from crohn s disease with a single surgical procedure , and as a result , patients with crohn s disease fear surgery so much that their hrqol is reduced .
it is now well accepted that tumor necrosis factor ( tnf ) plays a pivotal role in the underlying inflammatory pathophysiology of crohn s disease .
furthermore , neutralization of tnf has been shown to improve the symptoms of this debilitating illness [ 13 , 16 ] .
infliximab has been shown to improve hrqol , as measured by the ibdq and the short - form health survey ( sf-36 ) [ 8 , 17 ] , but the occurrence of infusion reactions along with the risk of developing anti - infliximab antibodies , which can reduce long - term efficacy , could negatively impact hrqol over time .
patients may feel that subcutaneous treatment has advantages over treatments requiring infusion ; a long stay at the clinic may negatively impact a patient s daily life .
this increased freedom in patient treatment management may lead to improved treatment compliance with a consequent impact on therapeutic outcomes .
thus , there is a need for alternative long - term therapies for moderate - to - severe crohn s disease that have minimal negative impact on patients everyday life while also reducing the impact of the disease on patients hrqol .
certolizumab pegol is the first polyethylene glycolated ( pegylated ) anti - tnf antibody fragment ( fab ) to be studied in crohn s disease . unlike conventional anti - tnf agents
, certolizumab pegol lacks the crystallizable fragment ( fc ) region , thus potentially avoiding unwanted effects .
certolizumab pegol is administered by subcutaneous injection once every 4 weeks ( more frequent dosing may be used during an induction phase ) .
certolizumab pegol 400 mg administered subcutaneously demonstrated efficacy and was well tolerated in patients with moderate - to - severe active crohn s disease in phases ii and iii ( precise 1 and precise 2 [ 22 , 23 ] ) studies .
certolizumab pegol has also demonstrated clinical efficacy and good tolerability in patients with rheumatoid arthritis [ 24 , 25 ] .
this is the first paper to report the effect of certolizumab pegol , a monthly subcutaneous treatment , on the hrqol of patients with moderate - to - severe active crohn s disease in a phase ii placebo - controlled study .
this study was approved by the independent ethics committee / institutional review board for each center before initiation .
it was conducted in accordance with the international conference for harmonization guidelines for good clinical practice and the declaration of helsinki ( revised 1996 ) .
written informed consent was obtained from each patient at screening before any study procedures were performed .
adult patients ( age 18 years ) with moderate - to - severe active crohn s disease , as defined by a cdai score of 220450 points during the week before the administration of the first dose of study drug , were eligible for the study .
exclusion criteria included suspected or diagnosed abscess , a bowel perforation or evidence of non - inflammatory obstruction in 6 months before screening , extensive bowel resection , a functional colostomy or ileostomy , or a known history of tuberculosis .
concomitant medication was allowed , provided that doses were stable and could be continued for the 12-week double - blind period and the 8-week follow - up .
this was a multicenter , randomized , double - blind , placebo - controlled , 12-week study conducted at 58 international centers between february 2001 and march 2002 .
an 8-week follow - up period allowed collection of additional safety data . at screening , patients with moderate - to - severe active crohn s disease were stratified according to concomitant steroids , immunosuppressants , or long - term anti - infectives .
eligible patients were randomized ( 1:1:1:1 ) to receive certolizumab pegol ( 100 , 200 , or 400 mg ) or placebo ( 0.9% w / v saline ) by subcutaneous injection at weeks 0 , 4 , and 8 ( fig . 1 ) .
before dosing , patients demography , medical history , and concomitant diseases were recorded .
1study design ( showing the total numbers of patients recruited into each treatment group ) study design ( showing the total numbers of patients recruited into each treatment group ) the primary endpoint of the study was the proportion of patients achieving a clinical response [ 100-point decrease in cdai score or cdai score 150 points ( remission ) ] at week 12 .
hrqol was a secondary endpoint evaluated at baseline ( week 0 , pre - injection ) and at weeks 2 , 4 , 6 , 8 , 10 , and 12 using the 32-item self - administered ibdq .
the questionnaire assesses the four aspects of a patient s life that are affected by ibd : symptoms directly related to the primary bowel symptoms , systemic symptoms , emotional function , and social function .
the reliability , validity , and responsiveness of the ibdq have been demonstrated by guyatt et al . [ 26 , 27 ] and irvine et al . [ 7 , 28 ] .
total ibdq scores range from 32 to 224 , with a higher score indicating better hrqol .
remission is generally indicated by a score of at least 170 points , and a change of 16 points is regarded as clinically meaningful .
in addition to a total score , domain scores can be calculated for the bowel symptoms , systemic symptoms , emotional function , and social function domains .
ibdq measurements were performed in parallel with determination of cdai score and serum concentrations of c - reactive protein ( crp ) .
official ibdq versions translated into danish , french , german , and swedish were used .
our own translations were used for russian , serbian , and afrikaans . to assess whether our own translated versions of the ibdq provided a valid measure of hrqol , we compared scores of the translated versions with those of the official translated versions and evaluated those scores in relation to the patient s cdai scores .
a sensitivity analysis was also carried out to check for influence of specific item scores on the resulting total scores .
the results of these analyses demonstrated that our own translated versions of the ibdq provided a valid measure of hrqol , comparable to the official , translated versions .
a sample size of 260 patients was calculated to give approximately 83% power to detect a true difference between treatment groups of 23% for the primary endpoint of the study , based on a placebo response rate of 12% . after screening ,
patients were to be randomized in a 1:1:1:1 ratio ( 65 patients to each of the four treatment groups ) .
therefore , 195 patients were to receive certolizumab pegol and 65 were to receive placebo .
efficacy was assessed for the intent - to - treat ( itt ) population , which included all patients who received at least one injection and had at least one efficacy measurement after the first injection .
patients who terminated the trial prematurely were advanced to the end - of - study visit .
relationships between cdai and ibdq global scores at week 12 were assessed for the itt population using kendall s tau coefficient , a measure of the strength of dependence showing to what extent these two variables move in the same direction .
changes from baseline in ibdq total score and individual domain scores for each treatment group were formally compared with the placebo group .
least - squares means , adjusted for stratum , pooled country , and baseline ibdq score , were compared across treatment groups .
fisher s exact test was used to compare the proportion of patients who achieved remission ( defined as an ibdq total score of 170 points ) in each active treatment group with that for the placebo group at each week .
the analyses were performed based on both the overall itt population and in the subgroup of patients with elevated baseline crp ( 10 mg
this study was approved by the independent ethics committee / institutional review board for each center before initiation .
it was conducted in accordance with the international conference for harmonization guidelines for good clinical practice and the declaration of helsinki ( revised 1996 ) .
written informed consent was obtained from each patient at screening before any study procedures were performed .
adult patients ( age 18 years ) with moderate - to - severe active crohn s disease , as defined by a cdai score of 220450 points during the week before the administration of the first dose of study drug , were eligible for the study .
exclusion criteria included suspected or diagnosed abscess , a bowel perforation or evidence of non - inflammatory obstruction in 6 months before screening , extensive bowel resection , a functional colostomy or ileostomy , or a known history of tuberculosis .
concomitant medication was allowed , provided that doses were stable and could be continued for the 12-week double - blind period and the 8-week follow - up .
this was a multicenter , randomized , double - blind , placebo - controlled , 12-week study conducted at 58 international centers between february 2001 and march 2002 .
an 8-week follow - up period allowed collection of additional safety data . at screening , patients with moderate - to - severe active crohn s disease were stratified according to concomitant steroids , immunosuppressants , or long - term anti - infectives .
eligible patients were randomized ( 1:1:1:1 ) to receive certolizumab pegol ( 100 , 200 , or 400 mg ) or placebo ( 0.9% w / v saline ) by subcutaneous injection at weeks 0 , 4 , and 8 ( fig . 1 ) . before dosing ,
1study design ( showing the total numbers of patients recruited into each treatment group ) study design ( showing the total numbers of patients recruited into each treatment group )
the primary endpoint of the study was the proportion of patients achieving a clinical response [ 100-point decrease in cdai score or cdai score 150 points ( remission ) ] at week 12 .
hrqol was a secondary endpoint evaluated at baseline ( week 0 , pre - injection ) and at weeks 2 , 4 , 6 , 8 , 10 , and 12 using the 32-item self - administered ibdq .
the questionnaire assesses the four aspects of a patient s life that are affected by ibd : symptoms directly related to the primary bowel symptoms , systemic symptoms , emotional function , and social function .
the reliability , validity , and responsiveness of the ibdq have been demonstrated by guyatt et al . [ 26 , 27 ] and irvine et al . [ 7 , 28 ] .
total ibdq scores range from 32 to 224 , with a higher score indicating better hrqol .
remission is generally indicated by a score of at least 170 points , and a change of 16 points is regarded as clinically meaningful .
in addition to a total score , domain scores can be calculated for the bowel symptoms , systemic symptoms , emotional function , and social function domains .
ibdq measurements were performed in parallel with determination of cdai score and serum concentrations of c - reactive protein ( crp ) .
official ibdq versions translated into danish , french , german , and swedish were used .
our own translations were used for russian , serbian , and afrikaans . to assess whether our own translated versions of the ibdq provided a valid measure of hrqol , we compared scores of the translated versions with those of the official translated versions and evaluated those scores in relation to the patient s cdai scores .
a sensitivity analysis was also carried out to check for influence of specific item scores on the resulting total scores .
the results of these analyses demonstrated that our own translated versions of the ibdq provided a valid measure of hrqol , comparable to the official , translated versions .
a sample size of 260 patients was calculated to give approximately 83% power to detect a true difference between treatment groups of 23% for the primary endpoint of the study , based on a placebo response rate of 12% . after screening ,
patients were to be randomized in a 1:1:1:1 ratio ( 65 patients to each of the four treatment groups ) .
therefore , 195 patients were to receive certolizumab pegol and 65 were to receive placebo . efficacy was assessed for the intent - to - treat ( itt ) population , which included all patients who received at least one injection and had at least one efficacy measurement after the first injection .
patients who terminated the trial prematurely were advanced to the end - of - study visit .
relationships between cdai and ibdq global scores at week 12 were assessed for the itt population using kendall s tau coefficient , a measure of the strength of dependence showing to what extent these two variables move in the same direction .
changes from baseline in ibdq total score and individual domain scores for each treatment group were formally compared with the placebo group .
least - squares means , adjusted for stratum , pooled country , and baseline ibdq score , were compared across treatment groups .
fisher s exact test was used to compare the proportion of patients who achieved remission ( defined as an ibdq total score of 170 points ) in each active treatment group with that for the placebo group at each week .
the analyses were performed based on both the overall itt population and in the subgroup of patients with elevated baseline crp ( 10 mg
the clinical efficacy and safety assessments of this phase ii dose - finding study have been reported in detail elsewhere . because the 400 mg dose was identified as being the most appropriate dose
, this paper focuses on data for patients receiving certolizumab pegol 400 mg . in total
, 372 patients were screened of whom 292 patients were enrolled ( cdai score < 220 points was the most common reason for screening failure ) . of these 292 patients , 291 were included in the itt population used to measure the efficacy endpoints according to cdai scores [ 1 patient ( 400 mg group ) was excluded because of missing efficacy data ] .
baseline ibdq scores were not obtained for one patient in the certolizumab pegol 100 mg treatment group .
seventy - five patients ( 25.7% ) withdrew from the study by week 12 .
baseline crp measurements were obtained for 290 of the 291 patients in the itt population . elevated baseline crp levels ( 10 mg
one of these patients was the patient in the certolizumab pegol 100 mg treatment group who was excluded from analyses as a result of baseline ibdq scores not being obtained .
the subgroup analyses of ibdq by baseline crp status were therefore performed on data from 118 patients with an elevated baseline crp concentration .
the four treatment groups were generally well matched in terms of demographic and clinical characteristics ( including baseline ibdq total score and concomitant medication profile ; table 1 ) . in the itt population ,
21.6% of patients had received previous anti - tnf therapy , and 15.5% had concomitant medication with both immunomodulators and steroids .
table 1baseline demographic and clinical characteristics ( intent to treat population used to measure efficacy endpoints according to crohn s disease activity index scores ) placebo(n = 73)certolizumab pegol100 mg ( n = 74)200 mg ( n = 72)400 mg ( n = 72)mean cdai score ( range)291.5 ( 206448)299.2 ( 194520)310.7 ( 184446)304.5 ( 204461)mean ibdq score ( range)122.9 ( 61190)132.2 ( 66189)122.9 ( 74189)126.5 ( 71177)geometric mean plasma crp , mg / l ( range)7.268 ( 0.2786.10)6.231 ( 0.15141.00)6.475 ( 0.17127.00)7.738 ( 0.35128.28)crp 10 mg / l , n ( % ) 28 ( 38.4)31 ( 42.5)28 ( 38.9)32 ( 44.4)concomitant medications , n ( % ) aminosalicylates29 ( 39.7)37 ( 50.0)32 ( 44.4)28 ( 38.9 ) anti - infectives7 ( 9.6)6 ( 8.1)7 ( 9.7)6 ( 8.3 ) antidiarrheals10 ( 13.7)19 ( 25.7)16 ( 22.2)12 ( 16.7 ) steroids29 ( 39.7)24 ( 32.4)29 ( 40.3)22 ( 30.6 ) codeine and derivatives6 ( 8.2)5 ( 6.8)5 ( 6.9)2 ( 2.8)immunomodulators azathioprine17 ( 23.3)13 ( 17.6)23 ( 31.9)22 ( 30.6 ) 6-mercaptopurine4 ( 5.5)9 ( 12.2)2 ( 2.8)2 ( 2.8 ) methotrexate5 ( 6.8)4 ( 5.4)4 ( 5.6)3 ( 4.2)cdai crohn s disease activity index ; crp c - reactive protein ; ibdq inflammatory bowel disease questionnaire ; n number of patientsmaximum score is 224 points.data missing for one patient .
baseline demographic and clinical characteristics ( intent to treat population used to measure efficacy endpoints according to crohn s disease activity index scores ) cdai crohn s disease activity index ; crp c - reactive protein ; ibdq inflammatory bowel disease questionnaire ; n number of patients maximum score is 224 points .
the onset of effect ( clinical response ) of certolizumab pegol was evident at week 2 , when certolizumab pegol treatment produced a statistically significant improvement in cdai score compared with placebo ( certolizumab pegol 400 mg 33.3% vs placebo 15.1% ; p = 0.010 ) .
clinical response rates were highest in the certolizumab pegol 400 mg group at all time points , with maximal response at week 10 ( certolizumab pegol 400 mg 52.8% vs placebo 30.1% ; p = 0.006 ) .
lower cdai score ( representing lower disease severity ) was associated with a higher ibdq global score ( representing better hrqol ) , as reflected by a high negative kendall s tau value ( 0.48 ; p <
patients demonstrated hrqol improvements from baseline , as measured by change in ibdq total score , by the first assessment point , week 2 [ certolizumab pegol 100 mg , 16.6 points ; certolizumab pegol 200 mg , 21.8 points ( p 0.05 vs placebo ) ; certolizumab pegol 400 mg , 22.8 points ( p 0.05 vs placebo ) ; placebo , 10.6 points ] ( fig . 2 ) .
patients receiving certolizumab pegol 400 mg experienced statistically significantly greater improvements from baseline in ibdq total score at all time points ( p 0.05 ) compared with those receiving placebo .
the greatest increase in ibdq total score was recorded at week 10 ( certolizumab pegol 400 mg 32.2 points vs 18.6 points for placebo ; p 0.05 ) . approximately half of the patients treated with certolizumab pegol 400 mg demonstrated clinically meaningful improvements in ibdq total score ( 16-point increase ) at week 2 ( 52.8% ) and at week 12 ( 66.7% ) .
2mean change from baseline in inflammatory bowel disease questionnaire ( ibdq ) total score during 12 weeks subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate - to - severe crohn s disease ( intent - to - treat population ) ; least - squares means ( adjusted for stratum , pooled country , and baseline score ) mean change from baseline in inflammatory bowel disease questionnaire ( ibdq ) total score during 12 weeks subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate - to - severe crohn s disease ( intent - to - treat population ) ; least - squares means ( adjusted for stratum , pooled country , and baseline score ) as early as week 2 , patients receiving certolizumab pegol 400 mg demonstrated statistically significant improvements in the bowel symptoms and emotional function domains of the ibdq relative to placebo ( fig . 3 ) .
improvement in the systemic symptoms domain score approached statistical significance at week 2 ( p = 0.054 ) .
interestingly , patients in the certolizumab pegol 200 mg group achieved statistically significantly greater improvements than those receiving placebo in ibdq total score and all four ibdq domains at week 2 ( figs . 2 and 3a d ) .
3mean change from baseline in inflammatory bowel disease questionnaire ( ibdq ) domain scores during 12 weeks subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate - to - severe crohn s disease ( intent - to - treat population ) ; least - squares means ( adjusted for stratum , pooled country , and baseline score ) : a bowel symptoms domain , b systemic symptoms domain , c emotional function domain , and d social function domain mean change from baseline in inflammatory bowel disease questionnaire ( ibdq ) domain scores during 12 weeks subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate - to - severe crohn s disease ( intent - to - treat population ) ; least - squares means ( adjusted for stratum , pooled country , and baseline score ) : a bowel symptoms domain , b systemic symptoms domain , c emotional function domain , and d social function domain emotional function and systemic symptoms were the domains that patients reported as being most improved by certolizumab pegol treatment . at every assessment ,
patients receiving certolizumab pegol 400 mg showed statistically significantly ( p 0.05 ) greater improvements in emotional function domain scores than those receiving placebo ( fig . 3c ) .
they also demonstrated statistically significantly greater improvements in systemic symptoms domain scores than those receiving placebo at weeks 4 , 8 , 10 , and 12 ( fig .
the improvements patients experienced in social function domain scores approached statistical significance ( p = 0.054 ) at week 4 ( fig .
the remission rate in the certolizumab pegol 400 mg group was highest at week 10 .
this was statistically significantly greater than in the placebo group ( table 2 ) .
the proportion of patients achieving remission in the certolizumab pegol 400 mg group was also statistically significantly greater than in the placebo group at weeks 6 , 8 , and 12 .
the subgroup of patients with elevated crp showed similar improvements in ibdq scores to those observed in the itt population .
table 2proportion of patients achieving remission defined by inflammatory bowel disease questionnaire total score ( 170 points)patients ( % ) , intent - to - treat population ( n = 290)weekplacebo ( n = 73)certolizumab pegol100 mg ( n = 73)200 mg ( n = 72)400 mg ( n = 72)217.832.919.427.8419.237.0 * 26.433.3617.835.6
* 30.638.9 * 821.935.629.238.9 * 1027.443.836.147.2 * 1223.338.423.638.9*the higher remission rate at week 2 in the certolizumab pegol 100 mg group compared with higher - dose groups may reflect the slightly higher mean inflammatory bowel disease questionnaire ( ibdq ) score at baseline in this group . however , certolizumab pegol 400 mg resulted in the greatest mean change in ibdq at all time points.*p
0.05 vs placebo proportion of patients achieving remission defined by inflammatory bowel disease questionnaire total score ( 170 points ) the higher remission rate at week 2 in the certolizumab pegol 100 mg group compared with higher - dose groups may reflect the slightly higher mean inflammatory bowel disease questionnaire ( ibdq ) score at baseline in this group
. however , certolizumab pegol 400 mg resulted in the greatest mean change in ibdq at all time points .
in total , 372 patients were screened of whom 292 patients were enrolled ( cdai score < 220 points was the most common reason for screening failure ) . of these 292 patients , 291 were included in the itt population used to measure the efficacy endpoints according to cdai scores [ 1 patient ( 400 mg group ) was excluded because of missing efficacy data ] .
baseline ibdq scores were not obtained for one patient in the certolizumab pegol 100 mg treatment group .
seventy - five patients ( 25.7% ) withdrew from the study by week 12 .
baseline crp measurements were obtained for 290 of the 291 patients in the itt population . elevated baseline crp levels ( 10 mg
one of these patients was the patient in the certolizumab pegol 100 mg treatment group who was excluded from analyses as a result of baseline ibdq scores not being obtained .
the subgroup analyses of ibdq by baseline crp status were therefore performed on data from 118 patients with an elevated baseline crp concentration .
the four treatment groups were generally well matched in terms of demographic and clinical characteristics ( including baseline ibdq total score and concomitant medication profile ; table 1 ) . in the itt population ,
21.6% of patients had received previous anti - tnf therapy , and 15.5% had concomitant medication with both immunomodulators and steroids .
table 1baseline demographic and clinical characteristics ( intent to treat population used to measure efficacy endpoints according to crohn s disease activity index scores ) placebo(n = 73)certolizumab pegol100 mg ( n = 74)200 mg ( n = 72)400 mg ( n = 72)mean cdai score ( range)291.5 ( 206448)299.2 ( 194520)310.7 ( 184446)304.5 ( 204461)mean ibdq score ( range)122.9 ( 61190)132.2 ( 66189)122.9 ( 74189)126.5 ( 71177)geometric mean plasma crp , mg / l ( range)7.268 ( 0.2786.10)6.231 ( 0.15141.00)6.475 ( 0.17127.00)7.738 ( 0.35128.28)crp 10 mg /
l , n ( % ) 28 ( 38.4)31 ( 42.5)28 ( 38.9)32 ( 44.4)concomitant medications , n ( % ) aminosalicylates29 ( 39.7)37 ( 50.0)32 ( 44.4)28 ( 38.9 ) anti - infectives7 ( 9.6)6 ( 8.1)7 ( 9.7)6 ( 8.3 ) antidiarrheals10 ( 13.7)19 ( 25.7)16 ( 22.2)12 ( 16.7 ) steroids29 ( 39.7)24 ( 32.4)29 ( 40.3)22 ( 30.6 ) codeine and derivatives6 ( 8.2)5 ( 6.8)5 ( 6.9)2 ( 2.8)immunomodulators azathioprine17 ( 23.3)13 ( 17.6)23 ( 31.9)22 ( 30.6 ) 6-mercaptopurine4 ( 5.5)9 ( 12.2)2 ( 2.8)2 ( 2.8 ) methotrexate5 ( 6.8)4 ( 5.4)4 ( 5.6)3 ( 4.2)cdai crohn s disease activity index ; crp c - reactive protein ; ibdq inflammatory bowel disease questionnaire ; n number of patientsmaximum score is 224 points.data missing for one patient .
baseline demographic and clinical characteristics ( intent to treat population used to measure efficacy endpoints according to crohn s disease activity index scores ) cdai crohn s disease activity index ; crp c - reactive protein ; ibdq inflammatory bowel disease questionnaire ; n number of patients maximum score is 224 points .
the onset of effect ( clinical response ) of certolizumab pegol was evident at week 2 , when certolizumab pegol treatment produced a statistically significant improvement in cdai score compared with placebo ( certolizumab pegol 400 mg 33.3% vs placebo 15.1% ; p = 0.010 ) .
clinical response rates were highest in the certolizumab pegol 400 mg group at all time points , with maximal response at week 10 ( certolizumab pegol 400 mg 52.8% vs placebo 30.1% ; p = 0.006 ) .
lower cdai score ( representing lower disease severity ) was associated with a higher ibdq global score ( representing better hrqol ) , as reflected by a high negative kendall s tau value ( 0.48 ; p <
patients demonstrated hrqol improvements from baseline , as measured by change in ibdq total score , by the first assessment point , week 2 [ certolizumab pegol 100 mg , 16.6 points ; certolizumab pegol 200 mg , 21.8 points ( p 0.05 vs placebo ) ; certolizumab pegol 400 mg , 22.8 points ( p 0.05 vs placebo ) ; placebo , 10.6 points ] ( fig . 2 ) .
patients receiving certolizumab pegol 400 mg experienced statistically significantly greater improvements from baseline in ibdq total score at all time points ( p 0.05 ) compared with those receiving placebo .
the greatest increase in ibdq total score was recorded at week 10 ( certolizumab pegol 400 mg 32.2 points vs 18.6 points for placebo ; p 0.05 ) .
approximately half of the patients treated with certolizumab pegol 400 mg demonstrated clinically meaningful improvements in ibdq total score ( 16-point increase ) at week 2 ( 52.8% ) and at week 12 ( 66.7% ) .
2mean change from baseline in inflammatory bowel disease questionnaire ( ibdq ) total score during 12 weeks subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate - to - severe crohn s disease ( intent - to - treat population ) ; least - squares means ( adjusted for stratum , pooled country , and baseline score ) mean change from baseline in inflammatory bowel disease questionnaire ( ibdq ) total score during 12 weeks subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate - to - severe crohn s disease ( intent - to - treat population ) ; least - squares means ( adjusted for stratum , pooled country , and baseline score ) as early as week 2 , patients receiving certolizumab pegol 400 mg demonstrated statistically significant improvements in the bowel symptoms and emotional function domains of the ibdq relative to placebo ( fig . 3 ) .
improvement in the systemic symptoms domain score approached statistical significance at week 2 ( p = 0.054 ) .
interestingly , patients in the certolizumab pegol 200 mg group achieved statistically significantly greater improvements than those receiving placebo in ibdq total score and all four ibdq domains at week 2 ( figs .
3mean change from baseline in inflammatory bowel disease questionnaire ( ibdq ) domain scores during 12 weeks subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate - to - severe crohn s disease ( intent - to - treat population ) ; least - squares means ( adjusted for stratum , pooled country , and baseline score ) : a bowel symptoms domain , b systemic symptoms domain , c emotional function domain , and d social function domain mean change from baseline in inflammatory bowel disease questionnaire ( ibdq ) domain scores during 12 weeks subcutaneous treatment with either certolizumab pegol or placebo in patients with moderate - to - severe crohn s disease ( intent - to - treat population ) ; least - squares means ( adjusted for stratum , pooled country , and baseline score ) : a bowel symptoms domain , b systemic symptoms domain , c emotional function domain , and d social function domain emotional function and systemic symptoms were the domains that patients reported as being most improved by certolizumab pegol treatment . at every assessment
, patients receiving certolizumab pegol 400 mg showed statistically significantly ( p 0.05 ) greater improvements in emotional function domain scores than those receiving placebo ( fig . 3c ) .
they also demonstrated statistically significantly greater improvements in systemic symptoms domain scores than those receiving placebo at weeks 4 , 8 , 10 , and 12 ( fig .
the improvements patients experienced in social function domain scores approached statistical significance ( p = 0.054 ) at week 4 ( fig .
the remission rate in the certolizumab pegol 400 mg group was highest at week 10 .
this was statistically significantly greater than in the placebo group ( table 2 ) .
the proportion of patients achieving remission in the certolizumab pegol 400 mg group was also statistically significantly greater than in the placebo group at weeks 6 , 8 , and 12 .
the subgroup of patients with elevated crp showed similar improvements in ibdq scores to those observed in the itt population .
table 2proportion of patients achieving remission defined by inflammatory bowel disease questionnaire total score ( 170 points)patients ( % ) , intent - to - treat population ( n = 290)weekplacebo ( n = 73)certolizumab pegol100 mg ( n = 73)200 mg ( n = 72)400 mg ( n = 72)217.832.919.427.8419.237.0 * 26.433.3617.835.6 * 30.638.9 * 821.935.629.238.9 * 1027.443.836.147.2 * 1223.338.423.638.9*the higher remission rate at week 2 in the certolizumab pegol 100 mg group compared with higher - dose groups may reflect the slightly higher mean inflammatory bowel disease questionnaire ( ibdq ) score at baseline in this group .
however , certolizumab pegol 400 mg resulted in the greatest mean change in ibdq at all time points.*p
0.05 vs placebo proportion of patients achieving remission defined by inflammatory bowel disease questionnaire total score ( 170 points ) the higher remission rate at week 2 in the certolizumab pegol 100 mg group compared with higher - dose groups may reflect the slightly higher mean inflammatory bowel disease questionnaire ( ibdq ) score at baseline in this group . however , certolizumab pegol 400 mg resulted in the greatest mean change in ibdq at all time points .
patients with crohn s disease often have poor hrqol , with disease - related concerns , such as worry about needing surgery , being the main causal factor .
it therefore follows that treatments relieving the symptoms of crohn s disease and/or reducing the risk of surgery should improve hrqol in patients with crohn s disease .
certolizumab pegol administered subcutaneously every 4 weeks is a rapid and clinically effective treatment of moderate - to - severe crohn s disease with the 400 mg dose ( highest dose tested ) inducing the highest response rates [ 21 , 22 , 23 ] .
certolizumab pegol 400 mg appears to exert a rapid , consistently positive effect on hrqol in patients with moderate - to - severe crohn s disease .
patients who received certolizumab pegol 400 mg demonstrated statistically significantly greater improvements in ibdq total score than those in the placebo group as early as week 2 and at all subsequent time points .
as a reflection of the lower baseline ibdq scores in the 400 mg group , patients in this group experienced the greatest improvement , despite the remission rate ( which is the proportion of patients reaching a pre - defined score ) being higher in the 100 mg group .
improvements in hrqol were not dependent on baseline crp level . higher baseline crp concentration was associated with greater improvement in clinical variables in this phase ii study , but such a relationship has not been evident in phase iii studies . in addition , patients receiving certolizumab pegol 200 mg showed a statistically significantly greater change from baseline in ibdq total score than those receiving placebo at weeks 2 and 4 . collectively , these findings suggest that certolizumab pegol has an early treatment effect in patients with moderate - to - severe crohn s disease .
the benefits of certolizumab pegol on hrqol were also apparent in the phase iii studies [ 22 , 23 , 31 , 32 ] .
improvements in emotional well - being ( e.g. depression , anxiety , irritability , and anger resulting from bowel problems ) and systemic symptom domains were particularly marked with certolizumab pegol treatment .
it appears that the improvements in these two domains of the ibdq are largely responsible for the reported beneficial effect of certolizumab pegol on hrqol .
overall , these findings suggest that certolizumab pegol alleviates the psychological symptoms associated with crohn s disease and improves a patient s general well - being , in addition to providing clinical benefits in terms of cdai response .
hrqol provides an integral picture of the interactions between a patient s disease , their treatment , and quality of life positive effects resulting from the efficacy of a treatment and negative effects caused by adverse events are evaluated .
few trials have examined the effects of conventional agents upon hrqol in patients with crohn s disease . while corticosteroids are effective in controlling acute crohn s disease and are reported to improve ibdq scores , the potentially serious adverse events associated with corticosteroids may negate any positive effects on hrqol gained through control of symptoms . additionally , corticosteroids are not suitable for maintenance treatment .
improvements in hrqol have been observed with methotrexate treatment in a large , randomized , double - blind , placebo - controlled study . however , 17% of patients ( 16/94 ) receiving methotrexate were withdrawn from the study because of adverse events compared with 2% of placebo - treated patients ( 1/47 ) . the improvements in hrqol observed with certolizumab pegol in this study appear to be similar to those reported in patients with crohn s disease after treatment with infliximab [ 8 , 17 , 35 ] .
similarly , natalizumab has been shown to improve hrqol in patients with crohn s disease .
given the detrimental effects of crohn s disease on physical and psychosocial functioning , additional research is needed to assess the impact of currently available and newer therapies on hrqol . although hrqol was evaluated using the self - administered ibdq ,
which has proved to be a valid and reliable disease - specific instrument for determining health status in patients with crohn s disease , the analyses presented in this paper were purely exploratory in nature , and firm conclusions can not be drawn from the results described . however
, clinical benefit was also accompanied by improved hrqol in the phase iii studies [ 22 , 23 , 31 , 32 ] .
further benefits may be gained through the good tolerability of certolizumab pegol and convenience of subcutaneous dosing with certolizumab pegol once every 4 weeks .
this study demonstrates that the clinical efficacy of certolizumab pegol in patients with moderate - to - severe crohn s disease , which is evident within 2 weeks of injection , is paralleled by improvements in hrqol .
the benefits of certolizumab pegol were most pronounced at the highest dose tested ( 400 g ) .
improvements in emotional well - being and systemic symptoms appear to be largely responsible for the observed beneficial effect of certolizumab pegol on hrqol . | background and aimscertolizumab pegol , a polyethylene glycolated fc - free fab was efficacious and well tolerated in patients with moderate - to - severe crohn s disease in a previously reported randomized , placebo - controlled study . in this paper , we report the effect of certolizumab pegol on health - related quality of life ( hrqol).materials and methodspatients with moderate - to - severe active crohn s disease ( n = 292 ) received subcutaneous certolizumab pegol 100 , 200 , or 400 mg or placebo at weeks 0 , 4 , and 8 .
a post hoc analysis of the intent - to - treat population ( 290 patients with hrqol data ) assessed hrqol by evaluating patients responses to the self - administered inflammatory bowel disease questionnaire ( ibdq ) at baseline and weeks 2 , 4 , 6 , 8 , 10 , and 12.resultspatients receiving certolizumab pegol 400 mg at weeks 0 , 4 , and 8 demonstrated , via their ibdq total score , significantly ( p 0.05 ) greater improvement in hrqol from baseline to week 12 and at all other time points compared with placebo . moreover , hrqol improved over time in all certolizumab pegol groups , irrespective of baseline c - reactive protein levels .
emotional well - being ( ibdq emotional function domain ) improved throughout the study for patients receiving certolizumab pegol 400 mg .
this improvement was significantly ( p 0.05 ) greater than for patients receiving placebo at all time points . in addition , systemic symptoms ( ibdq systemic symptoms domain ) improved significantly more in patients receiving certolizumab pegol 400 mg than in those receiving placebo at weeks 4 , 8 , 10 , and 12 ( p 0.05 ) and approached statistical significance at week 2 ( p = 0.054).conclusionthis analysis suggests that certolizumab pegol 400 mg improves hrqol in patients with moderate - to - severe crohn s disease . |
obesity is a severe social problem in korea ( kang , 2013 ) . not only social and economic factors ( chung et al . , 2016 ) but also other various risk factors are important factors affecting the pathophysiologic and developing process of obesity . and life style and psychological factors are also related to hormone changes in obesity ( azuma et al . , 2015 ; kim et al . , 2016 ; nordkap et al . ,
growth hormone ( gh ) is affected by psychological stress and many physiological stresses ( he et al . , 2015 ; karaca et al . ,
2016 ) . and gh action is related to body energy expenditure and body weight ( glad et al . , 2015 ;
therefore , gh is widely studied in many field of studies ( huang et al . , 2016 ; imran et al .
however , hormone signaling of gh needs growth hormone receptor ( ghr ) ( carter - su et al . , 2016 ) because gh is not a steroid hormone ( carter - su et al . , 2016 ) , and its main function is related to signaling of ghr into a cell .
it may show a kind of decreased pituitary hormone situation , which cause higher risk of metabolic syndrome ( khang et al . , 2016 ) , because the studies suggest that gh function is related with ghr function .
one of the famous example is ghr - null ( ghr[/ ] ) mouse ( sackmann - sala et al . , 2014 ) , which showed insulin sensitivity and increased adiposity .
insulin sensitivity is another problem in obesity , and gh is a major hormone in insulin sensitivity ( galescu et al . , 2016 ; poggiogalle et al . , 2016 ;
those are closely related keywords to obesity , and these studies suggest that polymorphisms of ghr gene may associate with obesity .
however , genetic effect such as common variation of functional gene may also affect its function ( barrie et al .
therefore , in this study , we investigated the relationship between genetic polymorphisms of ghr gene and obese vs. nonobese in a korean population , to compare polymorphism effect of ghr on obesity .
three hundred sixty - eight subjects who participated in a general health check - up program were enrolled in this study .
the biochemical characteristics including fasting plasma glucose , fasted glycated hemoglobin , total cholesterol , and low - density lipoprotein cholesterol were measured .
body mass index ( bmi ) is calculated based on the weight ( kg ) divided by the square of height ( m ) ( yang , 2015 ) .
all subjects were classified according to the classification of korean society for the study of obesity ( underweight , bmi<18 kg / m ; normal , bmi 18 to < 23 kg / m ; moderately obese , bmi 23 to < 25 kg / m ; obesity i , bmi 25 to < 30 kg / m ; obesity ii , bmi30 kg / m ) .
two hundred eleven subjects with a bmi23 kg / m were included in overweight / obese group ( mean agestandard deviation , 44.76.4 years ) and 157 subjects with a bmi of 18.523.0 kg / m in nonoverweight / obese control group ( mean agestandard deviation , 43.66.2 years ) .
genomic dnas were extracted from peripheral bloods of all subjects by roche dna extraction kit ( roche diagnostics corp . ,
we selected seven snps including the rs6451620 ( intron ) , rs4130114 ( intron ) , rs4410646 ( intron ) , rs6898743 ( intron ) , rs4394131 ( intron ) , rs6182 ( cys440phe ) , and rs6184 ( pro579thr ) and rs2229765 in ghr gene .
after that , each genotype of snps was analyzed using targeted genotyping analysis software gcos software ( affymetrix ) .
to evaluate the odds ratio ( or ) , 95% confidence interval ( ci ) , and p - value , snpstats ( http://bioinfo.iconcologia.net/index.php ) and ibm spss statistics ver .
genetic models ( dominant [ major homozygous vs. heterozygous+minor homozygous ] , recessive [ major homozygous+heterozygous vs. minor homozygous ] , and log - additive [ major homozygous vs. heterozygous vs. minor homozygous ] models ) were applied ( kim et al . , 2014 ; zare et al . , 2013 ) .
three hundred sixty - eight subjects who participated in a general health check - up program were enrolled in this study .
the biochemical characteristics including fasting plasma glucose , fasted glycated hemoglobin , total cholesterol , and low - density lipoprotein cholesterol were measured .
body mass index ( bmi ) is calculated based on the weight ( kg ) divided by the square of height ( m ) ( yang , 2015 ) .
all subjects were classified according to the classification of korean society for the study of obesity ( underweight , bmi<18 kg / m ; normal , bmi 18 to < 23 kg / m ; moderately obese , bmi 23 to < 25 kg / m ; obesity i , bmi 25 to < 30 kg / m ; obesity ii , bmi30 kg / m ) .
two hundred eleven subjects with a bmi23 kg / m were included in overweight / obese group ( mean agestandard deviation , 44.76.4 years ) and 157 subjects with a bmi of 18.523.0 kg / m in nonoverweight / obese control group ( mean agestandard deviation , 43.66.2 years ) .
genomic dnas were extracted from peripheral bloods of all subjects by roche dna extraction kit ( roche diagnostics corp . , indianapolis , in , usa ) and quantified using nanodrop machine .
we selected seven snps including the rs6451620 ( intron ) , rs4130114 ( intron ) , rs4410646 ( intron ) , rs6898743 ( intron ) , rs4394131 ( intron ) , rs6182 ( cys440phe ) , and rs6184 ( pro579thr ) and rs2229765 in ghr gene .
after that , each genotype of snps was analyzed using targeted genotyping analysis software gcos software ( affymetrix ) .
to evaluate the odds ratio ( or ) , 95% confidence interval ( ci ) , and p - value , snpstats ( http://bioinfo.iconcologia.net/index.php ) and ibm spss statistics ver .
genetic models ( dominant [ major homozygous vs. heterozygous+minor homozygous ] , recessive [ major homozygous+heterozygous vs. minor homozygous ] , and log - additive [ major homozygous vs. heterozygous vs. minor homozygous ] models ) were applied ( kim et al . , 2014 ; zare et al . , 2013 ) .
to find genetic factors involved in the development of obesity , we performed an association study between polymorphisms of ghr gene and overweight / obese .
firstly , we calculated hardy - weinberg equilibrium ( hwe ) in tested polymorphisms of ghr gene .
the genotype distributions of the tested seven snps of ghr gene were consistent with the hwe ( rs6451620 , p=1.00 ; rs4130114 , p=1.00 ; rs4410646 , p=0.16 ; rs6898743 , p=1.00 ; rs4394131 , p=1.00 ; rs6182 , and rs6184 , p=1.00 ; rs2229765 , p=1.00 , data not shown ) .
tables 2 and 3 showed genotype and allele distributions of seven snps in ghr gene . among tested seven snps of ghr gene , two snps ( rs4410646 and rs6898743 ) revealed the significant association with obesity ( rs4410646 , or , 1.68 , 95% ci , 1.092.60 , p=0.02 in dominant model [ a / a genotype vs. a / c genotype+c / c genotype ] and or , 1.38 , 95% ci , 1.021.87 , p=0.036 in log - additive model [ a / a genotype vs. a / c genotype vs. c / c genotype ] rs6898743 , or , 0.63 , 95% ci , 0.410.98 , p=0.039 in dominant model [ c / c genotype vs. c / g genotype+g / g genotype ] and or , 0.73 , 95% ci , 0.540.99 , p=0.044 in log - additive model [ c / c genotype vs. c / g genotype vs. g / g gentoype ] ) .
however , the other five snps did not show any statistical significant association with susceptibility of obesity . in haplotype analysis , we found two haplotypes ( gc and ta haplotype ) of one ld block in ghr gene , which consisted of rs6182 and rs6184 .
but two haplotypes of ghr gene showed no statistical significant difference between the control group and the overweight / obese group ( gc haplotype , p=0.22 and ta haplotype , p=0.30 ) ( table 4 ) .
in present study , seven snps in ghr gene were studied , however , only two intron snps of them were weakly associated with obesity ( rs4410646 and rs6898743 ) .
the two snps were not similar in major / minor allele frequencies , however , both of them showed association to obesity in the dominant and log - additive models , and not in recessive model . among the other snps , four snps ( rs4130114 , rs4394131 , rs6182 , and rs6184 ) displayed similar major / minor allele frequencies , and all of them showed no association with obesity .
association between rs4410646 snp of ghr gene and obesity was previously found in previous study ( mong et al . , 2010 ) .
they reported that rs4410646 was significantly associated with the body composition ( p=0.0044 ) and blood pressure factor scores ( p=0.00017 ) ( mong et al . , 2010 ) .
in addition , the association between rs6898743 snp of ghr gene and cancer were studied , and rs6898743 was associated with esophageal adenocarcinoma ( mcelholm et al . , 2010 ; ong et al . , 2014 ) .
but there was no obesity study was found . in general , our result may show only weak association of ghr snps and obesity susceptibility .
a variation in ghr gene was studied in various anthropometry ( glad et al . , 2015 ) , and relationship to central obesity was found .
however , in their discussion , deficit in ghr does not always directly show gh deficiency , because they found responses to exogeneous gh in deficient ghr .
they suggested the complex and continuous lifelong relationship among gh sensitivity , gh secretion , and other metabolic mechanisms in normal gh
another example of complex gh sensitivity may be the white adipose tissue of ghr - null mice ( sackmann - sala et al . , 2014 ) .
they presented the insulin , leptin , adiponectin , and protein changes in enhanced insulin sensitivity ( sackmann - sala et al . , 2014 ) .
in addition , beattie et al . ( 2002 ) reported that decreased binding affinity may not reflect biological activities in the protein of living organism . to our knowledge
, this is the first study to report the relationship between snps in the ghr gene and obesity by bmi .
our findings support that ghr may play a role in pathophysiologic signaling of obesity or other related diseases . and
further study in a large numbers with more anthropometry will be needed to verify our findings . | a main target of growth hormone ( gh ) is adipose tissue in human body .
the gh secretion in obesity patients is impaired .
it is needless to say that growth hormone receptor ( ghr ) is necessary in gh hormone signaling .
the purpose of the present study is to examine the association between single nucleotide polymorphisms ( snps ) and the development of obesity .
a total of 211 overweight / obese subjects with a body mass index ( bmi ) 23 kg / m2 and 157 nonoverweight / obese controls with a bmi of 18.523.0 kg / m2 were involved in this study .
seven snps including the rs6451620 ( intron ) , rs4130114 ( intron ) , rs4410646 ( intron ) , rs6898743 ( intron ) , rs4394131 ( intron ) , rs6182 ( cys440phe ) , and rs6184 ( pro579thr ) and rs2229765 snps of ghr gene were genotyped .
genotyping was performed using custom dna chip .
snpstats was used to calculate the odds ratio , 95% confidence interval , and p - value . the link - age disequilibrium block and haplotypes among seven snps
were determined using haploview version 4.2 .
dominant , recessive , and log - additive genetic models were conducted for genetic analyzing . among tested snps in ghr gene ,
rs4410646 and rs6898743 showed significant association with obesity ( rs4410646 , p=0.02 in dominant model and p=0.036 in log - additive model ; rs6898743 , p=0.039 in dominant model and p=0.044 in log - additive model ) . in summary
, these results suggest that ghr gene polymorphisms might play a role in the development of obesity in the korean population . |
mycophenolate mofetil ( mpm ) , chemically known as 2-morpholinoethyl ( e)-6-(1,3-dihydro-4-hydroxy-6-methoxy-7-methyl-3-oxo-5-isobenzofuranyl)-4-methyl-4-hexenoate , is an immunosuppressive agent , inosine monophosphate dehydrogenase ( impdh ) inhibitor .
mpm is rapidly absorbed following oral administration and hydrolysed to its metabolite , mycophenolic acid ( mpa ) .
a survey of literature revealed that a few chromatographic methods have been reported to quantify mycophenolic acid ( mpa ) and its degradation products in body fluids [ 26 ] .
have developed high - performance liquid chromatographic ( hplc ) method for the determination of mpm in biological samples including rat and human body fluids . however , there are only three reports on the determination of mpm in pharmaceuticals .
have applied hplc for the simultaneous determination of mpm and its degradation product mpa in dosage form . an hplc method for detecting dissolution of mpm capsules and assay of the drug
the method is reported to be applicable over a concentration range 62621 g ml mpm .
recently , two uv - spectrophotometric methods to determine mpm in 540 g ml range have been reported by verma et al . .
the absorbance of mpm in either 0.1 m hcl or acetate buffer of ph 4.9 was measured at 250 nm . molecular interactions between electron donors and acceptors are generally associated with the formation of intensely colored charge - transfer ( ct ) complexes which absorb radiation in the visible region [ 11 , 12 ] . substituted quinones such as 2,3-dichloro-5,6-dicyano-1,4-benzoquinone ( ddq ) and p - chloranilic acid ( p - ca ) have been used as -acceptors with various donors to form c - t complexes and radicals .
applications of p - ca and ddq for the determination of a number of pharmaceutical compounds [ 1318 ] have recently been described . to the best of our knowledge
this paper describes two visible spectrophotometric methods based on the interaction between the amine moiety of mpm as the n - donor and p - ca as well as ddq as -acceptors resulting in the formation of intensely coloured c - t complexes .
the selectivity , sensitivity , accuracy , and precision of the developed methods , and their application to the determination of mpm in tablets were studied .
all absorption measurements were made using a systronics model 106 digital spectrophotometer ( systronics ltd . ,
, bangalore , india , and certified to be 99.5% pure . it was used without further purification .
cellcept 500 ( roche , mumbai , india ) tablets were purchased from local market .
1,4-dioxane and acetonitrile ( spectroscopic grade ) were purchased from merck , mumbai , india .
all other chemicals used were of analytical reagent grade . a 0.5% p - chloranilic acid ( p - ca ) and 0.25% 2,3-dichloro-5,6-dicyano benzoquinone ( ddq ) ( both from s. d. fine chem .
, mumbai ) , respectively , were prepared freshly in 1,4-dioxane . for p - ca method ,
a 500 g ml mpm stock solution was prepared by dissolving 50 mg of pure drug in acetonitrile in a 100 ml volumetric flask , the solution was diluted to the mark with the same solvent , and the above 500 g ml mpm stock solution was diluted with acetonitrile to get 150 g ml and used for the assay in ddq method .
varying aliquots of standard mpm solution ( 0.44.0 ml of 500 g ml ) equivalent to 40400 g ml were accurately transferred into a series of 5 ml calibrated flasks and the total volume in each flask was brought to 4 ml by adding acetonitrile .
after the addition of 1 ml of 0.5% p - ca solution , the content was mixed well and the absorbance was measured after 5 min at 520 nm against a reagent blank similarly prepared without adding mpm solution . into a series of 5 ml calibration flasks , aliquots ( 0.24.0 ml ) of standard mpm solution ( 150 g ml ) equivalent to 6120 g ml mpm
were accurately transferred , and to each flask 1 ml of 0.25% ddq solution was added and mixed .
after 5 min , the absorbance of the purple coloured was measured at 580 nm against the reference blank similarly prepared without drug .
standard graph was prepared by plotting the absorbance versus mpm concentration , and the concentration of the unknown was read from the calibration graph or computed from the respective regression equation derived using the absorbance - concentration data .
an amount of tablet powder equivalent to 50 mg mpm was transferred into a 100 ml volumetric flask and about 70 ml of acetonitrile was added to the flask .
the content was shaken well for 20 min and diluted to the mark with the same solvent .
the resulting solution was filtered through whatmann number 42 filter paper and used for the assay by following the general procedure described for p - ca method .
this tablet extract ( 500 g ml ) was diluted to 150 g ml with acetonitrile and a suitable aliquot was used for the assay in ddq method .
a placebo blank containing starch ( 15 mg ) , acacia ( 10 mg ) , sodium citrate ( 10 mg ) , hydroxyl cellulose ( 10 mg ) , magnesium stearate ( 10 mg ) , talc ( 15 mg ) , and sodium alginate ( 10 mg ) was prepared by mixing all the components into a homogeneous mixture .
a 5 mg of the placebo blank was accurately weighed and its solution was prepared as described under
procedure for commercial dosage forms and then subjected to analysis following the general procedures .
an accurately weighed 100 mg of mpm was added to 200 mg of placebo blank and homogenized .
an amount of synthetic mixture containing 50 mg mpm was accurately weighed and transferred into a 100 ml volumetric flask and the extract equivalent to 500 g ml mpm was prepared as described under
required volume of the previous extract was diluted to 150 g ml in mpm with acetonitrile and used for ddq method following the general recommended procedure .
the charge transfer complex forming reactions occur when -acceptors react with the basic nitrogenous compounds which act as n - donors .
charge - transfer complex formation is characterized by electronic transition(s ) to an excited state in which there is a partial transfer of electronic charge from the donor to the acceptor moiety . as a result
, the excitation energy of this resonance occurs very frequently in the visible region of the electromagnetic spectrum .
therefore , mpm , a nitrogenous base acting as n - donor , was made to react with p - ca and ddq ( -acceptors ) to produce a coloured charge transfer complexes in 1,4-dioxane - acetonitrile solvent system according to the following equation :
( 1)mpm+ampm - ac - t complexmpm++aradical anion in the p - ca method , mpm reacts with the reagent and gives a red chromogen that exhibits a strong absorption maximum at 520 nm in dioxane - acetonitrile medium ( figure 1(a ) ) .
this can be attributed to the formation of charge - transfer complex between mpm and p - ca followed by the formation of radical ions which probably was due to the dissociation of the original ( mpm - p - ca ) complex promoted by the high ionizing power of the acetonitrile solvent . in the second method ,
the interaction of mpm with ddq in dioxane - acetonitrile at room temperature gave a red - colored chromogen with strong absorption maxima at 460 , 540 , and 580 nm due to the formation of the free radical anion and the wavelength 580 was selected for further studies because of higher sample absorbance and lower blank absorbance readings ( figure 1(b ) ) .
optimum conditions were established by measuring the absorbance of c - t complexes at 520 and 580 nm , for p - ca and ddq method , respectively , by varying one and fixing other parameters . to establish optimum concentrations of the reagents for the sensitive and rapid formation of the mpm charge transfer complexes ,
the drug ( mpm ) was allowed to react with different volumes of the reagents ( 0.52.5 ml of 0.5% p - ca and 0.53 ml of 0.25% ddq ) . in both the cases , maximum and minimum absorbance values
were obtained for sample and blank , respectively , only when 1 ml of the reagent was used .
therefore , 1 ml of each reagent in a total volume of 5 ml was used throughout the investigation ( figure 2 ) . to dissolve mpm ,
acetonitrile was preferred to chloroform , dichloromethane , acetone , 2-propanol , 1,2-dichloroethane , 1,4-dioxane , methanol , and ethanol because as the complex formed in these solvents either had very low absorbance values or precipitated upon dilution , whereas in the case , of reagents , highly intense - coloured products were formed when 1,4-dioxane medium was maintained as solvent to dissolve p - ca and ddq .
therefore , acetonitrile and 1,4-dioxane were chosen as solvents to dissolve mpm and the reagents , respectively .
the optimum reaction times were determined by measuring the absorbance of the formed complex upon the addition of reagent solution to mpm solution at room temperature ( figure 3 ) . in both methods ,
the formation of c - t complex was completed within 5 min and the absorbance values of the measured species were stable for 5 h and 20 min for p - ca and ddq methods , respectively .
the composition of the c - t complexes with either p - ca or ddq was evaluated by following job 's continuous variations method .
the experiments were performed by preparing and mixing equimolar solutions of drug and reagent ( p - ca method : 4.61 10 m mpm and p - ca ; ddq method : 2.31 10 m ) by maintaining the total volume at 2.5 ml .
the plots of the molar ratio between drug and reagent versus the absorbance values were prepared ( figures 4(a ) and 4(b ) ) , and the results revealed that the formation of c - t complex between drug and reagent followed a 1 : 1 reaction stoichiometry .
this finding was anticipated by the presence of one basic electron donating center ( nitrogen atom ) in the mpm structure .
based on this fact , the following reaction pathway for the formation of c - t complex is proposed and shown in scheme 1 .
the conditional stability constants ( kf ) of the charge transfer complexes were calculated from the data of continuous variations method and found to be 9.46 10 and 7.48 10 for mpm - p - ca and mpm - ddq complexes , respectively .
the absorbances of mpm - p - ca and mpm - ddq complexes can be used to calculate the association constant using the benesi - hildebrand equation which depends on the experimental condition that one of the two component species should be present in large excess , so that its concentration is virtually unaltered on formation of the c - t complex . in this study , the donor - acceptor systems were studied within a limited range of concentration .
the acceptor concentrations were not low enough to be considered negligible with respect to donor concentration ; the ross and labes equation was therefore used to calculate the association constant of the charge transfer complexes :
( 2)[a][d][a]+[d]1a=1k1[a]+[d]+1 ,
where [ a ] and [ d ] refer to the molar concentration of acceptor and donor , respectively , a is the absorbance at the wavelength , and k and are the association constant and molar absorptivity , respectively .
the association constants ( k ) for mpm - p - ca and mpm - ddq complexes were calculated and found to be 1.096 10 and 1.117 10 , respectively .
the standard free energy change of the complexation ( g ) was calculated from the association constant ( k ) values according to the following well - established equation :
( 3)g0=2.303rtlogk ,
where g is the standard free energy of the c - t complex ( kj mol ) , r is the gas constant ( 8.314 j molk ) , and t is the absolute temperature ( 298.15 k ) .
the standard free energy change was calculated to be 23.06 and 23.11 kj mol for mpm - p - ca and mpm - ddq complexes , respectively .
a linear correlation was found between absorbance at max and concentration of mpm in the ranges given in table 1 .
regression analysis of beer 's law data using the method of least squares was made to evaluate the slope ( b ) , intercept ( a ) , standard deviations of y - axis , slope and intercept , and correlation coefficient ( r ) for each system and the values are presented in table 1 . the optical characteristics such as beer 's law limits , molar absorptivity , and sandell sensitivity values of both methods are also given in table 1 .
the limits of detection ( lod ) and quantitation ( loq ) calculated according to ich guidelines are also presented in table 1 .
the moderate values of and sandell sensitivity and lod indicate the sensitivity of the proposed methods .
the assays described under the construction of calibrations curves were repeated seven times within the day to determine the intraday precision and five times on different days to determine the interday precision of the methods .
the percentage relative standard deviation ( % rsd ) values were 0.99% ( intraday ) and 1.78% ( interday ) indicating high precision of the methods .
accuracy was evaluated as percentage relative error ( % re ) between the measured mean concentrations and taken concentrations for mpm .
the percentage relative error was calculated at each concentration and these results are also presented in table 2 . percent relative error ( % re ) values of 3.04% demonstrates the high accuracy of the proposed methods .
the results obtained from placebo blank and synthetic mixture analyses revealed that the inactive ingredients used in the preparation did not interfere in the assay of active ingredient .
the absorbance values obtained from the placebo blank solution were almost equal to the absorbance of the blank which revealed no interference from the adjuvants . to study
the role of additives added to the synthetic sample , 3 ml of the resulting solution prepared using synthetic mixture ( 500 and 150 g ml in mpm from p - ca method and ddq method ) was assayed ( n = 4 ) .
the percentage recoveries of 97.63106.1 with % rsd values in the range 1.221.85 demonstrated the accuracy as well as the precision of the proposed methods and complement the findings of the placebo blank analysis with respect to selectivity .
the robustness of the methods was evaluated by making small incremental changes in the volume of reagent and contact time , and the effect of the changes was studied on the absorbance of the measured species .
the changes had negligible influence on the results as revealed by small intermediate precision values expressed as % rsd ( 1.36% ) .
method ruggedness was demonstrated having the analysis done by four analysts and also by a single analyst performing analysis on four different instruments in the same laboratory .
intermediate precision values ( % rsd ) in both instances were in the range 0.543.15% indicating acceptable ruggedness .
the proposed methods were applied to the quantification of mpm in commercially available cellcept 500 tablets .
the results obtained were compared with those obtained using a reference non aqueous potentiometric method which is an official potentiometric titration of mpm with 0.1 m perchloric acid in anhydrous acetic acid medium .
statistical analysis of the results did not detect any significant difference in the performance of the proposed method to the reference method with respect to accuracy and precision as revealed by the student 's t - value and variance ratio f - value .
to further assess the accuracy of the proposed methods , recovery experiment was performed by applying the standard - addition technique .
the recovery was assessed by determining the agreement between the measured standard concentration and added known concentration to the sample .
the test was done by spiking the pre - analyzed tablet powder with pure mpm at three different levels ( 50 , 100 , and 150% , of the content present in the tablet powder ( taken ) and the total was found by the proposed method .
, the percentage recovery values were found in the range of 97.37104.1 with standard deviation values from 0.74 to 1.08% .
closeness of the results to 100% showed the fairly good accuracy of the proposed methods .
two simple , moderately sensitive , extraction - free , rapid , and cost - effective spectrophotometric methods based on charge transfer complex formation reaction were developed and validated for the determination of mpm .
the reagents utilized in the proposed methods are cheap , readily available , and the procedure does not involve any critical reaction conditions or tedious sample preparation . the proposed methods are characterized by the wide linear dynamic ranges relative to narrow ranges ( 1030 g ml ) of the reported uv - spectrophotometric methods .
also , the proposed methods are subject to no interference since the measurements are made at 520 and 580 nm compared to the reported methods where the absorbance is measured at 250 nm and the chances of interferences from inactive ingredients are more there by affecting the accuracy of the results .
the statistical parameters and the recovery data reveal good accuracy and precision of the methods .
these methods can be used as general methods for the determination of mpm in bulk powder and tablets .
the methods have many advantages over the separation techniques such as hplc and include reduced cost and speed with high accuracy . hence , the methods can be used in routine analysis of drug in quality control laboratories . | two simple , selective , and rapid spectrophotometric methods are described for the determination of mycophenolate mofetil ( mpm ) in pure form and in tablets . both methods
are based on charge - transfer complexation reaction of mpm with p - chloranilic acid ( p - ca ) or 2,3-dichloro-5,6-dicyano-1,4-benzoquinone ( ddq ) in dioxane - acetonitrile medium resulting in coloured product measurable at 520 nm ( p - ca ) or 580 nm ( ddq ) .
beer 's law is obeyed over the concentration ranges of 40400 and 12120 g ml1 mpm for p - ca and ddq , respectively , with correlation coefficients ( r ) of 0.9995 and 0.9947 .
the apparent molar absorptivity values are calculated to be 1.06 103 and 3.87 103 l mol1 cm1 , respectively , and the corresponding sandell 's sensitivities are 0.4106 and 0.1119 g cm1 . the limits of detection ( lod ) and quantification ( loq ) are also reported for both methods .
the described methods were successfully applied to the determination of mpm in tablets .
statistical comparison of the results with those of the reference method showed excellent agreement .
no interference was observed from the common excipients present in tablets .
both methods were validated statistically for accuracy and precision .
the accuracy and reliability of the methods were further ascertained by recovery studies via standard addition procedure . |
the contribution of nephrolithiasis related to end stage renal disease ( esrd ) in patients requiring renal replacement therapy has never been specifically evaluated .
nephrolithiasis still remains a too frequent and underappreciated cause of esrd and this is all the most unfortunate since such prognosis is now preventable in most cases .
infection stones , especially when bilateral developing a stag horn configuration are universally considered the most frequent cause of urolithiasis associated esrd.1,2 extensive stone development also has been observed with calcium - oxalate , uric acid or cystine stones and in patients with anatomic abnormalities of the urinary tract.3 in addition to the well known obstructive and infections mechanisms of kidney injury crystal deposition in the tubules and interstitium of both kidneys constitutes another , often underappreciated mechanism of progressive renal failure .
the potential risk of degradation of renal function justifies the etiological investigation of all lithiasis associated pathologies .
therefore calculus analysis of the crystalline phases and morphological characteristics is an important factor in the etiological diagnosis of the disease . in the present study we reviewed the charts of all patients for whom
urinary stone disease was recorded as the exclusive or preponderant cause of esrd in the entire cohort of patients who started maintenance dialysis therapy at our nephrology department from 1992 to 2006 .
we analyzed : the frequency of nephrolithiasis associated esrd ; distribution of various types of nephrolithiasis ; their respective cause ; treatment ; and evolution .
of the entire cohort of 7128 consecutive patients who started maintenance dialysis therapy at our nephrology department between january 1992 and december 2006 , a total of 45 patients ( 26 women , 19 men ) had renal stone disease as the cause of esrd and constitute the patients in this study . in all 45 patients
urinary stone disease was the exclusive or preponderant cause of esrd assessed by means of a detailed patient history , renal biopsy and exclusion of other possible causes of interstitial nephropathy and renal insufficiency .
the cause of stone disease was identified on the basis of history , laboratory data and urinary crystal examination .
stones that developed in patients who were on dialysis therapy were not considered in our study .
a renal failure is defined by a creatinine clearance lower than 80 ml / minute and the end stage is retained if the creatinine clearance is lower than 10 ml / minute .
the patient s survival rates were compared using the actuarial method and comparison was performed using logrank test .
the overall proposition of nephrolithiasis related esrd among the total number of incident esrd cases was 0.63% ( ie , 45 of 7128 patients ) . annual incidence was 3 .
of the 45 patients , there were 19 men ( 42.22% ) and 26 women ( 57.78% ) with a gender ratio of 0.73 .
the mean age of the patients was 48.4 17.63 years ( range of 1780 ) .
the majority of patients ( 32 ) originated from the north and the north west of tunisia , ( 74.11% ) ; ie , 20 ( 44.44% ) and 12 ( 16.67% ) respectively .
eight patients originated from the central tunisia ( 17.78% ) and 5 from the south ( 11.11% ) .
eleven patients had family antecedents of urinary lithiasis ( 24.45% ) and six of primary hyperparathyroidism type 1 .
twenty four patients had hypertension ( 53.33% ) , six had diabetes ( 13.33% ) and four cardiopathy ( 8.89% ) .
the diagnosis of nephrolithiasis stemmed from painful symptomatology , hematuria or urinary infection and was confirmed by radiology ( x - ray tomography
morphoconstitutional analysis , supplemented by biochemical and metabolic evaluation of nephrolithiasis , confirmed the nature and etiology of calculi .
thirty one patients were treated surgically : 15 had a lombotomy with lithotomy ; 10 a unilateral nephrectomy , 3 a percutaneous nephrolithotomy and 3 treated with extracorporeal lithotripsy .
the median of evolution of the nephrolithiasis from nephropathy to chronic renal failure ( crf ) was 24 months ( range 1408 months ) .
the evolution to crf was faster in men than women ( 68 versus 109 months , p = ns ) .
the evolution of the condition in patients originating from the center of tunisia rather than those from north ( 35.1 versus 124.8 months , p = ns ) or north west ( 35.1 versus 72.7 , p = ns ) .
patients with hypertension versus others without ( 59.1 versus 116.2 months p = 0.08 ) without any statistically signification .
the condition was also significantly faster in the event of diabetes ( 19.3 versus 96 months , p = ns ) or cardiopathy ( 18.75 versus 92.3 , p = ns ) .
obesity defined by a body mass index ( bmi ) 30 kg / m was correlated with a faster evolution to crf ( 14.8 versus 99.7 months , p = 0.03 ) .
surgical treatment slowed down the evolution of crf compared to medical treatment ( 97 versus 61 months ) without any statistically significance .
alkaline phosphatases higher than 128 iu / l were significantly correlated with a faster evolution to crf than those lower than 128 iu / l ( 57 versus 142.9 months , p = 0.011 ) no correlation was found with the other phosphocalcic parameters ( calcemia phosphate , calcium phosphate product and parathormone ) .
the final stage of crf was reached after a median evolution of 12 months ( range 1156 months ) . the age , gender and geographic origin had no significant impact on the evolution to esrd .
obesity was significantly correlated to the evolution to esrd ( 71 versus 18 months , p = 0.015 ) .
the patients having a parathyroid hormone ( pth ) between 150 and 300 pg / ml evolved more quickly to esrd than patients having secondary hyperparathyroidism ( 19.66 versus 36.6 months , p = ns ) .
the nephrolithiasis etiology had no impact on the evolution to esrd , nevertheless patients having primary hyperoxaluria type 1 evolved more quickly to esrd than those having infectious ( 15.45 versus 31.7 months , p = ns ) or calcic nephrolithiasis ( 15.45 versus 41.3 months , p = ns ) .
the medical treatment of the lithiasis was significantly correlated to a faster evolution to esrd compared to urological treatment ( 8.75 versus 53.2 months , p = 0.04 ) .
osteoarticular complications occurred in 91.11% of patients ( secondary hyperparathyroidism = 55.56% and adynamic bone disease in 35.56% of patients ) and cardiovascular in 66.67% ( cardiac failure : 22.22% , coronaropathy 20% and heartbeat rate disorders 22.3% ) .
the table 4 illustrates the causes of death . the patient s survival rate at 1 ,
2 , 3 and 5 years was : 97.6% ; 95% ; 92.8% ; and 69% respectively and this was 96% ; 95.2% ; 90% ; and 71% for all dialyzed patients .
age , gender , geographic origin , hypertension , bmi had no impact on patient s survival or nephrolithiasis etiology .
patient survival at 5 years was significantly combined with cardiopathy ( 33% versus 72% , p = 0.06 ) , hypocalcemia ( 56% versus 100% .
p = 0.014 ) , normal calcium phosphorus product ( 53% versus 92% p = 0.0084 ) .
. the faster evolution to the esrd ( 31.5 months ) was significantly correlated with longer survival at 7 years compared to slower evolution ( > 31.5 months ) .
end stage renal disease caused by urinary stone disease , although accounting for a limited proportion of cases , still remains a problem that should not be overlooked .
the principal mechanisms involved in the development of renal failure in patients with nephrolithiasis were recurrent stone formation , obstruction of the excretory tract and extensive crystal deposition in the renal parenchyma .
irreversible renal impairment was observed essentially in patients with a delayed diagnosis , suboptimal urological and medical management , or intrinsically severe forms of stone disease.4,5 severe forms of nephrolithiasis remain an underestimated cause of potentially avoidable esrd and the need renal replacement therapy.6 in our experience , nephrolithiasis related esrd contributed 0.63% to the total series of patients who started maintenance dialysis therapy at our department of nephrology during a 15 year period .
these figures are in agreement with those extracted from recent the united states renal data system data .
among 228,332 white patients who started renal replacement therapy in the us from 1993 to 1997 , a total of 1.5% were categorized as having nephrolithiasis , obstruction , gouty as the primary renal disease , thus leading to an estimated incidence of 2.9 patients / year .
paul jungers and colleagues have reported that the overall proportion of nephrolithiasis related esrd was 3.2%.6 another estimation can be extracted from a prospective epidemiological inquiry conducted at all nephrology and dialysis units in the ile de france area , which has a total population of 10.7 million inhabitants . of 1060 adult patients who started maintenance dialysis therapy in 1998 , nephrolithiasis was recorded as the cause of esrd in 1.8% of cases.7 in a cohort of 171 patients with severe idiopathic calcium stone disease reported by marangella and colleagues 18% had mild renal insufficiency but no patients reached esrd during a mean follow up of 3.5 years.8 of 2000 consecutive unselected stone formers reported by gupta and colleagues only 33 patients ( 1.7% ) had mild to moderate renal failure.9 a very low frequency of progressive renal failure was reported in other studies .
of 3266 patients with nephrolithiasis reported recently by worcester and colleagues none developed esrd and there was no significant decline in creatinine clearance between the first and the last visit.2 jungers reported that the frequency of nephrolithiasis related esrd significantly decreased with time ; from an average proportion of 4.7% from 1989 to 1991 to 2.2% from 1998 to 2000.6 in our study , the rate of esrd , due to nephrolithiasis , was the prerogative of the fifty years old adult patients with female prevalence . in a moroccan study including
183 patients , oussama and colleagues reported that the average age was 60 years with clear male prevalence.10 coe and colleagues found the similar results.11 the potential risk of degradation of renal function highlights the crucial importance of accurate stone analysis and metabolic evaluation to provide early diagnosis and the correct therapy for conditions that may lead to esrd . in our study ,
morphoconstitutional analysis of calculi from 45 patients showed that struvite was the main component in 40% of cases .
in all these patients , disease onset occurred a long time ago , before the modern era of urological treatment .
this cause of stones has been reported in other studies : jungers in 36%6 and gupta in 64% of cases.9 however many authors reported that the frequency of this cause will decrease in the future because of earlier diagnosis and easier urological management.12 struvite accounted for 15.3% of nephrolithiasis among 183 patients for oussama and colleagues.10 the female prevalence noted in our series was also found by gupta9 although not by oussama.10 in children , daudon and colleagues noted that struvite frequency was 9.9% of cases.13 although responsible for esrd in nearly 26.67% of our patients , calcium stones appear to be an infrequent cause of esrd compared with the very large number of calcium stone formers .
its frequency was 26.7% for jungers and even higher for oussama in 58.5% of cases.6,10 in our experience ,
such an outcome is mainly caused by late diagnosis in patients with primary hyperparathyroidism ( 58.83% ) and idiopathic hypercalciuria ( 16.66% ) resulting in multiple stone episodes ; with the aggravating influence of recurrent urinary tract infection in several patients . among 12 calcium stones reported by jungers , heavy hypercalciuria was present in 33% of cases , primary hyperparathyroidism in 16.66% and medullary sponge kidney in 41.67% of cases.6 in our study the etiology was not identified in 6.66% of cases . this frequency was much less than that of gupta with 59% of cases.9 uric acid nephrolithiasis accounted for 15.56% of cases in our study ; however in western literature , it represented by 7.2% to 15.3% of cases.1416 this figure was 17.8% for jungers and 18% for oussama.6,10 it does seem that this type of calculi is more frequent in north africa than in europe or us , suggesting the influence of nutritional factors.10 there was a male prevalence in our series and also for oussama.10 primary hyperoxaluria type 1 ( ph1 ) is frequently misdiagnosed , especially when urinary stones manifest first in adult patients or if early symptoms are misinterpreted.6 it accounted for 17.78% of nephrolithiasis in our study .
this frequency was largely higher than that of jungers.6 a very low frequency of progressive renal failure especially esrd was reported .
of 3266 patients with nephrolithiasis reported by worcester and colleagues none developed esrd and there was no significant decline in creatinine clearance between the first and the last visit ( 2 ) . in our experience , the evolution of nephrolithiasis nephropathy to chronic renal failure ( crf ) was paradoxically very fast , with a median of 24 months .
this figure was of 7 years for teichmann17 and 4 years for coe.11 in agreement with the results of giovanni,18 the median evolution was shorter among women .
there was no significant correlation found with calcemia , phosphatemia , calcium phosphate product , parathormone and hemoglobin .
however alkaline phosphatases levels superior to 128 iu / l were correlated with faster evolution to crf . in agreement with
steven s results.19 urological treatment allowed the slackening of the evolution to crf compared to the symptomatic treatment in our series .
the end stage of renal insufficiency was reached after a median of 12 months in our patients .
singh and colleagues reported that 14 patients among 20 with nephrolithiasis evolved to esrd in a time lower that one year.20 contrary to the age , gender and geographical origin which had no impact on the evolution to esrd , obesity was significantly associated with a slower evolution .
this result was also reported by daniel.21 secondary hyperparathyroidism and urological treatment were associated with slower evolution to esrd . in the experience of teichmann , all
patients treated medically suffered a deterioration in their renal function against only 28% of surgically treated patients.17 the etiology of nephrolithiasis had no impact on the evolution to esrd among our patients , nevertheless those having primary hyperoxaluria type 1 ( pha1 ) developed faster esrd than those having a struvite or a primary hyperparathyroidism .
the patient s survival rate at 1 , 2 , 3 and 5 years was respectively 97.6% , 95% , 92.8% and 69% , which was comparable with the survival rate of the totality of dialyzed patients .
no data concerning patient s survival was found in the literature . the age , gender and geographical origin had no impact on the patient s survival rate .
oconnor and colleagues showed that advanced age is a risk factor of renal function deterioration and mortality among patients having nephrolithiasis related esrd.22 hypertension had no impact on the patient s survival rate , whereas cardiopathy was associated with lower rate at 5 years .
contrary to the results of daniel21 we did not find any statistical correlation between the bmi and the patient survival rate .
the fast evolution to esrd ( 31.5 months ) was significantly associated with a better survival compared to the slow evolution ( > 31.5 months ) .
the evolution to crf can be prevented with an early etiologic diagnosis of nephrolithiasis and an appropriate treatment .
however severe forms of renal stone disease are much less frequent that the common benign forms .
this must be kept in mind in view of the disastrous consequences that may result from delayed or improper diagnosis and treatment.23 in this regard , adequate analysis of stones and/or crystalluria together with proper metabolic evaluation are of crucial diagnosis value .
they should never be neglected , especially in patients with the onset of nephrolithiasis in childhood , severely recurrent stones , nephrocalcinosis or incipient renal insufficiency as noted by gambaro and colleagues.3 to this end the patient should be involved in the care of the nephrolithiasis .
the installation of medical software containing different clinical , biological and morphologic data accessible to all physicians from all specialties allows better coordination of the patient s care .
nephrolithiasis remains the cause of potentially avoidable esrd in too many patients despite recent major advances in the diagnosis and management of patients forming stones .
these findings highlight the crucial importance of accurate stone analysis and metabolic evaluation to provide early diagnosis and proper therapy for a condition that may lead to esrd through recurrent stone formation and/or crystal infiltration .
esrd and renal replacement therapy should be prevented in the future in most patients with renal stone disease . | introduction : nephrolithiasis still remains a too frequent and underappreciated cause of end stage renal disease ( esrd).methods and patients : of the entire cohort of 7128 consecutive patients who started maintenance dialysis in our nephrology department between january 1992 and december 2006 , a total of 45 patients ( 26 women , 19 men ) had renal stone disease as the cause of esrd . the type of nephrolithiasis was determined in 45 cases and etiology in 42 .
the treatment and evolution of stone disease and patient s survival were studied.results:the overall proportion of nephrolithiasis related esrd was 0.63% .
the mean age was 48.4 years .
infection stones ( struvite ) accounted for 40% , calcium stones , 26.67% ( primary hyperparathyroidism:15.56% ; familial hypercalciuria : 4.44% , unknown etiology : 6.66% ) , primary hyperoxaluria type 1 , 17.78% and uric acid lithiasis in
15.56% of cases.the mean delay of the evolution of the stone renal disease to chronic renal failure was 85.8 months .
the feminine gender , obesity and elevated alkaline phosphatases > 128 iu / l were significantly correlated with fast evolution of esrd .
the median evolution to esrd was 12 months .
the normal body mass index ( bmi ) , medical treatment of stone and primary hyperoxaluria type 1 were correlated with fast evolution to esrd.all patients were treated by hemodialysis during a mean evolution of 60 months .
sixteen patients died .
the patient 's survival rate at 1 , 3 and 5 years was 97.6 , 92.8 and 69% respectively .
hypocalcemia , cardiopathy and normal calcium - phosphate product were significantly correlated with lower survival rate.conclusion:severe forms of nephrolithiasis remain an underestimated cause of esrd .
these findings highlight the crucial importance of accurate stone analysis and metabolic evaluation to provide early diagnosis and efficient treatment for conditions leading to esrd . |
it has been previously established that lung cancer could be induced in rats by exposure to radon and radon daughters .
although the oat - cell carcinomas that are common in humans were not found in rats , other histological types of lung carcinomas , especially squamous cell carcinomas and primitive lung adenocarcinomas , were similar to those observed in humans .
a dose - effect relationship was established for cumulative doses varying from 25 to 3000 working - level - months ( wlm ) , which was similar for medium and high cumulative doses to that observed in uranium miners .
this experimental protocol was also used to study the potential cocarcinogenic effects of other environmental or industrial airborne pollutants such as tobacco smoke , mineral fibers , diesel exhausts , or minerals from metallic mine ores that may act synergistically with radon exposure . in rats exposed to radon and tobacco smoke combined , the incidence of lung cancers was higher by a factor of 2 - 4 according to the cumulative radon exposure and the duration of tobacco smoke exposure . when mineral fibers were injected intrapleurally ,
an increased incidence of malignant thoracic tumors was observed in rats exposed to radon and fibers combined , but synergistic effects resulted in additivity . with diesel exhausts or minerals from metallic ores , a slight , nonsignificant increase in the incidence of lung carcinomas was observed compared with rats exposed to radon alone .
these results demonstrated that it is possible to establish the potential cocarcinogenic action , showing either multiplicative , additive , or no effect of various environmental or industrial airborne pollutants combined with radon exposure .
this radon model is valid for investigating possible interactions between two occupational exposures.imagesp64-afigure 1.figure 2.figure 3.figure 4.figure 5.figure 6 . | |
in oral and maxillofacial surgery , facial bone fracture is a common trauma , resulting from traffic accident ( ta ) , assault , slip down , or industrial accident .
if the facial bone fracture is not appropriately treated , the result may be functional disability and an aesthetic problem . in some cases
bell reported that mortality rate due to head and neck injuries is the highest cause of death under 34 years old .
since there are many emergency circumstances requiring appropriate and immediate treatment , many clinical studies have investigated the trends and characteristics .
generally , the risk of facial bone fracture is increased by population , traffic , exercise , and leisure activity with differences by age , gender , cause , and site .
the research literature is rich on this topic , and results vary by social , regional , and cultural conditions of study populations .
this study examined patients with facial bone fracture visiting a dental hospital of pusan national university hospital after january 2010divided into the dental clinic center of pusan national university hospital at ami - dong , busan and pusan national university dental hospital at yangsan and investigated the distribution by period , age , gender , site , and cause .
we compared these data with previous data to understand the trends , and performed an observational study to enhance appropriate care and treatment for patients with facial bone fracture .
the targets of this study were the patients with facial bone fracture who visited the department of oral and maxillofacial surgery of pusan national university dental hospital located in yangsan and the dental clinic center in pusan national university hospital located in busan from january 2010 to december 2013 . based on their charts , the patients were classified by year , month
the sites were categorized by maxilla , mandible , zygoma , nasal bone , and orbital cavity following schultz .
exclusion criteria included fracture with limitation of alveolar bone , fracture of teeth , trauma with limitation of soft tissue , and death of patient without any treatment for the injuries of facial bone before the death . and
, this study protocol was reviewed and approved by pusan national university dental hospital institutional review board .
this study investigated 531 patients with facial bone fracture in yangsan and 802 patients in busan .
the number of patients continuously increased after 2010 in yangsan , while a similar number of patients was counted in each year in busan ( fig . 1 , 2 ) .
the number of patients by month in yangsan was the highest in march at 11.5% , followed by june and september ( fig .
the highest number was in april at 10.5% , followed by june and october ( fig .
the number of males was the highest at both yangsan and busan , and the gender ratio of male and female was 3.58:1 in yangsan and 4.31:1 in busan ( fig . 5 , 6 ) .
regarding the distribution by age , the number in their 20s was the highest at 21.8% , followed by those in their 30s at 17.3% and in their 10s at 16.2% in yangsan ( fig . 7 ) .
in busan , the number in their 20s was the highest at 22.2% , followed by those in their 30s at 16.3% and in their 50s at 15.5% ( fig . 8) . regarding the distribution by site , mandible fracture was most common in yangsan ( 46.7% ) , and in busan ( 41.8% ) ( fig . 9 , 10 ) .
in yangsan , the order occurrence was mandible , nasal bone , maxilla , zygoma , and orbital cavity ; in busan , the order was mandible , nasal bone , zygoma , orbital cavity , and maxilla . regarding the causes of fractures ,
slip down was most common ( 36.5% ) , followed by collision , ta , assaults , and fall down in yangsan , and no patient had pathologic fracture ( fig .
11 ) . in busan , slip down was most common ( 33.7% ) , followed by collision , ta , assault , and fall down , the same as in yangsan , and there was one patient with pathologic fracture ( fig .
this study investigated 531 patients with facial bone fracture in yangsan and 802 patients in busan .
the number of patients continuously increased after 2010 in yangsan , while a similar number of patients was counted in each year in busan ( fig . 1 , 2 ) .
the number of patients by month in yangsan was the highest in march at 11.5% , followed by june and september ( fig .
the highest number was in april at 10.5% , followed by june and october ( fig .
the number of males was the highest at both yangsan and busan , and the gender ratio of male and female was 3.58:1 in yangsan and 4.31:1 in busan ( fig . 5 , 6 ) .
regarding the distribution by age , the number in their 20s was the highest at 21.8% , followed by those in their 30s at 17.3% and in their 10s at 16.2% in yangsan ( fig .
the number in their 20s was the highest at 22.2% , followed by those in their 30s at 16.3% and in their 50s at 15.5% ( fig . 8) .
regarding the distribution by site , mandible fracture was most common in yangsan ( 46.7% ) , and in busan ( 41.8% ) ( fig . 9 , 10 ) . in yangsan , the order occurrence was mandible , nasal bone , maxilla , zygoma , and orbital cavity ; in busan , the order was mandible , nasal bone , zygoma , orbital cavity , and maxilla .
regarding the causes of fractures , slip down was most common ( 36.5% ) , followed by collision , ta , assaults , and fall down in yangsan , and no patient had pathologic fracture ( fig .
11 ) . in busan , slip down was most common ( 33.7% ) , followed by collision , ta , assault , and fall down , the same as in yangsan , and there was one patient with pathologic fracture ( fig .
our investigation and comparison of patients with facial bone fracture who visited pusan national university hospital at yangsan and busan from 2010 to 2013 found differences in the total number of patients at each hospital but the trends were not significantly different .
the number of patients who visited the hospitals investigated in this study was 531 in yangsan and 802 in busan .
generally , patients visited the hospital in busan more than the hospital in yangsan , presumably caused by difference of standard population in 2013 ( 3.56 million in busan and 0.27 million in yangsan )
. however , other explanations could be the difference in the number of hospitals , or accessibility to yangsan from other areas of gyeongsangnam - do province . comparing the patients with facial bone fracture visiting the hospitals from 1992 to 1996 and from 1999 to 2003
, the yearly average number of patients increased over two times ( table 1 ) .
presumably this increase stemmed from the development of a regional emergency clinic center , a 1,339 emergency medical information center , and increased awareness of the citizens .
since the population of busan was 3.81 million in 2000 and the total population decreased , we surmise that the number of patients was not proportional to the population . comparing the monthly distribution to other publications from different regions
, it was highest in july at 13.4% in chuncheon according to kim et al . , in september at 12.7% in seongnam according to oh et al .
, in march at 10.7% in masan according to lee and ro , and in july at 10.8% , and in daegu according to lee and moon . in this study
, it was the highest in march at 11.5% in yangsan and in april at 10.5% in busan . according to breytenbach
, fractures occur in warm seasons , seen in several studies that found highest numbers in spring ( when temperatures are rising ) or summer ( the hottest season ) .
the gender ratio in the standard population of 2013 was 1.01:1 in yangsan and 0.98:1 in busan , indicating that the rate of male in busan was dominant over female although more females inhabit busan , but the rate of female patients were higher than the male in yangsan . compared to previous data , facial bone fractures occur more often in males ( table 2 ) .
the gender ratio was similar to other studies indicating the ratio of facial bone fracture between male and female as 4:1 according to kim et al .
presumably there are more male drivers and males are more prone to violence , resulting in fractures , social activity , and drinking .
as females take jobs with high risk and their social activity increases , they are exposed to more risks of facial fracture , increasing the female ratio of fracture .
we noted that of the age groups , the highest numbers were patients in their 20s , followed by those in their 30s at both yangsan and busan , then those in their 10s in yangsan and 50s in busan .
and oh et al . , and overseas studies of olson et al . and nakamura and gross .
presumably social activities increased in the 20s , with more exposure to beating , violence , drinking , driving that might cause fracture .
our old data also shows patients in their 20s with the highest number of fractures ( table 3 ) .
the number of patients with fracture in 10s , 30s , 40s , and 50s was similar at both yangsan and busan . by sites ,
the mandible was the most frequent , followed by nasal bone at both yangsan and busan . in the older data ,
the mandible was the highest but the patients with nasal bone fracture were few ( table 4 ) .
jang and jang reported that the cases of mandible were 59.3% but there were no isolated nasal bone fractures .
their explanation was that patients with isolated nasal bone fracture went to plastic surgery or otorhinolaryngology .
the rate of patients with the isolated nasal bone fracture visiting this hospital was high since many came to this maxillofacial surgery .
comparative frequency of mandibular and maxillary fracture was variously reported as 9:1 according to lee and ro , 6.1:1 according to rim et al .
in domestic studies ; and it was 3.56:1 in yangsan and 4.33:1 in busan according to this study . in the distribution by causes , slip down was the highest at both yangsan ( 36.5% ) and busan ( 33.7% ) , and collisions and ta showed similar rate . compared to our older data , assault was significantly less and slip down increased noticeably ( table 5 ) .
our explanation is that the general psychological atmosphere of this society became more non - violent , but there may be many cases providing inaccurate medical history to hide the cause related to violence due to the medical insurance system . also , ta was reported as the highest cause in many domestic and overseas studies but park et al . , reported the highest causes in order of slip down , fall down , assault , and ta ; oh et al . summarized the causes in order of slip down , fall down , ta , and assaults . in concentrated residential areas or with patients aged 10s to 20s ,
this study investigated and targeted patients with facial bone fracture visiting the department of oral and maxillofacial surgery of the dental clinic center in pusan national university hospital located at busan and of pusan national university dental hospital located at yangsan for outpatient department and emergency room from january 2010 to december 2013 .
five - hundred and thirty - one patients with facial bone fracture visited yangsan and 802 patients visited busan .
the gender ratio was 3.58:1 in yangsan and 4.31:1 in busan , showing that the male was dominant .
the number of patients with fractures was the highest in 20s at both yangsan and busan and the next was 30s .
the site of fracture was highest in order of mandible , nasal bone , maxilla , zygoma , and orbital cavity in yangsan and mandible , nasal bone , zygoma , orbital cavity , and maxilla in busan .
the commonest cause of fracture was slip down at both yangsan and busan and the next was in order of collision , ta , assaults , and fall down . | purpose : this study examined patients with facial bone fracture visiting pusan national university dental hospital to understand the trends , and to enhance appropriate care and treatment for patients with facial bone fracture.methods:we investigated 531 patients presenting with facial bone fracture in yangsan and 802 patients in busan from january 2010 to december 2013 .
we divided the patients by year , month , gender , age , site , and cause to compare with historic data and other studies.results:the gender ratio was 3.58:1 in yangsan and 4.31:1 in busan .
patients aged in their 20s had the highest number of facial bone fractures in both yangsan and busan .
the most frequent fracture site was the mandible , and the most frequent cause was slip down in both yangsan and busan.conclusion:the investigation and comparison of patients with facial bone fracture who visited pusan national university hospital located at yangsan and busan from 2010 to 2013 found a difference in the total number of patients at each hospital , but the trends were not significantly different . |
though many patients suffer from peripheral arterial occlusive disease ( paod ) , diagnosis is often delayed .
if paod is not diagnosed and treated immediately , it is highly probable that the disease will progress to critical limb ischemia ( cli ) .
although treatment options , especially endovascular treatment modalities , have improved in recent decades , mortality and amputation rates remain high .
various scoring systems have been established to identify patients at high risk for cardiovascular disease .
the most renowned is the framingham risk score , which distinguishes individuals at low , moderate , and high risk for coronary heart disease .
this score includes clinical ( eg , blood pressure ) and laboratory parameters ( eg , high density lipoprotein cholesterol ) . recently , the procam score was published to evaluate the 10-year myocardial infarction ( mi ) risk to identify patients who might profit from more intense prevention and cardiovascular risk factor management .
this score includes laboratory parameters ( eg , cholesterol ) , clinical parameters ( eg , blood pressure ) , and concomitant medication ( antihypertensives ) .
although cli is a common disorder , there is still no risk score to evaluate the cli risk in paod patients .
we therefore evaluated cli risk factors in our paod cohort and developed a risk score , the graz cli score , which is easy to use with paod patients .
for our cross - sectional study , we formed a first cohort of 1000 patients with paod treated at our department between 2003 and 2007 to identify cli risk factors and to develop our scoring model .
we validated our scoring system with a second cohort , consisting of 1124 paod patients we treated between 2007 and 2011 .
the inclusion criterion for our study was treatment at our institution for paod during the given time period ; there were no exclusion criteria .
the study was approved by the institutional review board of the medical university of graz , austria ( irb number 24 - 506 ex 11/12 ) . as this was a retrospective analysis of blinded data , the board agreed that neither written nor verbal consent would be required .
paod was diagnosed and graded in our outpatient clinic on the basis of clinical evaluation , ankle brachial index , and duplex scan according to the tasc ii criteria .
successive paod patients presenting at our outpatient angiology clinic were scheduled for admission to our ward for further treatment of their atherosclerotic disease .
cli was defined as paod presenting with ischemic rest pain and/or skin ulceration / gangrene in accordance with current guidelines and corresponding to fontaine classes 3 and 4 . upon hospital admission ,
the patient 's medical records were analyzed systematically for cardiovascular risk factors , comorbidities , and medication .
groups were compared with the test for categorical values , the t test for normally distributed continuous variables , and the mann
univariate regression analysis was used to assess the influence of individual demographic data and comedication parameters on cli .
variables exhibiting a significance level of p < 0.05 on univariate analysis were then entered into a multivariate binary logistic regression model .
for all tests , the threshold p value for significance was p 0.05 .
terpstra test performed to detect any statistical trend indicating that cli rates increase with the cli risk score .
groups were compared with the test for categorical values , the t test for normally distributed continuous variables , and the mann
univariate regression analysis was used to assess the influence of individual demographic data and comedication parameters on cli .
variables exhibiting a significance level of p < 0.05 on univariate analysis were then entered into a multivariate binary logistic regression model .
for all tests , the threshold p value for significance was p 0.05 .
terpstra test performed to detect any statistical trend indicating that cli rates increase with the cli risk score .
there were statistically significant differences between cohort 1 and cohort 2 with respect to the incidence of coronary artery disease ( cad ; 326 [ 32.6% ] vs 420 [ 37.4% ] , p = 0.03 ) and atrial fibrillation ( 149 [ 14.9% ] vs 219 [ 19.4% ] , p = 0.01 ) . with regard to concomitant medication we found differences in antiplatelet therapy ( aspirin : 414 [ 41.4% ] vs 379 [ 33.7% ] , p = 0.001 ) and clopidogrel ( 152 [ 15.2% ] vs 287 [ 25.5% ] , p = 0.001 ) .
this might be because of the differences in the time periods when patients presented at our outpatient clinic , where cohort 1 was treated between 2004 and 2007 and cohort 2 was treated between 2007 and 2011 . during the latter period , patients were increasingly treated with clopidogrel .
furthermore , cad frequency differed significantly between groups also leading to a difference in concomitant medication ( angiotensin receptor blocker and -blocker : 112 [ 11.2% ] vs 171 [ 15.2% ] , p = 0.01 , and 381 [ 38.1% ] vs 495 [ 44% ] , p = 0.01 ) .
patients characteristics of peripheral arterial occlusive disease patients included in the study ( cohort 1 and cohort 2 ) parameters of medical history and concomitant medication were first evaluated in a univariate regression analysis for the occurrence of cli in cohort 1 ( table 2 ) .
variables exhibiting a significance level of p < 0.05 on univariate analysis were then entered into a multivariate binary logistic regression model . in cohort 1 , the strongest risk factors for cli in the multivariate regression model were age > 75 years ( or 2.0 [ 95% confidence interval [ ci ] 1.33.0 ] , p < 0.001 ) , type 2 diabetes ( or 3.1 [ 95% ci 2.24.4 ] , p < 0.001 ) , prior mi ( or 2.5 [ 95% ci 1.15.5 ] , p = 0.03 ) , and therapy with low molecular weight heparin ( lmwh ) ( 2.8 [ 95% ci 1.74.4 ] , p < 0.001 ) . for those conditions we scored 2 points each .
one point was given for age between 65 and 75 years ( or 1.6 [ 95% ci 1.12.5 ] , p = 0.03 ) as well as for use of cardiac glycosides ( or 1.9 [ 95% ci 1.33.4 ] , p = 0.003 ) or loop diuretics ( or 1.5 [ 95% ci 1.12.1 ] , p = 0.01 ) .
as statin therapy was associated with an or of 0.5 ( 95% ci 0.40.8 , p < 0.001 ) , we subtracted 1 point for those patients ( table 3 ) .
risk factors for critical limb ischemia in peripheral arterial occlusive disease patients ( cohort 1 ) in univariate regression analysis adjusted risk factors for critical limb ischemia in peripheral arterial occlusive disease patients ( cohort 1 ) in the second group of patients , we could prove that the frequency of cli was significantly higher in patients with a high cli score ( table 4 ) .
the high - risk group ( score 610 ) was very small and we actually were not able to show a linear progression for cli in these patients , but we nonetheless were able to define 3 different risk groups ( low cli risk : score 1 to 1 , n = 434 ; intermediate cli risk : score 24 , n = 569 ; and high cli risk : score 510 , n = 121 ) according to the number of score points ( table 1 ) .
the cli rate was significantly higher in patients with a higher score , especially those with a score of > 4 points ( figure 1 ) .
the statistical trend we saw for the increase in the cli rate with increasing score points was statistically significant ( p < 0.001 ) .
critical limb ischemia patients according to the critical limb ischemia risk score in cohort 2 percentage of patients with critical limb ischemia stratified by critical limb ischemia risk score .
data in the figure are % ( cli patients / all patients in the group ) .
we analyzed the correlation of the graz cli score with the inflammatory parameters that reflect the activity of the underlying atherosclerotic disease .
we calculated pearson 's correlation and spearman 's for the correlation of c - reactive protein ( crp ) and fibrinogen with the cli score and found a highly significant statistical correlation for both parameters ( for crp : pearson 's r = 0.3 ; spearman 's = 0.4 ; for fibrinogen : pearson 's r = 0.2 ; spearman 's = 0.3 ; p value for all calculations < 0.001 ) .
this study demonstrated that the risk for cli in paod patients can be determined easily on the basis of their clinical parameters and concomitant medication .
our score correlates well with inflammatory parameters that reflect the activity of atherosclerosis in these patients . in general
, scoring systems can be useful for identifying patients at high risk for vascular endpoints .
the framingham risk score can be used to highlight patients at high risk for cardiovascular disease and those with a high - risk score might profit from intense management of their vascular risk factors , as might those with a high graz cli score .
we hypothesize that those paod patients should be treated immediately as their elevated inflammatory parameters suggest a more aggressive atherosclerosis . with our score ,
age and diabetes were among the most aggressive risk factors , with an or of 2.0 and 3.1 , respectively .
we were able to show that an elevated cha2ds2-vasc score is associated with an elevated cli risk . with this scoring system ,
there are signs of chronic inflammation in both , diabetes and insulin resistance , a typical feature of type 2 diabetes .
concerning age there are similar data on age - related inflammation increase in atherosclerotic patients .
the reason for this age - related systemic chronic inflammation is mainly attributed to the progressive activation of immune cells over time . when the graz cli score was analyzed ,
much research has been conducted to evaluate the protective effect of statin therapy , especially in cli patients .
westin et al recently showed that statins were associated with lower mortality , a reduction in major adverse cardiovascular and cerebrovascular events , and longer amputation - free survival in cli patients . even in patients who have undergone endovascular treatment , statin therapy is associated with superior clinical outcome . especially in combination with angiotensin - converting enzyme inhibitors ,
statins may have even reduced mortality in diabetic cli patients during a 2-year follow - up .
the beneficial findings of statins in cli patients were further underlined in large databases , like the reach registry .
a recent publication on symptomatic paod patients revealed that among them , statin use was associated with an 18% lower rate of adverse limb outcomes , including worsening of symptoms , peripheral revascularization , and ischemic amputations .
statin therapy should by all means be initiated for all paod patients , especially those with diabetes .
these recommendations are also reflected in current guidelines , although adherence to them is rather poor .
as this medication is mainly given to patients with congestive heart failure , their low cardiac output causes hemodynamic alterations that include a low flow state , increased peripheral resistance , endothelial cell dysfunction , and vascular smooth muscle cell dysfunction that could exacerbate ischemic symptoms .
we already know that patients with paod have a higher risk of fatal and nonfatal cardiovascular events than do patients with symptomatic atherosclerosis in other vascular beds such as the coronary , carotid , and cerebral arteries .
the higher values for inflammatory parameters with higher cli scores might reflect a more active underlying atherosclerosis that in turn could increase their mi rate . in elderly patients ,
diurectics are often prescribed for the many geriatric patients who suffer from congestive heart failure , and loop diuretics were implicated as a further cli risk factor in our cohort .
dehydration reduces peripheral perfusion , leading to vascular occlusion and more severe paod symptoms , reflected in a higher cli rate .
antiplatelet therapy is mandatory for atherosclerosis patients , especially after endovascular treatment , but for some patients , such as those with atrial fibrillation , vitamin k antagonist ( vka ) is indicated .
when vka therapy is contraindicated because of a high bleeding risk , lmwh is an alternative .
this therapy was a risk factor for cli in our cohort with an or of 2.8 .
one explanation is that these patients might tend to be multimorbid , with highly active underlying atherosclerosis leading to a high cli rate .
this activation of platelets might explain the increase in vascular endpoints including the development of cli .
furthermore , there were statistically significant differences between cohort 1 and cohort 2 concerning incidence of cad , atrial fibrillation , and comedication ( clopidogrel , angiotensin receptor blocker , and -blocker therapy ) .
as clopidogrel is beneficial especially in pad patients , at least some impact on our results can not be excluded . with the graz cli score
, we have developed a simple risk stratification scheme on the basis of information that can be easily acquired from the medical history , without any laboratory parameters .
this score proved to be a promising tool to identify paod patients at high risk for cli and should be further evaluated in an upcoming prospective study . | abstractcritical limb ischemia ( cli ) , a frequently encountered disorder , is associated with a high rate of limb amputation and mortality . to identify patients at high risk for cli
, we developed a simple risk score for peripheral arterial occlusive disease ( paod).in our cross - sectional study , we first evaluated 1000 consecutive paod patients treated at our institution from 2005 to 2007 , documenting clinical symptoms , comorbidities , and concomitant medication .
we calculated odds ratios ( or ) in a binary logistic regression model to find possible risk factors for cli .
we then verified the score in a second step that included the 1124 paod patients we treated between 2007 and 2011.in the first patient group , the greatest risk factors for cli were age 75 years ( or 2.0 ) , type 2 diabetes ( or 3.1 ) , prior myocardial infarction ( or 2.5 ) , and therapy with low molecular weight heparins ( 2.8 ) .
we scored 1 point for each of those conditions .
one point was given for age between 65 and 75 years ( or 1.6 ) as well as for therapy with cardiac glycosides ( or 1.9 ) or loop diuretic therapy ( or 1.5 ) .
as statin therapy was protective for cli with an or of 0.5 , we subtracted 1 point for those patients.in the second group , we could prove that frequency of cli was significantly higher in patients with a high cli score .
the score correlated well with inflammatory parameters ( c - reactive protein and fibrinogen ) .
we were also able to define 3 different risk groups for low ( score 1 to 1 ) , intermediate ( score 24 ) , and high cli risk ( score > 4).we developed a simple risk stratification scheme that is based on conditions that can be easily assessed from the medical history , without any laboratory parameters .
this score should help to identify paod patients at high risk for cli . |
burn injury is a major cause of trauma to the human body , causing death and disability , with a long healing period and high health care costs .
the mortality rate of burn injury is decreasing with new treatment modalities , although secondary infections and long healing periods still affect mortality rates .
early debridement and skin grafting are successful , but insufficient graft donor area and unsuitable patient circumstances for surgery hinder skin grafting . in these circumstances , using products that increase the wound - healing process often affect patient morbidity and mortality . for this purpose , different kinds of dressings and pharmacotherapies have been developed , but most of them are costly , and the mechanisms underlying these therapies have not been fully documented .
growth factors have been implicated in mediating the normal healing process as well as playing a role in impaired wound healing .
certain growth factors , such as platelet - derived endothelial cell growth factor ( pdgf ) and insulin - like growth factor - i ( igf - i ) , inhibit the apoptosis pathways necessary for the rapid turnover of cells required to facilitate the different stages of physiologic wound healing .
the wound healing effects of growth hormone ( gh ) appear to be directly and indirectly associated with igf-1 expression .
poffenbarger and haberal reported that a glycoprotein named nonsuppressible insulin - like activity ( nsila ) exhibits insulin - like activity on fibroblasts in tissue and modulates the fibroblastic response in wound healing .
platelet - rich plasma ( prp ) is a new adjunct that is increasingly used to treat soft and bony tissue defects to accelerate bone formation , especially in oral and maxillofacial bone reconstructive surgery , and in managing chronic nonhealing wounds [ 25 ] .
platelet - rich plasma is produced from centrifuged blood combined with thrombin and calcium chloride as a viscous coagulum gel , which is a rich source of growth factors released by activated platelets .
they release multiple growth factors and cytokines , including transforming growth factor- ( tgf-1 and 2 ) , transforming growth factor- ( tgf- ) , vascular endothelial growth factor ( vegf ) , platelet - derived endothelial cell growth factor ( pdgf ) , epidermal growth factor ( egf ) , platelet thromboplastin , thrombospondin , coagulation factors , fibroblast growth factor ( fgf ) , platelet activating growth factor-4 , calcium , serotonin , histamine , and hydrolytic enzymes with degranulation triggered by proteins such as thrombin .
it has been demonstrated that platelet - derived growth factors are successful in treating nonhealing cutaneous human wounds .
growth factors attract cells to the wound site ; they stimulate cell proliferation and increase differentiation and extracellular matrix deposition . transforming growth
factor- is important in tissue repair , and it was demonstrated that tgf- has therapeutic value in the treatment of chronic , nonhealing wounds .
platelet - derived endothelial cell growth factor improves dermal regeneration , promotes protein and collagen synthesis causing endothelial migration or angiogenesis , and increases expression of tgf- . transforming growth factor- and pdgf levels are higher in platelet - rich plasma compared with platelet - poor plasma ( ppp ) . using the effects of tgf- on wound healing ( e.g. , promoting differentiation and proliferation ) , we aimed to achieve enhanced wound healing and more - organized collagen in burn wounds treated with prp gel compared to controls , without excessive deposition of connective tissue or scar formation .
although the positive effects of prp on graft maturation and wound healing are well known , a similar effect on burn healing is not clear .
this study sought to investigate the effects of prp on burn wound healing in an experimental partial - thickness hot water burn injury .
this study was performed at the experimental research center of baskent university with the permission of the experimental research ethical committee of baskent university faculty of medicine .
all rats were brought to the experimental research center 1 week before the experiment and kept in special cages under controlled temperature ( 22c ) , humidity , and lighting conditions ( 12-hour day , 12-hour night ) .
group 1 rats ( study group ; n=10 ) were exposed to prp application after burn injury ; group 2 rats ( control group ; n=10 ) were exposed to burn injury only ; and group 3 rats ( blood donor group ; n=10 ) were used as blood donors for the preparation of prp .
all animals were anesthetized by an intraperitoneal injection of 50 mg / kg ketamine hydrochloride and 10 mg / kg xylazine hydrochloride . under sterile conditions , a 333 cm sponge ( vileda , procter & gamble company , cincinnati , oh )
was kept in boiling water and touched to the rats shaved dorsum for 35 seconds with its own weight to induce a partial - thickness burn injury .
the burn site was covered with opsite drape ( smith & nephew , inc . , wound management , st .
after experimentation , all rats were kept in special cages under controlled temperature , humidity , and lighting conditions .
rats were given 0.002 g / kg fentanyl citrate twice a day for postoperative analgesia .
all rats were killed after administration of anesthesia ( 60 mg / kg intraperitoneal ketamine ) on the seventh day after burn injury .
platelet - rich plasma was derived after 2-step centrifugation of blood taken from the rats in group 3 . with the first soft , short spin ( 1500 rpm , 20c , 10 minutes ) ,
then , the plasma fraction was separated with a hard , long spin ( 2000 rpm , 20c , 15 minutes ) into the prp and the plasma - poor platelets .
after this , the prp was combined with thrombin ( diathrombin , diamed , switzerland ) and calcium chloride ( calcium chloride , diamed , switzerland , 0.02 mol / l ) to activate platelets to produce sufficient clot formation that could be applied on the burn site .
the mixture ratio was 1 ml : 0.1 ml : 1 ml ( prp - thrombin - ca chloride ) .
after the rats were killed on the seventh day , the burn site was excised and tissue samples were sent to the biochemistry department for detection of tissue hydroxyproline levels and to the pathology department for histopathologic examinations .
who was included in the study and blinded to all information regarding the groups performed the histopathologic examination of the tissues .
tissue specimens were fixed in a 10% formaldehyde solution , and then embedded in paraffin .
histopathologic examinations for wound healing were assessed by light microscope with hematoxylin - eosin staining , and collagen deposition was assessed by light microscopy with trichrome staining in 100 and 200 magnification areas .
the wound healing process was assessed with a modified ehrlich - hunt numeric scale for polymorphonuclear leucocytes ( pmnl ) , fibroblasts , vessel proliferation , and collagen deposition as follows : 0 ( none ) , 1 ( focal ) , 2 ( mild ) , 3 ( moderate ) , and 4 ( significant ) .
all animals were anesthetized by an intraperitoneal injection of 50 mg / kg ketamine hydrochloride and 10 mg / kg xylazine hydrochloride . under sterile conditions , a 333 cm sponge ( vileda , procter & gamble company , cincinnati , oh )
was kept in boiling water and touched to the rats shaved dorsum for 35 seconds with its own weight to induce a partial - thickness burn injury .
the burn site was covered with opsite drape ( smith & nephew , inc . , wound management , st .
after experimentation , all rats were kept in special cages under controlled temperature , humidity , and lighting conditions .
rats were given 0.002 g / kg fentanyl citrate twice a day for postoperative analgesia .
all rats were killed after administration of anesthesia ( 60 mg / kg intraperitoneal ketamine ) on the seventh day after burn injury .
platelet - rich plasma was derived after 2-step centrifugation of blood taken from the rats in group 3 . with the first soft , short spin ( 1500 rpm , 20c , 10 minutes ) ,
then , the plasma fraction was separated with a hard , long spin ( 2000 rpm , 20c , 15 minutes ) into the prp and the plasma - poor platelets .
after this , the prp was combined with thrombin ( diathrombin , diamed , switzerland ) and calcium chloride ( calcium chloride , diamed , switzerland , 0.02 mol / l ) to activate platelets to produce sufficient clot formation that could be applied on the burn site .
the mixture ratio was 1 ml : 0.1 ml : 1 ml ( prp - thrombin - ca chloride ) .
after the rats were killed on the seventh day , the burn site was excised and tissue samples were sent to the biochemistry department for detection of tissue hydroxyproline levels and to the pathology department for histopathologic examinations .
who was included in the study and blinded to all information regarding the groups performed the histopathologic examination of the tissues .
tissue specimens were fixed in a 10% formaldehyde solution , and then embedded in paraffin .
histopathologic examinations for wound healing were assessed by light microscope with hematoxylin - eosin staining , and collagen deposition was assessed by light microscopy with trichrome staining in 100 and 200 magnification areas .
the wound healing process was assessed with a modified ehrlich - hunt numeric scale for polymorphonuclear leucocytes ( pmnl ) , fibroblasts , vessel proliferation , and collagen deposition as follows : 0 ( none ) , 1 ( focal ) , 2 ( mild ) , 3 ( moderate ) , and 4 ( significant ) .
the others were compared using the t test . values for p less than 0.05 were accepted as significant .
two rats from each group died during the experiment , so the statistical analysis was performed with 8 rats in each group . in the study group ,
mean tissue hydroxyproline ( hyp ) level was detected as 0.950.35 g hyp / g , whereas in the control group mean tissue hyp level was detected as 0.690.08 g hyp / g .
the hyp levels were significantly higher in the study group than in the control group ( p=.03 ) ( figure 1 ) . in both groups , partial - thickness burn injury
statistically , except for the inflammatory cell ( pmnl ) infiltration , there was no significant difference between the groups ( figure 2 ) . in the study group ,
the mean pmnl score was 10.53 , whereas in the control group it was 2.3750.51 ( mean sem ) . therefore , inflammatory cell infiltration was statistically higher in the control group ( p=.005 ) .
moderate inflammatory cell infiltration from the control group and mild inflammatory cell infiltration from the study group are shown in figures 3 and 4 . in the study group ,
the mean fibroblast score was 0.6250.51 , whereas in the control group it was 0.3750.51 ( mean sem ) .
although the fibroblast score was higher in the study group , there was no significant difference between the groups ( p=.31 ) .
focal fibroblast infiltration from the study group is shown in figure 5 . in the study group the mean vessel proliferation score was 0.750.7 , whereas in the control group it was 0.250.46 ( mean sem ) .
vessel proliferation score was higher in the study group ; however , no significant difference was detected between the groups ( p=.26 ) .
mild vessel proliferation from the study group is shown in figure 6 . in the study group ,
the mean collagen deposition score was 0.6250.51 , while in the control group it was 0.3750.51 ( mean sem ) .
collagen deposition scores were higher in the study group but no significant difference was detected between the groups ( p=.31 ) .
mild collagen deposition from the study group is shown in figure 7 . in the study group
the epithelization rates were higher in the study group , but there was no significant difference between the groups ( p>.05 ) .
in the study group , mean tissue hydroxyproline ( hyp ) level was detected as 0.950.35 g hyp / g , whereas in the control group mean tissue hyp level was detected as 0.690.08 g hyp / g .
the hyp levels were significantly higher in the study group than in the control group ( p=.03 ) ( figure 1 ) .
in both groups , partial - thickness burn injury was confirmed with macroscopic and microscopic examination .
statistically , except for the inflammatory cell ( pmnl ) infiltration , there was no significant difference between the groups ( figure 2 ) .
in the study group , the mean pmnl score was 10.53 , whereas in the control group it was 2.3750.51 ( mean sem ) .
therefore , inflammatory cell infiltration was statistically higher in the control group ( p=.005 ) .
moderate inflammatory cell infiltration from the control group and mild inflammatory cell infiltration from the study group are shown in figures 3 and 4 .
in the study group , the mean fibroblast score was 0.6250.51 , whereas in the control group it was 0.3750.51 ( mean sem ) . although the fibroblast score was higher in the study group , there was no significant difference between the groups ( p=.31 ) .
in the study group the mean vessel proliferation score was 0.750.7 , whereas in the control group it was 0.250.46 ( mean sem ) .
vessel proliferation score was higher in the study group ; however , no significant difference was detected between the groups ( p=.26 ) .
in the study group , the mean collagen deposition score was 0.6250.51 , while in the control group it was 0.3750.51 ( mean sem ) .
collagen deposition scores were higher in the study group but no significant difference was detected between the groups ( p=.31 ) .
in the study group the epithelization rate was 37.5% , while in the control group it was 25% .
the epithelization rates were higher in the study group , but there was no significant difference between the groups ( p>.05 ) .
patient age , burn depth , and size of burn area are the most - important factors that influence mortality and morbidity after thermal injuries .
burn depth is also the most important factor affecting patients long - term appearance and functioning .
malnutrition , immunosuppression , and extensive burn area provide a suitable environment for microorganisms ; such infections are the most important cause of mortality and morbidity . in laboratory studies
it has been shown that topical administration of bone marrow cells increases dermal fibroblast population , vessel proliferation , and wound healing .
for this reason , wound covering materials and artificial skin substitutes were developed and are used , but their indications are not yet clear .
the first laboratory products used for this purpose were epithelial cells produced in a laboratory environment , but approximately 3 weeks are needed to produce the few thousand cm area required in extensive burns .
desired properties of wound cover materials include forming a barrier against fluid loss and microorganisms , supporting increasing cells in wound healing , and allowing vessel proliferation , keratinocyte adhesion , and differentiation ; unfortunately , no such material currently exists .
platelet - rich plasma contains a high level of platelets , clotting factors , cytokines , and growth factors .
they start releasing these activated factors 10 minutes after clotting , and more than 95% of growth factors are released in the first hour .
several in vivo and in vitro studies showed that all cells in the wound healing process are sensitive to the growth factors .
epidermal growth factor is a chemotactic factor for fibroblasts , and topical application of egf increases epidermal regeneration and strength of wound tension .
platelet - derived endothelial cell growth factor stimulates fibroblast and smooth muscle cell migration and proliferation , increases collagen deposition , and is also a chemotactic factor for neutrophils and monocytes .
furthermore , it has been shown that chondrocyte , osteoblast , and periosteal cell proliferation is promoted by pdgf and bfgf .
transforming growth factor-1 regulates cell differentiation , proliferation , chemotaxis , and synthesis of several extracellular matrix proteins . in animal models
, it has been shown that topical application of tgf-1 increases collagen synthesis , granulation tissue , and strength of wound tension .
transforming growth factor- triggers collagen synthesis and quickens maturation of collagen in the early period of wound healing .
in addition , using tgf- and pdgf together increases collagen deposition more than using tgf- alone .
it has been demonstrated that prp increases wound healing in acute trauma wounds , chronic nonhealing wounds , and incisional wounds , and is effective in soft and hard tissue reconstructions [ 2,3,5,6,1820 ] .
it has been shown that the inflammatory phase is reduced , and prolonged inflammation ( which often leads to bacterial infections and scar formation ) does not exist in wounds treated with prp .
. showed that using prp with acellular xenogeneic dermal matrix for treatment of deep second - degree burns decreased infection rate and increased wound healing .
furthermore , an animal study and several case reports showed improved burn wound - healing time after the application of prp .
a deep dermal burn could benefit from prp through its hemostatic antimicrobial abilities and the positive effects seen in wound healing .
a recent review showed a significant benefit of platelet - rich plasma on wound healing in acute wounds and it improved long - term outcome of laser therapies .
there are several studies suggesting that addition of prp to the graft site enhances wound healing , promoting epithelization and angiogenesis in split - thickness skin grafts and donor sites .
. demonstrated that the viscoelastic properties of scars treated with the combination of split - thickness skin grafting ( stsg ) plus autologous platelet concentrate return more rapidly to the plateau state than areas treated with stsg only . in a recent study ,
prp increased the speed of repair of the extracellular matrix and its components in deep second - degree burn wounds in horses , and treatment with 2 applications of prp accelerated formation of extracellular matrix during the first half of wound healing .
we have shown that in the study group , pmnl infiltration was significantly lower than in the control group , whereas there were no statistically significant differences between groups in terms of fibroblast infiltration , vessel proliferation , collagen deposition , or epithelization .
the difference between the wound healing processes in the burn wounds versus regular burn wound healing may be the cause of this result . as opposed to regular wound healing , in burn wounds
this hemorrhagic infill constructs a skeleton structure for cells coming into the wound site , including neutrophils , monocytes , fibroblasts , and endothelial cells .
moreover , in burn wounds , blood supply is reduced because of the damaged vessels . at the burn site it is hard to define healed parts because of this lack of hemorrhage , which determines the borders of the wound .
although in partial - thickness burn injuries cellular damage generally exists in the upper dermis , in the lower dermis the structural matrix damage is minimal .
thus , structural matrix replacement is limited , and wound healing in partial - thickness wounds usually does not involve replacement of damaged structural matrix .
further evidence supporting our conclusion is the statistically higher tissue hydroxyproline levels in the prp - treated group , which is an important indicator in wound healing . in the vertebrata ,
nearly all the hydroxyproline is present in collagen ; therefore , determining the hydroxyproline level is an adequate method for assessing collagen metabolism .
hydroxyproline levels were statistically higher in the prp group than in the control group ( p=.03 ) .
histopathologically , there was significantly less inflammatory cell infiltration ( p=.005 ) and there were no statistically significant differences between groups in fibroblast development , collagen production , vessel proliferations , or epithelization .
platelet - rich plasma seems to partially improve burn wound healing in this experimental burn injury model .
use of platelet - rich plasma can be an option for patients who have extensive burn areas with no chance for grafting to accelerate the burn healing process . | backgroundto investigate the effects of platelet - rich plasma on tissue maturation and burn healing in an experimental partial - thickness burn injury model.material/methodsthirty wistar albino rats were divided into 3 groups of 10 rats each .
group 1 ( platelet - rich plasma group ) was exposed to burn injury and topical platelet - rich plasma was applied .
group 2 ( control group ) was exposed to burn injury only .
group 3 ( blood donor group ) was used as blood donors for platelet - rich plasma .
the rats were killed on the seventh day after burn injury .
tissue hydroxyproline levels were measured and histopathologic changes were examined.resultshydroxyproline levels were significantly higher in the platelet - rich plasma group than in the control group ( p=.03 ) .
histopathologically , there was significantly less inflammatory cell infiltration ( p=.005 ) and there were no statistically significant differences between groups in fibroblast development , collagen production , vessel proliferations , or epithelization.conclusionsplatelet-rich plasma seems to partially improve burn healing in this experimental burn injury model . as an initial conclusion , it appears that platelet - rich plasma can be used in humans , although further studies should be performed with this type of treatment . |
malaria occurs often in poor , tropical and subtropical areas and still took an estimated 627 000 lives in 2012 , mostly those of children under five years of age in africa .
the culicidae mosquitoes are responsible for transmission of the worm parasites of heart dogs such as dirofileria immitis , dirofileria repense , wuchereria bancrofti , some arboviral diseases such as japanese encephalitis , rift valley fever , western equine encephalitis and eastern equine encephalitis , tahyna , sagiyama , trivitatus , lymphocytic choriomanangitis , west nile virus , st .
about 70 anopheles species in the world are able to transmit malaria and of these 40 species have been identified as the major vectors ( azari - hamidian and harbach 2009 ) . by now ,
64 species of culicidae mosquitoes due to 3 sub genera and 7 genera are reported from iran ( azari - hamidian and harbach 2009 ) .
now , seven species of anopheles reported as the malaria vectors in the country including : an .
2004c , vatandoost and borhani 2004a , vatandoost and moinvaziri 2004b , vatandoost et al . 2005b , sedaghat and harback 2005 , hanafi - bojad et al .
2006a , 2006b , 2007 , 2008 , 2009a , 2009b , moosa - kazemi et al .
pulcherrimus as secondary vectors of malaria in the south east of iran ( zaim et al . 1993 ) .
twelve insecticides recommended by who for irs currently , which belong to four chemical groups including one organochlorine , six pyrethroids , three organophosphates and two carbamates ( pluess et al . 2010 ) .
stephensi was reported in iran in 1957 , subsequently to dieldrin in 1960 , and then to malathion in 1976 ( eshghy 1978 , manouchehri et al .
release of larvivorous fish , using the bacillus thuringiensis , and larviciding by chlorpyrifos - methyl are the main larval control measures and pyrethroid as new insecticides are being used as irs and llins in iran ( vatandoost and hanafi bojd 2005a , moosa - kazemi et al .
2007 ) . in spite of more than 50 years malaria - control programming , 42%60%of the total malaria cases reported from sistan and baluchistan province . out of 112 malaria cases representing 79 males and 33 females have been reported in chabahar county , southeastern iran in 2010 .
out of 38 cases were from bangladesh , pakistan and afghanistan and the rest were the local aborigines ( moh and me 2010 ) .
chabahar is commercial and industrial free zone as well as important due to agricultural and husbandry in sistan and baluchistan province in southeastern iran and also the border line of pakistan .
there are scatter data about susceptibility level of malaria vectors in iran , the susceptibility level of culicinae mosquitoes in southeastern country had not been reported .
therefore , this study was carried out to determine susceptibility level of mosquitoes to monitoring and evaluation of insecticides resistance .
this information could provide an essential clue for judicious use of insecticides and will be very useful to health authorities for future planning of vector control in this endemic malarious area .
the study was carried out in chabahar city , siatan and baluchistan province , southeastern iran ( 2517gn , 6037ge ) ( fig .
1 ) . the people engaged to agriculture , horticulture , livestock , fishing sailing , and hand crafts including needlework , making carpet and musical instruments .
the absolute maximum and average of temperature was reported 36 c and 16.5 c , respectively ( meteorological organization of iran , 2010 ) .
chabahar county with 24 , 729 square kilometers ( 19.3% ) comprised 3 cities , 5 distracts and 11 rural districts .
the population of chabahar reported 211,081 including 143,535 rural ( 68% ) and 67,546 urban ( 32% ) .
naval base near the 22 bahman squar ( 25 18gn,6037ge ) in chabahar city selected as fix station including , 30 households , and 114 population .
baluchi - adam village ( 25 47gn , 6116ge ) with 71 households , and 379 populations , and bahoo - kalat village ( 2543gn , 6125ge ) , 198 households and 1064 population selected randomly as fixed stations .
sampling methods such as larval collection , hand catch was carried out during january to december 2010 ( who 1975 , who 1975 ) .
these studies were conducted once every 15 days and collected mosquitoes were identified by specific systematic keys ( shahgudian 1960 , zaim and cranston 1986 ) .
location of study area in southeastern iran , 2010 in each fixed and variable station larvae was collected from january to december 2010 .
larval mosquitoes picked up from the water using a dropper , pipette or fine net and inserted into the bulb .
the related data such as water temperature , larval type , number and date sampling was recorded .
larvae and pupae in holding container filled with water were transferred to the laboratory for rearing .
adult mosquitoes live quite well on bowl of sucrose 10% in bottom of the cage .
the adults were kept in the temperature ( 2224 c ) , relative humidity ( 6070% ) and 12l-12d photoperiod ( who 1975a , 1975b ) .
culicidae mosquitoes were collected from the villages that spraying was not performed before , between 06.30 and 09.30 am .
sampling was carried out in each human dwelling , cattle and goat sheds for 15 min using suction tube and torch ( who 1975b ) .
the mosquitoes were inserted in the cage as dimensions of 40 40 cm and sent to the laboratory .
total of 200 to 250 mosquitoes were entered in each cage and covered with wet towel .
the mosquitoes were kept in standard condition ( 2 5c , 75% h ) . impregnated papers with ddt 4 % , malathion 5 % , propoxur 0.1 % , deltamethrin 0.05 % ,
lambda - cyhalothrin 0.05 % , cyfluthrin 0.15 % , and control papers were supplied by world health organization .
the adult susceptibility test kit including holding and exposure tubes , slides , copper and silver rings and related covers were washed with detergent , and then washed three times with tap water to be free of detergents and insecticides . after drying of the pipe holders , standard paper impregnated with insecticides fixed inside the tubes marked with red dot and copper ring . for holding tube used paper control ( no insecticide ) tube with a green sign and silver pipe fixed to the wall ( who 2006 ) . each time
45 mosquito collected and insert to holding tube overall 2025 mosquito were kept into holding tube .
the susceptibility tests performed on their standard condition ( 2226 c , 60% h ) .
the susceptibility of the wild strain of culicidae mosquitoes was assessed to the insecticides impregnated papers .
the mosquitoes were exposed to different insecticides by different interval times and 24 hours recovery period .
the following criteria have been used for interpretation and classification of results , based on who recommendations ( african network on vector resistance 2005 ) .
susceptibility test carried out with less than 20 mosquitoes per test have not been consideredat least 80 mosquitoes used per test , three resistance classes as defined .
resistant when the mortality was lower than 80 % , susceptible when mortality was 98 % or higher and possible resistant or tolerant when mortality was between 97 % and 80 % .three resistance classes as defined when twenty to 79 mosquitoes used per test , susceptible when mortality was 98 % or higher , resistance suspected , when mortality was between 95 % and 97 % , and resistant when the mortality was lower than 95 % .
susceptibility test carried out with less than 20 mosquitoes per test have not been considered at least 80 mosquitoes used per test , three resistance classes as defined .
resistant when the mortality was lower than 80 % , susceptible when mortality was 98 % or higher and possible resistant or tolerant when mortality was between 97 % and 80 % .
three resistance classes as defined when twenty to 79 mosquitoes used per test , susceptible when mortality was 98 % or higher , resistance suspected , when mortality was between 95 % and 97 % , and resistant when the mortality was lower than 95 % .
the samples were recorded in the special forms by and the appropriate time of deaths associated with history of collection , relative humidity and temperature ( shahgudian 1960 , zaim and cranston 1986 , azari - hamidian and harbach 2009 ) .
results were considered reliable if the control mortality was less than 5 % and rejected if more than 20 % .
results were corrected by abbott 's formula when mortality rates of control group were between 5 to 20 % ( abbott 1925 , who 2006 ) .
the lt50 and lt90 were calculated for plotting the regression line using microsoft excel software ver .
the study was carried out in chabahar city , siatan and baluchistan province , southeastern iran ( 2517gn , 6037ge ) ( fig .
1 ) . the people engaged to agriculture , horticulture , livestock , fishing sailing , and hand crafts including needlework , making carpet and musical instruments .
the absolute maximum and average of temperature was reported 36 c and 16.5 c , respectively ( meteorological organization of iran , 2010 ) .
chabahar county with 24 , 729 square kilometers ( 19.3% ) comprised 3 cities , 5 distracts and 11 rural districts .
the population of chabahar reported 211,081 including 143,535 rural ( 68% ) and 67,546 urban ( 32% ) .
naval base near the 22 bahman squar ( 25 18gn,6037ge ) in chabahar city selected as fix station including , 30 households , and 114 population .
baluchi - adam village ( 25 47gn , 6116ge ) with 71 households , and 379 populations , and bahoo - kalat village ( 2543gn , 6125ge ) , 198 households and 1064 population selected randomly as fixed stations .
sampling methods such as larval collection , hand catch was carried out during january to december 2010 ( who 1975 , who 1975 ) .
these studies were conducted once every 15 days and collected mosquitoes were identified by specific systematic keys ( shahgudian 1960 , zaim and cranston 1986 ) .
larval mosquitoes picked up from the water using a dropper , pipette or fine net and inserted into the bulb .
the related data such as water temperature , larval type , number and date sampling was recorded .
larvae and pupae in holding container filled with water were transferred to the laboratory for rearing .
adult mosquitoes live quite well on bowl of sucrose 10% in bottom of the cage .
the adults were kept in the temperature ( 2224 c ) , relative humidity ( 6070% ) and 12l-12d photoperiod ( who 1975a , 1975b ) .
culicidae mosquitoes were collected from the villages that spraying was not performed before , between 06.30 and 09.30 am .
sampling was carried out in each human dwelling , cattle and goat sheds for 15 min using suction tube and torch ( who 1975b ) .
the mosquitoes were inserted in the cage as dimensions of 40 40 cm and sent to the laboratory .
total of 200 to 250 mosquitoes were entered in each cage and covered with wet towel .
impregnated papers with ddt 4 % , malathion 5 % , propoxur 0.1 % , deltamethrin 0.05 % , lambda - cyhalothrin 0.05 % , cyfluthrin 0.15 % , and control papers were supplied by world health organization .
the adult susceptibility test kit including holding and exposure tubes , slides , copper and silver rings and related covers were washed with detergent , and then washed three times with tap water to be free of detergents and insecticides . after drying of the pipe holders , standard paper impregnated with insecticides fixed inside the tubes marked with red dot and copper ring . for holding tube used paper control ( no insecticide ) tube with a green sign and silver pipe fixed to the wall ( who 2006 ) .
each time 45 mosquito collected and insert to holding tube overall 2025 mosquito were kept into holding tube .
the susceptibility tests performed on their standard condition ( 2226 c , 60% h ) .
the susceptibility of the wild strain of culicidae mosquitoes was assessed to the insecticides impregnated papers .
the mosquitoes were exposed to different insecticides by different interval times and 24 hours recovery period .
the following criteria have been used for interpretation and classification of results , based on who recommendations ( african network on vector resistance 2005 ) .
susceptibility test carried out with less than 20 mosquitoes per test have not been consideredat least 80 mosquitoes used per test , three resistance classes as defined .
resistant when the mortality was lower than 80 % , susceptible when mortality was 98 % or higher and possible resistant or tolerant when mortality was between 97 % and 80 % .three resistance classes as defined when twenty to 79 mosquitoes used per test , susceptible when mortality was 98 % or higher , resistance suspected , when mortality was between 95 % and 97 % , and resistant when the mortality was lower than 95 % .
susceptibility test carried out with less than 20 mosquitoes per test have not been considered at least 80 mosquitoes used per test , three resistance classes as defined .
resistant when the mortality was lower than 80 % , susceptible when mortality was 98 % or higher and possible resistant or tolerant when mortality was between 97 % and 80 % .
three resistance classes as defined when twenty to 79 mosquitoes used per test , susceptible when mortality was 98 % or higher , resistance suspected , when mortality was between 95 % and 97 % , and resistant when the mortality was lower than 95 % .
the samples were recorded in the special forms by and the appropriate time of deaths associated with history of collection , relative humidity and temperature ( shahgudian 1960 , zaim and cranston 1986 , azari - hamidian and harbach 2009 ) .
results were considered reliable if the control mortality was less than 5 % and rejected if more than 20 % .
results were corrected by abbott 's formula when mortality rates of control group were between 5 to 20 % ( abbott 1925 , who 2006 ) . data were analyzed by probit analysis ( finney 1971 , 1978 ) .
the lt50 and lt90 were calculated for plotting the regression line using microsoft excel software ver .
culicifacies , the species composition of adult culicidae mosquitoes collected by different sampling methods in chabahar county , sistan and baluchistan province , southeastern iran , 2010 an .
mortality of culicidae mosquitoes exposed to ddt and other insecticides has shown in tables 1 and 2 .
this species was quite resistant to ddt while susceptible to deltamethrin and tolerant to other insecticides ( fig .
culicifacies is tolerant to ddt , malathion , and propoxure whereas susceptible to delta - methrin , cyfluthrin and lambda - cyhalothrin ( table 2 ) .
the lt50 and lt90 values of this species to ddt 4 % were 18.12 and 46.42 minutes ( table 1 , fig .
anopheles dthali was tolerant to ddt and propoxur whereas susceptible to deltamethrin , lamda - cyhalotrin , cyfluthrin and malathion ( table 2 , fig .
the lt50 and lt90 values to ddt 4 % were 17.86 and 48.42 minutes ( table 1 ) .
regression line parameters of various species exposed to ddt 4% in chabahar county , sistan and baluchistan province , southeastern iran , 2010 b se= slope and its standard error .
lt50 , 95 % ci= lethal time causing 50 % mortality and its 95 % confidence interval .
lt90 , 95 % ci= lethal time causing 90 % mortality and its 95 % confidence interval .
susceptibility level of various culicidae mosquitoes exposed to some insecticides in chabahar county , sistan and baluchistan province , southeastern iran , 2010 mortality rate error bar r resistance , s susceptible , t tolerance regression line of adult culicidae mosquitoes exposed to ddt 4 % in chabahar county , sistan and baluchistan province 2010 it is concluded that cx .
pipiens is quite resistance to ddt , propoxur , lambda - cyhalothrin , and cyfluthrin whereas susceptible to malathion ( table 2 , fig .
caspius found to be resistant to ddt whereas susceptible to other insecticides ( table 2 , fig .
the lt50 and lt90 values to ddt 4 % were 44.68 , and 164.01 minutes ( table 1 ) .
culiseta longiaerolata observed tolerant to ddt , propoxur , cyfuthrin , lambda - cyhalothrin whereas susceptible to deltamethrin ( table 2 , fig .
the lt50 and lt90 values to ddt 4 % were 17.82 and 51.26 minutes ( table 1 ) .
in our study , 4 genera and 9 species of mosquito larvae and adults were identified based on morphological characters .
culicidae species were belongs to the genus of anopheles , culex , culisita and ochleratatus . the species of cx .
( 2004c ) reported three biological forms of this species including type , intermediate and mysorensis in southern iran .
type and intermediate forms cited as vector in urban areas whereas , mysorensis form as vector in rural area ( vatandoost et al .
1976a , 1976b ) and subsequently to dieldrin in 19609 ( mofidi and saminmi 1960 ) and matlathion in 1976 ( eshghy 1978 ) . following the emergence of resistance of anopheles stephensi to ddt , other organophosphours , carbamate and pyrethroid insecticides were used .
stephensi to ddt and dieldrin was studied at various parts of iran bordered in persian gulf and oman sea during 198588 .
the results revealed that this species was resistant to ddt and it is quite susceptible to dieldrin ( manouchehri et al .
, the results showed that this species was resistant to ddt ( eshghy and janbakhsh 1976 ) .
stephensi was susceptible to bendiocarb , propoxur , malathion , fenitrothion , deltamethrin , permethrin , cyfluthrin and lambda - cyhalothrin , resistance to ddt and tolerant to dieldrin in hormozgan province , south of iran .
stephensi of bandar abbas strain , showed a relatively high adult resistance of 4.66-fold ( lt50= 108min ) to 4 % ddt ( davari et al .
2007 ) . in our study , the lt50 values of ddt 4.0 % against this species were 29.7 minutes , a manifestation of ddt resistance .
stephensi , the main malaria vector , resistant to pyrethroid insecticides in southeastern iran ( vatandoost and hanafi - bojd 2012 ) .
anopheles culicifacies comprised of five sibling species of a , b , c , d and e in north and south of india reported as a and b , in north east found d associated sympatric with a and b whereas in central area reported all of sibling species ( goswami et al .
polytene chromosome examination has been reported only available method that able to differentiate four members of this complex in areas where species e is not prevalent ( goswami et al .
species a has been reported in oman and iran ( zaim and javaherian 1991 ) .
this species is known as vector of malaria in east afghanistan ( zahar 1990 ) . in our study ,
in contrast , ddt resistance reported for the first time after residual spraying in iran by vatandoost et al .
culicifacies to pyrethroid and irritability to fenitrothion , cyfluthrin , and permethrin while high extremely resistance to ddt .
culicifacies in afghanistan , and also resistance to dieldrin , malathion and ddt in india ( goswami et al . 2006 ) . in our study ,
dthali found tolerant to ddt and propoxur while susceptible to deltamethrin , lambda - cyhalothrin , cyfluthrin and malathion .
in contrast , there are also some reports emphasizing to susceptibility levels of this species in different countries ( who 1992 , hanafibojd et al . 2007 ) . in spite , the existence of many reports about to susceptibility of an .
dthali to insecticides , resistance reported to malathion , chloropyriphos , bromophos , carbamate in egypt and temephos in jordan ( who 1992 ) . in our research , cx .
pipiens larvae with 1073 ( 51.14% ) collection , was found one of the most predominant species . in adult collection ,
quinquefasciatus found as 4.82 % of total collection gjullin and peters ( 1952 ) cited decrease of susceptibility level of cx .
pipiens was quite resistance to ddt , propoxur , lambda - cyhalothrin , and cyfluthrin , whereas tolerant to deltamethrin and susceptible to malathion . in parallel , there are many reports in relation to some organophosphorus and pyrethroid insecticides resistance in tunisia ( ben cheikh et al .
1991 ) , burkina faso ( chandre et al . 1998 ) , saudi arabia ( amin and hemingway 1998 ) and china ( jinfu 1999 ) .
lt50 value for propoxur and malathion calculated as 51 , and 31 minutes respectively ( nazari and janbakhsh 2000 ) . the species reported quit susceptible to dieldrin , propoxur and malathion ( nazari and janbakhsh 2000 ) . in our study , cx .
pipiens was highly resistance to ddt , propoxur , cyfluthrin and lambda - cyhalothrin as evidenced by the discriminating concentrations of times .
the results may be related to the present of the species complex , application of irs and distribution of llins in southern iran . in the present study , oc .
longiaerolata quite resistance to ddt and susceptible to other insecticides the lt50 and lt90 values to ddt 4 % were 17.82 and 50.26 minutes .
based on the literature , no reports were available on the susceptibility levels of oc .
iran is in the malaria elimination stage ( who 2011 ) . by now , irs in human dwellings and animal shelters , space - spraying , personal protection through distribution of llins and curtains ( icns ) , repellents measures used to control of malaria vectors in iran .
in addition , some biological and chemical agents against larval and adult stages of culicidae mosquitoes had been evaluated in the laboratory .
results obtained from susceptibility tests of the malaria vectors on ddt revealed that only an .
stephensi was highly resistance to ddt in chabahar area , precautionary measures should be taken in future vector control operations .
moreover , the status of resistance in other locations in malaria endemic area should be investigated . since the country relies on deltamethrin for irs operation , tolerant populations of cx .
pipiens implies careful consideration and regular monitoring of susceptibility level of mosquitoes in the future . | background : according to the national strategy plan on monitoring of insecticides resistance , this study was carried out to determine the base line susceptibility of the culicidae mosquitoes to the who - recommended insecticides in an endemic focus of malaria in southeastern iran.methods:larval collection was carried out by dipping method and adult collection occurred by suction tube from january to december 2010 .
the susceptibility test was assessed to ddt 4 % , malathion 5 % , propoxur 0.1 % , deltamethrin 0.05 % , lambda - cyhalothrin 0.05 % , and cyfluthrin 0.15 % at different interval times ( discriminative dose ) followed by 24 h recovery period .
the lt50 and lt90 values were calculated for plotting the regression line using microsoft office excel software ver .
2007.results:anopheles stephensi was quite resistant to ddt and showed susceptible or tolerant to other insecticides .
the lt50 and lt90 values to ddt in this species were 29.07 , and 98.26 minutes , respectively .
anopheles culicifacies and anopheles dthali were found susceptible or tolerant to insecticides .
culex pipiens was found resistance to ddt , propoxur , lambda - cyhalothrin and cyfluthrin whereas observed susceptible to malathion and tolerant to deltamethrin .
ochlerotatus caspius sl . was resistant to ddt , whereas found susceptible to other insecticides .
culisita longiareolata was susceptible to deltamethrin , whereas tolerant to other insecticides .
the lt50 and lt90 values of cs .
longiareolata to ddt were 17.82 , and 51.26 minutes.conclusion:we suggested the same study in different parts of the country for monitoring and evaluation of control measures . |
adolescent obesity is a major public health problem in the united states , with prevalence rates disproportionately high among certain youth populations . non - hispanic black and hispanic children and adolescents are consistently more likely to be obese ( body mass index [ bmi ] 95% ) compared with non - hispanic white youth .
this pattern is seen in both adolescent males ( hispanic 26.5% , non - hispanic black 22.6% , and non - hispanic white 17.5% ) and females ( hispanic 19.8% , non - hispanic black 24.8% , and non - hispanic white 14.7% ) aged 12 to 19 years .
obesity prevalence is overall significantly higher among males ( 16.1% ) than among females ( 11.7% ) , although this difference by sex is not significant in hispanic or non - hispanic black children and adolescents .
the lack of daily - recommended levels of adolescent physical activity ( pa ) , particularly among girls and members of ethnic populations , has been seen . a comprehensive review of 108 studies revealed that ethnic minority children were as active as non - hispanic whites ; however , non - hispanic whites were more physically active as adolescents than other ethnic groups .
despite the health benefits of pa , levels of inactivity are increasing among all youth over time .
this trend is disturbing because youth pa is a predictor of sport participation and physical fitness activities during early adulthood .
while youth from all backgrounds generally fail to meet national pa standards , low - income adolescents are less likely to adhere to these guidelines than their counterparts . understanding the impact of sports participation is essential since time for school physical education
declines in sport participation are particularly significant among girls and boys during the transition from middle school to high school .
youth from higher income families are more likely to participate in out - of - school sport programs than youth from lower income families , leaving lower income children particularly vulnerable to reduction in pa during school .
since physical inactivity and sedentary behavior are important risk factors for overweight and obesity in adolescence , considerable research attention is focused on creating policies that encourage pa and sport participation .
studies demonstrate that pa is positively associated with healthy weight , but the relationship between adolescent sport participation and obesity can be influenced by factors such as sociodemographic or the built environment ; thus , further research is necessary .
identifying associations between sport participation , pa , and patterns of obesity in adolescents is vital for the development of effective intervention strategies .
research on sport participation that explored activity involvement among early adolescents includes team sports , school / community clubs , and organizational activities .
students involved in organized sports alone or in combination with other activities had significantly higher odds for exercise and healthy self - image .
although the benefits of sport participation are clear , few studies report on sports program participation by weight status for adolescents . furthermore , race , gender , and socioeconomic status ( ses ) are rarely compared in sport participation studies of overweight and obese adolescents .
for example , in a study of australian adolescents , obese adolescents spent considerably less time than their normal - weight peers on team sports . over the past 3 decades
, scholars and public health officials have been challenged to understand the pronounced reduction in pa once children reach adolescence .
consequently , examining sport participation in this population is important , especially for adolescent males , a group whose obesity prevalence has steadily risen in recent years despite girls having reached a plateau . notwithstanding these differences among girls and boys , national health and nutrition examination survey ( nhanes )
data over the past 2 decades show a dramatic increase in overweight and obesity prevalence across all population groups and a declining disparity of obesity across ses .
this article uses a nationally representative sample of 12- to 19-year - old us adolescents to identify the relationship between obesity and leisure time sport participation , with a focus on differences by race , ethnicity , household income , and sex .
we used the nhanes from years 1999 to 2006 ; the activity portion of nhanes was discontinued in 2006 .
nhanes is a stratified , multistage probability sample of the civilian , noninstitutionalized us population .
it includes an in - home questionnaire on several demographic and health - related topics , a computer - assisted interview , and an examination component consisting of a thorough physical exam including measured height and weight .
we included 9152 adolescents age 12 to 19 years . because our goal was to identify adolescents in middle or high school
whose activities were most likely to be related to leisure time sport participation , we excluded 1220 who were not enrolled in school ( most were 18 - 19 years ) , 754 enrolled in college , and 513 in elementary school , for a final sample of 6675 ( n = 3345 females and 3330 males ) .
children aged 12 years and older were eligible for the survey portion that collected data on their participation in specific activities . they self - reported all activities during the previous 30 days , the number of times they participated in the activity , the average duration , and average intensity . nhanes does not provide information on whether the activity was part of an organized sports team .
so we chose to identify adolescents who listed their most frequent activity as 1 of the 5 most popular sports for high school athletes .
although some adolescents may have been classified as participating in a sport that were not in a school - based team or organized program outside of school , we can capture differences based on their activities using the frequency described below .
to operationalize participation in leisure time sports , we first identified the most common sports according to the national federation of state high school associations , based on total number of participants .
for girls these are running / track , * basketball , volleyball , soccer , and softball ; and for boys , football , running / track , basketball , baseball , and soccer . in order to create mutually exclusive categorizing , and to maximize the likelihood that reported pa was part of sport participation , we then assigned each individual to their most frequent common sport based on this hierarchy : ( a ) most amount of total time spent playing the sport , ( b ) most amount of vigorous time ( defined in nhanes as activity that causes heavy sweating or large increases in breathing or heart rate ) spent playing the sport , and ( c ) number of unique occasions playing the sport . for the small number of adolescents who participated in 2 sports equally using all criteria ( n = 84 , 1.2% of the sample ) , we assigned their sport based on the overall popularity ( eg , a boy playing equal amounts of football and basketball was assigned to football ) .
nonathlete / participant - in - other - activities , although it is possible they participated in another , less common , sport .
adolescents were categorized as obese , overweight , or healthy weight based on the bmi percentiles and current centers for disease control and prevention recommendations .
we used height and weight as measured during the examination component to calculate bmi percentile , using sas code developed for that purpose .
because of concerns that athletes with relatively low adiposity may be considered overweight based on standard definitions , we compared those who were obese to the combined group of overweight and healthy weight adolescents .
this is consistent with previous research that suggests participants in sports like american football and rugby tend to be more muscular , and thus a high bmi in these players does not necessarily reflect excessive adiposity .
race and ethnicity were categorized as non - hispanic white , non - hispanic black , hispanic , or other race .
income was based on the percentage of federal poverty level ( fpl ) of household income , from < 100% ( most deprived ) to 500% ( most affluent ) .
grade was categorized as middle school ( grades 6 - 8 ) and high school ( grades 9 - 12 ) .
we used adjusted wald tests to examine the differences in the prevalence of obesity by organized sport participation for girls and boys separately . because demographic factors such as race , ethnicity , and ses likely affect sports participation , we used logistic regression analyses to examine the relationship between sport participation and obesity , controlling for child s school level , race / ethnicity , and income .
all analyses were adjusted for the complex survey design of nhanes , as recommended by nchs .
analyses were performed using the survey estimation routines in stata 12.0 ( college station , tx ) . because this study used only de - identified secondary data , it was deemed exempt from further review by the institutional review board under federal regulation 45 cfr
table 1 shows the demographic characteristics of the sample , stratified by sex . there were no significant differences by sex for race , income , grade , or age . for girls , 48% did not participate in the 5 most common female high school sports ( table 2 ) , whereas only 29% of boys did not participate in the 5 most common male high school sports ( table 3 ) .
white girls ( table 2 ) were more likely to play softball or volleyball , while black girls were more likely to play basketball , and hispanic females more likely to play soccer .
higher income girls were more likely to play softball , and lower income girls were more likely to play basketball ; participation did not otherwise differ by income .
percentage of girls ( n = 3345 ) playing each sport , by demographic characteristics .
percentage of boys ( n = 3330 ) playing each sport , by demographic characteristics .
p value represents adjusted wald test for difference in participation by demographics . overall , black males ( table 3 ) were more likely than white males to participate in 1 of the 5 most common high school sports and were most likely to play basketball .
white males were more likely to play baseball or run track , and hispanic males were more likely to play soccer .
the only differences in income were that higher income boys were more likely to play baseball , while lower income boys were more likely to play basketball .
although leisure time sport participation declined from middle school to high school , football was the only sport for which this decline was statistically significant .
there were few differences in leisure time sport participation by weight for girls ( table 4 ) .
the prevalence of obesity did not differ overall among those who played organized sports compared to those who did not .
girls who reported they participate in running / track had a significantly lower prevalence of obesity than girls who did not participate in any leisure time sport activity .
in contrast , the prevalence of obesity among male nonathletes / participants - in - other - activities was significantly higher than among boys who did .
this finding was consistent for male baseball , basketball , running / track , and soccer athletes , although the prevalence of obesity among football players did not differ from nonathletes / participants - in - other - activities .
percentage of girls and boys who are obese ( body mass index > 95th percentile ) , by sport .
p value represents adjusted wald test comparing each sport to the population playing no sport / participant - in - other - activities .
girls were no more likely to be obese when playing any common sport , compared to female nonathletes / participants - in - other - activities .
girls participating in running / track were less likely to be obese , controlling for grade , race , and income .
however , lower income was significantly associated with greater likelihood of being obese for girls ( or = 3.17 for < 100% fpl ) .
male athletes were significantly less likely to be obese compared to male nonathletes / participants - in - other - activities , except for football players . in contrast to girls , income was not significantly associated with greater odds of obesity among boys ( see table 5 ) .
the results of this study are consistent with national trends for declining levels of pa among adolescent youth entering high school . while an estimated 30 to 45 million american youth play recreational and competitive organized sports , nearly two thirds of youth are estimated to withdraw from sport participation each year , with attrition rates being particularly high among adolescents . additionally , the results of this study correspond with national trends for sport participation among adolescent girls . while pa is important for the health of youth , adolescent girls reduce participation in sports more than adolescent boys in high school .
our analysis shows that rates of participation in the 5 most popular sports was 16.6% lower for girls in high school compared to middle school .
one potential explanation for the drop in sport participation among adolescent girls may be related to the timing of puberty and menarche .
prior research found that advanced pubertal girls had significantly lower self - reported pa and accumulated fewer minutes of moderate - to - vigorous and vigorous pa and accelerometer counts per day at age 13 years than girls that physically mature later
. this may be particularly important for girls who are overweight or obese , as they are more likely to begin puberty earlier than their healthy - weight peers .
however , we found virtually no differences in obesity prevalence between most common sports and nonathletes / participants - in - other - activities in girls . our analysis does show that runners are significantly less likely to be obese than their peers who play the most popular organized female team sports .
the differences in obesity rates among runners and 4 other organized team sport athletes are particularly important since previous research indicates that adolescent girls achieve pa primarily through sports .
it is difficult to surmise why obesity prevalence among adolescent girls who play basketball , softball , soccer , and volleyball is similar to nonathletes / participants - in - other - activities .
one explanation may be that these sports do not provide each participant with the sufficient level of daily moderate to vigorous pa .
the us department of health and human services recommends children and adolescents aged 6 to 17 should have 60 minutes of activity or more of pa each day , which includes moderate or vigorous intense aerobic exercise at least 3 days per week . by grade 12 only 32% of females
meet the recommended levels of pa , and less when using objectively measured activity . whereas running / track can be an individual activity within an organized team , the 4 remaining sports examined in this study are group - based activities , which might not offer adequate levels of vigorous pa for each team member .
perhaps core team members receive significantly higher levels of moderate to vigorous pa than injured athletes or teammates who are afforded less practice or game time
. another consideration may be that females with lower bmi percentile select to participate in track rather than the 4 remaining popular sports .
compared with income and racial differences , the type of leisure time sports adolescent boys played had the strongest , most consistent inverse relationship with obesity , except for football .
consistent with other research , obesity prevalence for football players was comparable to obesity prevalence of nonathletes / participants - in - other - activities .
the self - selection of heavier adolescents into playing football may contribute to the findings that their risk of obesity is equal to male nonathletes / participants - in - other - activities .
. a higher than normal bmi could potentially represent greater muscle density rather than fat .
an examination of 71 males from different high school football players determined the use of age - adjusted bmi percentile can lead to misclassification of overweight and obese status especially for bigger lineman do have muscular body composition .
further studies conclude that bmi calculations from body mass and weight may not accurately portray the modern nfl athlete , division - i ncaa , and high school football players regarding health status because bmi calculations do not distinguish between fat and lean tissue .
we are unaware of any existing longitudinal research or studies consisting of a larger sample size that examines the accuracy of bmi for high school aged or younger football players and our dichotomy of healthy weight versus obese ( as opposed to overweight ) should help with bmi accuracy .
as expected , boys who played baseball , basketball , soccer , and running / track had significantly lower obesity . aside from football , the obesity among male athletes is significantly lower than nonathletes / participants - in - other - activities .
similar to the female population in this study , 7% fewer boys played leisure time sport activity in high school , compared to middle school .
researchers estimate that 3.5 million kids play football in youth leagues ; that number dwindles to 1.3 million in high school .
boys who spent their childhood playing in soccer leagues may find other activities more appealing as teenagers .
other reasons for attrition cited by soccer players include a lack of free time and the need for more time to study .
consistent with studies measuring pa among adolescent females , household income was not associated with a decline in leisure time sport activity .
as family income levels increased , the girls in our study were more likely to participate in basketball , running / track , softball , and soccer .
however , differences in family income did not have a significant impact on volleyball participation . among the factors cited for influencing girls sport participation
is a lack of access , decreased quality of experience including poor facilities and nonoptimal playing times , and a shortage of positive role models . perhaps any one or all of these factors combined with other environmental issues may have a greater impact on participation rates for certain sports such as volleyball .
the present study proposes that household income has a greater impact on differences in female adolescent obesity than sport participation or race .
previous research suggests the prevalence of obesity among black girls was not related to household income , while the prevalence of obesity among white females was significantly higher in lowest income category than at higher income levels .
conversely , participation in sport participation has a bigger impact on obesity for boys than race or family income .
this income paradox may be explained by school - level income ( schools with median incomes that are substantially lower than the national or state median incomes ) or other environmental characteristics .
while previous studies have shown a robust association between household income and pa , richmond and colleagues found that after taking into account characteristics of the school that adolescent attended , individual household income was no longer significantly associated with adolescent pa in either males or females .
our data show that a greater proportion of male youth reporting leisure time sport participation are healthy weight than are female sport participants .
obesity prevalence is significantly lower among male athletes than nonathletes / participants - in - other - activities in all sports except football .
we are unaware of the reasons why differences in obesity among athletes persist along gender lines .
possibly , healthy weight boys self - select to play common sports at a higher rate than their female peers .
previous research has determined that certain women do not report weight management to be significantly important for sport and exercise participation .
it is also possible that overweight or obese girls may be encouraged to exercise more than overweight or obese boys .
our findings do not corroborate previous reports of an association between race / ethnicity , sex , sports participation , and the prevalence of obesity .
as expected , our results did indicate that football players and black and hispanic males were at greater odds of being obese than their white male counterparts who played the other popular sports , but these results were not statistically significant .
one significant limitation of these findings is that we are unable to determine if reports of sport participation were based on organized teams and leagues , or if they represent unorganized activities .
it is possible that levels of pa ( low , moderate , and vigorous ) differ when adolescents play unstructured games with friends rather than compete in organized leagues .
a second limitation is that the study relies on self - reports of pa , which suffer from significant bias .
estimating pa frequency and duration is believed to be particularly challenging for children . also , given that with nhanes one can only examine relationships cross - sectionally , it is not clear whether weight status influences organized sports participation or vice versa . to our knowledge , this is the first study to use multivariate analysis to examine the impact of leisure time sport activity , race , and household income on the likelihood of obesity .
our findings are that , on average , across a nationally representative sample , male and female adolescences who participate in common sports have lower rates of obesity than nonathletes / participants - in - other - activities . however , the effect of leisure time sport activity on obesity for female adolescents is not significant except for the negative relationship between obesity and running / track .
these results suggest adolescent athletes who participate in the 4 most popular organized female team sports may benefit from increased levels of vigorous activity .
physicians who counsel parents and girls toward sport participation should recognize that while leisure time sport activity may have significant benefits , the relationship between participation and healthy weight is not as clear .
additionally , nationally represented surveys such as nhanes will benefit by collecting future data on pa , recreational sport participation , and organized sport participation among adolescents . | objective . to understand the relationships between participation in different types of leisure time sport activity and adolescent obesity , and how those relationships might differ based on race , gender , and household income . methods .
data consisted of 6667 students that took part in the 1999 to 2006 national health and nutrition examination survey .
the authors used adjusted wald tests to examine differences in the prevalence of obesity ( body mass index > 95th percentile for age and sex ) by sport for boys and girls separately . results . among adolescent youth age 12 to 19 years , 16.6% of male leisure time sport participants and 15.3% of female sport participants were obese , compared with 23.6% for male nonathlete participant - in - other - activities and 17.0% obesity rate for female nonathlete / participant - in - other - activities .
for both males and females , reported participation in leisure time sports decreased between middle school and high school , and this reduction was associated with higher body mass index . |
it is believed that the number of handicapped individuals is increasing in proportion to the general population .
children and adolescents with disabilities appear to have poorer oral health than their non - disabled counterparts .
variable access to dental care , inadequate oral hygiene and disability - related factors may account for differences .
a study on dental health and dental care requirements of young handicapped adults in wessex demonstrated that the amount of untreated caries was higher and the periodontal status was worse in this group than would normally be expected in young adults in this age range .
arnrup demonstrated that gingivitis , disturbances in occlusal development and dental caries were the most commonly found diagnoses in medically compromised children .
the objectives of the present study on a selected group of disabled children in mashhad were ( 1 ) to determine the frequency and severity of their oral disease , ( 2 ) assess their treatment needs with reference to dental caries , periodontal disease and malocclusion , and ( 3 ) make recommendations for public dental health care for individuals with disabilities .
the present study was conducted between september and march 1996 , in 13 special schools in different socio - economic areas in mashhad , iran . from 3000 children attending special schools , 1621 individuals ( 609 females and 1012 males ) 5 to 16 years old , were eligible and examined .
all examinations were performed at schools while children were seated on a chair with a fixed artificial light directed towards the mouth .
the diagnosis of dental caries was undertaken by visual examination using a dental mirror and an explorer utilizing the criteria recommended by world health organization .
indices used to measure caries experience include dmft + dmft and dental treatment needs . the ohi - s by green & vermillion was used to assess oral hygiene .
this index has two components : the debris index ( di - s ) and the calculus index ( ci - s ) .
six surfaces from six teeth ( one surface of each tooth ) are examined to determine ohi - score :
debris and calculus scores were obtained through examining the buccal or lingual surface of the teeth .
the gingival condition of each case was scored using the gingival index ( jackson & james ) .
occlusion was recorded according to the angle classification system and in cases of primary dentition , it was recorded according to the terminal plane of the second primary molars .
the dental treatment needs of each child was assessed based on the following categories : none : no restorative treatment required ; simple : preventive treatment required , such as scaling and brushing , oral hygiene instruction ( ohi ) , application of topical fluoride and fissure sealant ; moderate : one or more teeth require one or two surface restoration ; and complex : one or more teeth require a three or four surface restoration / stainless steel crown , endodontic therapy and crown , and/or extraction .
the recommendations were based on dental treatment needs , classified as follows : no treatment required : child required no dental or periodontal treatment , but required assistance with oral hygiene ; simple treatment need : child required simple preventive or periodontal treatment ; moderate treatment need : child required moderate dental and/or periodontal treatment ; and complex treatment need : child required complex dental and/or periodontal treatment .
the present study was conducted between september and march 1996 , in 13 special schools in different socio - economic areas in mashhad , iran . from 3000 children attending special schools , 1621 individuals ( 609 females and 1012 males ) 5 to 16 years old , were eligible and examined .
all examinations were performed at schools while children were seated on a chair with a fixed artificial light directed towards the mouth .
the diagnosis of dental caries was undertaken by visual examination using a dental mirror and an explorer utilizing the criteria recommended by world health organization .
indices used to measure caries experience include dmft + dmft and dental treatment needs . the ohi - s by green & vermillion was used to assess oral hygiene .
this index has two components : the debris index ( di - s ) and the calculus index ( ci - s ) .
six surfaces from six teeth ( one surface of each tooth ) are examined to determine ohi - score :
debris and calculus scores were obtained through examining the buccal or lingual surface of the teeth .
the gingival condition of each case was scored using the gingival index ( jackson & james ) .
occlusion was recorded according to the angle classification system and in cases of primary dentition , it was recorded according to the terminal plane of the second primary molars .
the dental treatment needs of each child was assessed based on the following categories : none : no restorative treatment required ; simple : preventive treatment required , such as scaling and brushing , oral hygiene instruction ( ohi ) , application of topical fluoride and fissure sealant ; moderate : one or more teeth require one or two surface restoration ; and complex : one or more teeth require a three or four surface restoration / stainless steel crown , endodontic therapy and crown , and/or extraction .
the recommendations were based on dental treatment needs , classified as follows : no treatment required : child required no dental or periodontal treatment , but required assistance with oral hygiene ; simple treatment need : child required simple preventive or periodontal treatment ; moderate treatment need : child required moderate dental and/or periodontal treatment ; and complex treatment need : child required complex dental and/or periodontal treatment .
more than one half of the study populations ( 60% ) were mentally retarded .
the caries experience of children with hearing impairment was lower than that of the children with mental retardation and visual impairment ( d+d : 1.51 , 2.05 , and 2.28 , respectively ; dmft + dmft : 1.91 2 versus 2.27 1.90 and 2.68 2.30 ) . the filled component in mr children was lower than vi and hi children ( f+f : 0.077 , 0.14 , and 0.18 , respectively ) .
the number of missing teeth due to caries in mr children was
also lower than the hi and vi children ( m+m : 0.14 , 0.20 , and 0.24 , respectively ) .
the mean fd , fd indices for mr children was lower than those of vi and hi children ( mr = 0.037 , vi = 0.065 , and hi = 0.12 ) , indicating lower amount of restorative treatment relative to untreated caries in mr children .
the decayed component of mean dmft+dmft index was the largest component of the index in all three groups .
gingival index was fair in most children ( 66.37% ) and mentally retarded children had the worst status among the other groups .
di - s in mr children ( 67.29% ) and ci - s in hi children ( 5% ) were higher than the other group .
there was a significant difference in the distribution of the angle classification of occlusion , with a higher percentage of class i malocclusion in the handicapped subjects .
more than one half of the study populations ( 60% ) were mentally retarded .
the caries experience of children with hearing impairment was lower than that of the children with mental retardation and visual impairment ( d+d : 1.51 , 2.05 , and 2.28 , respectively ; dmft + dmft : 1.91 2 versus 2.27 1.90 and 2.68 2.30 ) .
the filled component in mr children was lower than vi and hi children ( f+f : 0.077 , 0.14 , and 0.18 , respectively ) . the number of missing teeth due to caries in mr children was
also lower than the hi and vi children ( m+m : 0.14 , 0.20 , and 0.24 , respectively ) .
the mean fd , fd indices for mr children was lower than those of vi and hi children ( mr = 0.037 , vi = 0.065 , and hi = 0.12 ) , indicating lower amount of restorative treatment relative to untreated caries in mr children . the decayed component of mean dmft+dmft index was the largest component of the index in all three groups .
gingival index was fair in most children ( 66.37% ) and mentally retarded children had the worst status among the other groups .
di - s in mr children ( 67.29% ) and ci - s in hi children ( 5% ) were higher than the other group .
there was a significant difference in the distribution of the angle classification of occlusion , with a higher percentage of class i malocclusion in the handicapped subjects .
a number of surveys and controlled studies have been conducted to determine the effect of some disabilities on the prevalence of oral diseases .
the mean dmft + dmft of 2.2 2.8 was reported by mala desai for 9 - 13-year - old children with disabilities .
this finding is consistent with our study ( mean dmft + dmft = 2.2 2 ) in which the decayed component was the major contributor to the dmf + dmf index .
nunn et al reported mean deciduous caries experience ( dft ) to be 0.9 and the mean permanent caries experience ( dmft ) 2.0 in a study on physically handicapped children in 1993 .
alavi et al reported mean dmft of 9.64 4.64 in diabetic children in 2006 .
dmft score and the frequency of decayed teeth were higher in boys than in girls .
our study showed that a considerable number of hi children ( 34% ) were caries free .
mean deciduous caries experience ( dft ) was 0.23 and the mean permanent caries experience ( dmft ) was 5.69 . the differences between our study and nunn 's can be attributed to variations in age and number of the cases ( 5 - 16 year olds versus 3 - 17 year olds ; 1621 versus 129 , respectively ) .
ivancic reported that the average dmft index in disabled children was 1.14 for mixed and 6.39 for permanent dentition .
they suggested reorganizing preventive care measurements and improving dental care for disabled children .
in this study ,
the dental caries prevalence in vi subjects was higher than mr and hi subjects , which is contrary to swallows results .
the lower prevalence of caries in hearing impaired subjects suggests that hi children have higher ability to learn oral hygiene in comparison with other groups .
in the present study the mean dmft of 12-year - old children with disabilities ( 2.59 ) was higher than those of the mala desai and nunn studies , but lower than the who goal of 3.0 for year 2000 .
al - qahtani reported that all 6 - 7-year - old blind children had caries with a mean dmft of 6.58 .
caries prevalence in 11 - 12-year - old blind children was 88.2% with a mean dmft of 3.89 .
a good oral hygiene was found in 8.3% of 6 - 7-year - old , and 29.4% of 11 - 12-year - old blind children .
the caries prevalence in the 6 - 7-year - old deaf children was 95.7% with a mean dmft score of 7.35 .
the caries prevalence in 11 - 12-year - old deaf children was 93% with a mean dmft of 5.12 .
less than one - fifth ( 17.4% ) of the 6 - 7-year - old deaf children and only 7.0% of 11 - 12-year - old deaf children had good oral hygiene .
the caries prevalence in 6 - 7-year - old mentally retarded children was 93.9% with a mean dmft of 8.00 .
all of the 11 - 12-year - old mentally retarded children had carious teeth with a mean dmft of 5.81 .
only 3.1% of 6 - 7-year - old and none of the 11 - 12-year - old mentally retarded population had good oral hygiene .
this has been supported by several studies.1
,
14 the preventive and restorative treatment needs of many children in the present study were unmet .
contributory factors include inadequate funding and resources , insufficient trained dentists to treat patients with disabilities , and complex treatment needs requiring special care or general anaesthesia .
the average individual or group di - s and ci - s scores are combined to obtain the simplified oral hygiene index ( ohi - s ) . in this study children had poor or fair ( 44% and % 42 ) di - s , but ci - s index was very low , even zero in some cases .
poorer oral hygiene and gingival health in the studied children are consistent with the findings of other studies , which can be attributed to environmental , systemic and local factors .17
,
10
,
16 local factors such as malocclusion , lack of normal masticatory functions , and attrition due to bruxism were more prevalent in those with severe disabilities ; these have been suggested to contribute to periodontal disease .
therefore , optimal oral hygiene is of particular importance in this group in order to limit disease onset .
the proportion of children requiring periodontal treatment in the present study was very high ( 74% ) and the major needs were prophylaxis plus oral hygiene advice .
malocclusion can complicate the child 's disability , resulting in dental trauma ( e.g. a large overjet predisposes the children to trauma in those with seizures ) , periodontal disease ( promoted by crowding or eruption problems ) , functional problems ( mastication , drooling ) , speech impairment and even temporomandibular joint dysfunction.16
,
18
according to this study , following recommendations are made concerning the public dental health care of children and adolescent with disabilities :
greater coordinated efforts should be made by the dental , medical , and social services to serve their needs .
individualized recall visits should be introduced to schools by dental teams to perform preventive measures , particularly for those at a high risk of caries and/or periodontal diseases .
trained dental auxiliaries should be utilized . following the examination , diagnosis and treatment planning by a dentist ,
a trained auxiliary could provide preventive care , simple restorations , fissure sealants , oral hygiene instructions and oral health promotion .
access to general practitioners , pedodontists , and orthodontists should be improved , and referral mechanisms established whereby high risk children can obtain priority treatment .
campaigns should be launched to decrease dental disease , through changing the school diet to limit cariogenic intake , instituting preventive training programs for staff , and advocating public health programs .
education on the subject of oral health care in disabled individuals should be expanded in dental schools and higher education should be established to upgrade professional knowledge .
a national epidemiological survey should be conducted by experts in public dental health , in order to prepare and implement a long - term public dental health care plan .
the efficacy of the implementation of this plan should be evaluated subsequently in terms of oral health of the recipients .
the rate of caries , periodontal disease and malocclusion in disabled children exceeds that of children without disabilities .
preventive , restorative and periodontal treatment needs are unmet in handicapped children , despite the fact that only 26% of the cases required simple treatment .
following diagnostic examinations and treatment planning by a dentist , a wide range of preventive , restorative and periodontal treatments could be performed by trained dental auxiliaries . an epidemiological survey followed by the implementation and evaluation of a long - range public dental health care plan for children and adolescents with disabilities is highly recommended . | background and aims
children and adolescents with disabilities appear to have poorer oral health than their non - disabled counterparts .
the aim of this study was to assess the frequency and severity of oral diseases and treatment needs using world health organization criteria of caries , periodontal disease and malocclusions in a selected population of children with disabilities in mashhad , iran .
materials and methods
a randomized study on 1621 children aged 5 - 16 was conducted in 13 special schools by six examiners , using a mouth mirror , explorer and enough lighting .
results
the caries frequency of hearing impaired children ( hi ) was lower than those mentally retarded ( mr ) and visually impaired ( vi ) ( dmft : 2 1.91 versus 2.27 1.97 and 2.68 2.30 , respectively ) . mr children appear to have poorer oral hygiene and periodontal status than their otherwise disabled counterparts . most children had class i malocclusion ( 57% ) .
conclusion according to this study , an epidemiological survey followed by the implementation and evaluation of long - term public dental health care plan for children and adolescents with disabilities is highly recommended . |
a total of 22 mycobacterial strains were included in this work . to study the sequence diversity of t cell antigens in mtbc , we used illumina next - generation dna sequencing to generate nearly complete genome sequences from 20 strains representative of the six main human mtbc lineages , and one strain of mycobacterium canettii which is the closest known outgroup of mtbc3,22 ( table 1 ) .
in addition , we used the published genome sequence of the h37rv laboratory strain of m. tuberculosis as a common reference23 . for each of the 21 strains newly sequenced , a mean of 6.8 million sequence reads with a mean length of 51 base pairs were generated and mapped to the h37rv reference genome .
on average , the reads covered 98.9% of the 4.4 mb reference genome ( table 1 ) .
the regions not covered primarily included members of the highly gc - rich and repetitive pe / ppe gene families24 .
a total of 32,745 snps were identified , corresponding to an average of 1 snp call for every 3 kb of sequence generated .
we used a total of 9,037 unique snps ( i.e. snps that occurred in one or several strains ) to derive a genome - wide phylogeny of 22 strains ( fig .
these lineages were completely congruent to the strain groupings previously defined based on genomic deletion analysis and multilocus sequencing3,7,10 .
the perfect congruence between these different phylogenetic markers further corroborates the highly clonal population structure of mtbc and lack of ongoing horizontal gene transfer in this organism25 . because of the comprehensive nature of genome - scale data , a higher degree of phylogenetic resolution
the brown and green lineages ( also known as mycobacterium africanum ) are the most basal groups when compared to the m. canettii outgroup .
however , the fact that the two m. africanum lineages represent the most ancestral forms of human mtbc reinforces the notion that human mtbc originated in africa3,7 .
we used these genome sequence data and the phylogeny derived from them to compare the genetic diversity in antigens and other experimentally determined gene classes . for comparisons across different gene categories
antigens were defined based on the presence of 491 experimentally confirmed human t cell epitopes ( supplementary table 3 ) , which were compiled through the immune epitope database ( iedb ) initiative26 .
essential gene category was defined based on genome - wide analyses of transposon insertion mutants that were defective for the ability to grow on middlebrook 7h11 agar , or in the spleens of intravenously - infected mice , published previously27 - 28 .
we excluded from this analysis genes belonging to the pe / ppe gene family24 and those related to mobile elements as they are difficult to study using current next - generation dna sequencing technologies ( total genes excluded : 273/3,990 ( 6.8% ) genes annotated in the h37rv reference genome ; supplementary table 4 ) .
based on evolutionary theory and findings in other bacteria29 , one would expect that in contrast to non - essential genes , the essential genes in mtbc will be under stronger purifying selection and thus more evolutionary conserved . in support of this notion
, we observed that on average essential genes harboured less nucleotide diversity than non - essential genes ( fig .
we then compared the rates of synonymous and non - synonymous snps in the essential and non - essential gene categories .
the synonymous and non - synonymous changes were derived by comparison to the most likely recent common ancestor of mtbc , which we inferred based on our new genome - wide phylogeny ( fig .
1 ) . because mtbc harbours little sequence diversity , it was necessary to analyze the distribution of synonymous and non - synonymous snps based on gene concatenates rather than individual genes .
the two measures of distribution we used were based on the number of non - redundant snps across all 21 mtbc strains ( dn / ds based on measure a in table 2 and fig .
3 ) , and on the individual pairwise comparisons between each strain and the inferred most likely recent common ancestor ( dn / ds based on measures b in table 2 ) . from these analyses
, we found that the dn / ds measures for essential genes were significantly lower than for non - essential genes ( measure a in fig .
3 ; measure b in table 2 , mann - whitney u test p<0.0001 ) . taken together ,
these data show that in mtbc essential genes are more evolutionary conserved than non - essential genes .
because mtbc interacts with humans through antigen - specific cd4 or cd8 t - cells , we would expect t cell antigens to be among the most diverse genes in the genome .
particularly when invoking a co - evolutionary arms - race and associated immune evasion , we would anticipate these antigens to be under diversifying selection and to be more variable than other genes in order to escape t cell recognition .
however , when we analyzed the nucleotide diversity in 78 experimentally confirmed human t cell antigens ( supplementary table 2 ) , we found that they were on average not more diverse than essential genes ( fig .
moreover , we found that the dn / ds measures in these antigens also resembled those of essential genes ( measure a in fig .
3 ; measure b in table 2 , mann - whitney u test p=0.77 ) . thus
, human t cell antigens in mtbc do not appear to be under diversifying selection .
t cell antigens consist of epitope regions that interact with human t cells , and non - epitope regions which are not targets of t cell recognition .
, we generated a separate concatenate of the epitope regions and another concatenate of all corresponding non - epitope regions . because little data is currently available in the iedb with respect to whether these 491 epitopes are recognized by cd4 or cd8 t cells
if immune escape was driving antigen evolution to evade t cell recognition in mtbc , we would expect non - synonymous changes to accumulate in epitope regions , leading to a high dn / ds .
contrary to this expectation however , the overall dn / ds of the epitope regions was 0.53 , which was still similar to essential genes and lower than non - essential genes ( table 2, fig .
3 ) . moreover , when we analyzed the distribution of amino acid replacements in individual epitopes we found that the large majority ( 95% ) of the 491 epitopes showed no amino acid change ( fig .
only five epitopes , contained in esxh , psts1 , and rv1986 , harboured more than one variable position ( supplementary table 5 ) .
the higher number of amino acid substitutions in these five epitopes may reflect ongoing immune evasion , but further investigation is needed to determine whether the observed changes are due to immune pressure , other selection pressure(s ) , or mere random genetic drift3 . because these five epitopes were clear outliers compared to the large majority of t cell epitopes analyzed here , we repeated our dn / ds analysis after excluding the three antigens harbouring the five outlier epitopes .
our analysis revealed that the epitope regions had the lowest dn / ds of all gene categories ( table 2 , fig .
furthermore , when we compared the proportion of non - redundant non - synonymous changes in epitope and non - epitope regions , we found that epitopes were less likely than non - epitopes to harbour changes at non - synonymous sites ( measure a in table 2 , , p<0.05 ) , whereas no difference was observed at synonymous sites ( table 2 , , p=0.89 ) . to further corroborate our finding of hyperconservation of human t cell epitopes in mtbc
, we repeated our analysis using a data set from a previous study in which 89 individual genes were sequenced in 99 human - adapted strains representative of the six major global lineages of mtbc3 .
sixteen of these 89 genes belonged to the t cell antigens analyzed here , including two of the three outlier antigens esxh and psts1 .
analysis of this additional dataset of 16 antigens in 99 mtbc strains revealed an overall dn / ds for the epitope regions of 0.74 .
however , after excluding the two outlier antigens , the dn / ds dropped to 0.46 , which was again lower than the genome - based dn / ds values for essential and non - essential genes ( fig .
, our findings strongly suggest that a large proportion of the mtbc genome known to interact with human t cells is highly conserved and under as strong , or perhaps even stronger , purifying selection than essential genes .
a total of 22 mycobacterial strains were included in this work . to study the sequence diversity of t cell antigens in mtbc , we used illumina next - generation dna sequencing to generate nearly complete genome sequences from 20 strains representative of the six main human mtbc lineages , and one strain of mycobacterium canettii which is the closest known outgroup of mtbc3,22 ( table 1 ) .
in addition , we used the published genome sequence of the h37rv laboratory strain of m. tuberculosis as a common reference23 . for each of the 21 strains newly sequenced , a mean of 6.8 million sequence reads with a mean length of 51 base pairs were generated and mapped to the h37rv reference genome .
on average , the reads covered 98.9% of the 4.4 mb reference genome ( table 1 ) .
the regions not covered primarily included members of the highly gc - rich and repetitive pe / ppe gene families24 .
a total of 32,745 snps were identified , corresponding to an average of 1 snp call for every 3 kb of sequence generated .
we used a total of 9,037 unique snps ( i.e. snps that occurred in one or several strains ) to derive a genome - wide phylogeny of 22 strains ( fig .
these lineages were completely congruent to the strain groupings previously defined based on genomic deletion analysis and multilocus sequencing3,7,10 .
the perfect congruence between these different phylogenetic markers further corroborates the highly clonal population structure of mtbc and lack of ongoing horizontal gene transfer in this organism25 . because of the comprehensive nature of genome - scale data , a higher degree of phylogenetic resolution
the brown and green lineages ( also known as mycobacterium africanum ) are the most basal groups when compared to the m. canettii outgroup .
however , the fact that the two m. africanum lineages represent the most ancestral forms of human mtbc reinforces the notion that human mtbc originated in africa3,7 .
we used these genome sequence data and the phylogeny derived from them to compare the genetic diversity in antigens and other experimentally determined gene classes . for comparisons across different gene categories
essential genes , non - essential genes , and antigens ( supplementary fig .
antigens were defined based on the presence of 491 experimentally confirmed human t cell epitopes ( supplementary table 3 ) , which were compiled through the immune epitope database ( iedb ) initiative26 .
the essential gene category was defined based on genome - wide analyses of transposon insertion mutants that were defective for the ability to grow on middlebrook 7h11 agar , or in the spleens of intravenously - infected mice , published previously27 - 28 .
we excluded from this analysis genes belonging to the pe / ppe gene family24 and those related to mobile elements as they are difficult to study using current next - generation dna sequencing technologies ( total genes excluded : 273/3,990 ( 6.8% ) genes annotated in the h37rv reference genome ; supplementary table 4 ) .
based on evolutionary theory and findings in other bacteria29 , one would expect that in contrast to non - essential genes , the essential genes in mtbc will be under stronger purifying selection and thus more evolutionary conserved . in support of this notion
, we observed that on average essential genes harboured less nucleotide diversity than non - essential genes ( fig .
we then compared the rates of synonymous and non - synonymous snps in the essential and non - essential gene categories .
the synonymous and non - synonymous changes were derived by comparison to the most likely recent common ancestor of mtbc , which we inferred based on our new genome - wide phylogeny ( fig .
1 ) . because mtbc harbours little sequence diversity , it was necessary to analyze the distribution of synonymous and non - synonymous snps based on gene concatenates rather than individual genes .
the two measures of distribution we used were based on the number of non - redundant snps across all 21 mtbc strains ( dn / ds based on measure a in table 2 and fig .
3 ) , and on the individual pairwise comparisons between each strain and the inferred most likely recent common ancestor ( dn / ds based on measures b in table 2 ) . from these analyses , we found that the dn / ds measures for essential genes were significantly lower than for non - essential genes ( measure a in fig .
3 ; measure b in table 2 , mann - whitney u test p<0.0001 ) . taken together ,
these data show that in mtbc essential genes are more evolutionary conserved than non - essential genes .
because mtbc interacts with humans through antigen - specific cd4 or cd8 t - cells , we would expect t cell antigens to be among the most diverse genes in the genome .
particularly when invoking a co - evolutionary arms - race and associated immune evasion , we would anticipate these antigens to be under diversifying selection and to be more variable than other genes in order to escape t cell recognition .
however , when we analyzed the nucleotide diversity in 78 experimentally confirmed human t cell antigens ( supplementary table 2 ) , we found that they were on average not more diverse than essential genes ( fig .
moreover , we found that the dn / ds measures in these antigens also resembled those of essential genes ( measure a in fig .
3 ; measure b in table 2 , mann - whitney u test p=0.77 ) . thus
, human t cell antigens in mtbc do not appear to be under diversifying selection . instead , purifying selection appears to be the driving selection pressure in these genes .
t cell antigens consist of epitope regions that interact with human t cells , and non - epitope regions which are not targets of t cell recognition .
hence , we decided to study these regions separately . to this end , we generated a separate concatenate of the epitope regions and another concatenate of all corresponding non - epitope regions . because little data is currently available in the iedb with respect to whether these 491 epitopes are recognized by cd4 or cd8 t cells , we analyzed them as one class .
if immune escape was driving antigen evolution to evade t cell recognition in mtbc , we would expect non - synonymous changes to accumulate in epitope regions , leading to a high dn / ds .
contrary to this expectation however , the overall dn / ds of the epitope regions was 0.53 , which was still similar to essential genes and lower than non - essential genes ( table 2, fig .
3 ) . moreover , when we analyzed the distribution of amino acid replacements in individual epitopes we found that the large majority ( 95% ) of the 491 epitopes showed no amino acid change ( fig .
only five epitopes , contained in esxh , psts1 , and rv1986 , harboured more than one variable position ( supplementary table 5 ) .
the higher number of amino acid substitutions in these five epitopes may reflect ongoing immune evasion , but further investigation is needed to determine whether the observed changes are due to immune pressure , other selection pressure(s ) , or mere random genetic drift3 . because these five epitopes were clear outliers compared to the large majority of t cell epitopes analyzed here , we repeated our dn / ds analysis after excluding the three antigens harbouring the five outlier epitopes .
our analysis revealed that the epitope regions had the lowest dn / ds of all gene categories ( table 2 , fig .
furthermore , when we compared the proportion of non - redundant non - synonymous changes in epitope and non - epitope regions , we found that epitopes were less likely than non - epitopes to harbour changes at non - synonymous sites ( measure a in table 2 , , p<0.05 ) , whereas no difference was observed at synonymous sites ( table 2 , , p=0.89 ) . to further corroborate our finding of hyperconservation of human t cell epitopes in mtbc
, we repeated our analysis using a data set from a previous study in which 89 individual genes were sequenced in 99 human - adapted strains representative of the six major global lineages of mtbc3 .
sixteen of these 89 genes belonged to the t cell antigens analyzed here , including two of the three outlier antigens esxh and psts1 .
analysis of this additional dataset of 16 antigens in 99 mtbc strains revealed an overall dn / ds for the epitope regions of 0.74 .
however , after excluding the two outlier antigens , the dn / ds dropped to 0.46 , which was again lower than the genome - based dn / ds values for essential and non - essential genes ( fig .
, our findings strongly suggest that a large proportion of the mtbc genome known to interact with human t cells is highly conserved and under as strong , or perhaps even stronger , purifying selection than essential genes .
in this study of 22 mtbc genomes , we demonstrate that , as expected , essential genes are more conserved than non - essential genes .
surprisingly , however , we found that the large majority of the currently known t cell antigens are as conserved as essential genes .
furthermore , the epitope regions of these antigen genes are the most highly conserved regions we studied . this observation , that the regions of the genome that interact with the human adaptive immune system appear to be under even stronger purifying selection than essential genes , is inconsistent with a classical model of an evolutionary arms - race .
it is possible that the known human t cell epitopes that we found to be hyperconserved represent a select subset of all of the human t cell epitopes encoded in the genome , and that certain approaches to epitope identification have favoured discovery of hyperconserved epitopes in mtbc .
for example , since most , if not all of the epitope discovery efforts to date have utilized proteins and/or peptide sequences of strains from one lineage ( lineage 4 ) and t cells from humans that are likely to have been infected by strains of other lineages , the assays used may have been especially suited to identification of hyperconserved and/or cross - reactive epitopes . while further investigation using alternative approaches to epitope discovery may reveal that variable epitopes that exhibit evidence of positive selection exist in the mtbc , it is likely that the large number of epitopes that we examined will remain a significant subset of the total , and that future vaccine development efforts will need to account for the possibility that immune recognition of certain epitopes may actually provide a net benefit to the bacteria .
lack of antigenic variation and immune evasion has been reported for a number of other human pathogens , including rna viruses such as measles , mumps , rubella , and influenza type c31 .
theoretical studies have suggested that the absence of immune escape variants in these viruses might be due to structural constraints in viral proteins or negative mutational effects leading to reduced infectivity or transmission31 .
while we can not exclude the possibility that structural and functional constraints that are independent of t cell recognition contribute to hyperconservation of the regions encoding mtbc peptides recognized by human t cells , one important characteristic of the aformentioned viral pathogens is that they spread among young and immunologically naive hosts , which might eliminate the need for immune evasion31 .
moreover , infection by these viruses usually results in acute disease , followed by elimination of the infection through adaptive immunity , and acquisition of lifelong immunity against re - infection .
by contrast , mtbc causes chronic and often lifelong infections , and adaptive immunity is usually unable to completely clear the infection18 .
furthermore , tuberculosis patients are prone to re - infection32 , and mixed infections are also increasingly recognized33 .
these observations suggest that the biological basis for the lack of antigenic variation in mtbc reported here differs from what has been proposed for antigenically homogeneous rna viruses31 .
in addition , we determined that the fraction of hyperconserved t cell epitopes of the mtbc that are derived from essential genes is indistinguishable from the frequency of essential genes in the mtbc genome as a whole ( 18% versus 21% , respectively ; = 0.28 , p = 0.59 ) , indicating that our results were not skewed by over - representation of t cell epitopes in essential genes .
moreover , the t cell epitopes that we analyzed are present in genes from diverse gene ontologies , and the representation of five main gene categories ( defined based on the ncbi categories of orthologous groups ( cog ) ) was no different in the t cell antigens when compared to the genome overall ( with 4 degrees of freedom = 5.8 , p = 0.21 ; supplementary table 6 ) .
hence the only identifiable common property of these regions is their recognition by human t lymphocytes .
these findings suggest that t lymphocyte recognition is an important factor in hyperconservation of these sequences , and that other structural or functional constraints are unlikely to fully account for the lack of sequence variation in these domains .
our data suggest that t cell epitopes in mtbc are under strong selection pressure to be maintained , perhaps because the immune response they elicit in humans , which are essential for survival of an individual host , might partially work towards the pathogen s benefit .
one potential mechanism of benefit to mtbc from human t cell recognition is that human t cell responses are essential for mtbc to establish latent infection .
this notion is supported by the fact that cd4 t cell - deficient hiv - positive individuals progress rapidly to active disease after infection , rather than to sustain prolonged periods of latent tuberculosis34 .
latent infection mediated by host t cell responses , with subsequent reactivation to active disease often occurring decades after initial infection , is a key characteristic of human tuberculosis , and might have evolved as a way for mtbc to transmit to later generations of susceptible hosts35 . in addition , there is evidence that t cell responses may contribute directly to human - to - human transmission of mtbc . in particular ,
cavitary tuberculosis , which generates secondary cases more efficiently than other disease forms36 , rarely occurs in cd4 t cell - deficient hiv - positive individuals , and the frequency of cavitary lung lesions in hiv - infected patients with tuberculosis is directly correlated with the number of peripheral cd4 t cells37 .
while the mechanisms of lung cavitation in tuberculosis are poorly understood , these observations suggest that cd4 t cells directly or indirectly mediate tissue damage in tuberculosis , and together with our finding of epitope hyperconservation indicate that certain t cell responses may be detrimental to the host and beneficial to the pathogen .
hence our findings suggest that mtbc takes advantage of host adaptive immunity to increase its likelihood of spread , and that the benefits of enhanced transmission exceed the costs of within - host cellular immune responses to these epitopes . in this manner , mtbc may resemble hiv , for which there is evidence that virulence has evolved , not to maximize replication of the virus within individual hosts , but to maximize the likelihood of its transmission38 . whether t cell responses to other epitopes , or whether specific t cell subsets ( e.g. th17 versus th1 ) that benefit the host and not the bacteria can be identified will require additional studies in humans .
one limitation of this study was the exclusion of pe / ppe genes because of technical reasons .
some of these genes are known to vary and to be cell - surface exposed , which has lead to the hypothesis they might be involved in antigenic variation24 . however , no direct evidence for this has yet been presented .
future work will need to clarify the function and evolution of pe / ppe genes .
by contrast , all the t cell antigens included in this study have been experimentally confirmed26
. furthermore , some of them are being targeted by new tuberculosis diagnostics and vaccines39 .
our findings thus have important implications for the development of these new tools . on the one hand , the fact that mtbc harbours little sequence diversity in t cell antigens will facilitate the development of diagnostics that are universally applicable across geographical regions where mtbc strains differ8 . on the other hand ,
the possibility that the immune responses induced by vaccine antigens might partially benefit the pathogen suggests current efforts in vaccine research should be broadened .
most disturbing is the suggestion that vaccine induced immunity against these conserved epitopes may perversely increase transmission . in this respect , it is interesting to note that the currently available tuberculosis vaccine bacille - calmette - guerin ( bcg ) , which is a live vaccine based on an attenuated from of m. bovis , offers no protection against pulmonary tuberculosis in adults40 .
more importantly , some clinical trials of bcg have even reported an increased risk of tuberculosis in vaccinees compared to unvaccinated individuals41 .
thus , in contrast to standard reverse vaccinology , in which the least variable antigens of a genome are targeted42 , research into new tuberculosis vaccines should explore more variable regions of the mtbc genome . while most of the t cell epitopes anlyzed here were highly conserved , five epitopes in three antigens harboured a larger number of amino acid changes .
the fact that the dn / ds measure dropped sharply after excluding these outlier antigens from the analysis further supports the notion that they are indeed outliers compared to the other antigens .
one of these outlier antigens , esxh ( rv0288 , also known as tb10.4 ) is a member of a gene family known to encode a type vii secretion system43 .
thus even though most of the other vaccine antigens analyzed here are conserved , our finding that this particular vaccine antigen harbours a comparatively high number of amino acid substitutions across a panel of global mtbc isolates , suggests that strain diversity should be considered during further development of the new vaccine candidates containing esxh8 .
we detected significant differences in dn / ds between essential , non - essential , and antigenic genes .
however , the individual dn / ds values remain high when compared to most other bacteria44 .
such a high dn / ds was reported previously for mtbc , and has been linked to reduced selective constraint against slightly deleterious mutations3 .
it was proposed that the serial transmission bottlenecks associated with patient - to - patient transmission in mtbc could lead to an increase in random genetic drift compared to the forces of natural selection .
our new data show that even though the strength of purifying selection in mtbc might be reduced overall compared to other bacteria , it clearly is still acting on and capable of differentiating between gene categories . in summary , we show that t cell epitopes of mtbc are highly conserved , and do not reflect any ongoing evolutionary arms - race or immune - evasion . instead , the patterns observed might be indicative of a distinct evolutionary strategy of immune - subversion developed by this highly successful pathogen . other intracellular bacteria such as salmonella enterica serovar typhi exhibit a similar lack of antigenic variation45 , suggesting
| mycobacterium tuberculosis is an obligate human pathogen capable of persisting in individual hosts for decades . to determine whether antigenic variation and immune escape contribute to the success of m. tuberculosis , we determined and analyzed 22 genome sequences representative of the global diversity of the m. tuberculosis complex ( mtbc ) .
as expected , we found that essential genes in mtbc were more evolutionarily conserved than non - essential genes .
surprisingly however , most of 491 experimentally confirmed human t cell epitopes showed little sequence variation and exhibited a lower ratio of non - synonymous to synonymous changes than essential and non - essential genes .
these findings are consistent with strong purifying selection acting on these epitopes , and imply that mtbc might benefit from recognition by human t cells . |
minimally invasive surgeries for gynecological conditions are becoming more common , especially since the wide - spread adoption of robotic surgery .
as surgeons grow increasingly comfortable with complex laparoscopic and robotic procedures , the rate - limiting step often becomes specimen retrieval through a small incision . in circumstances where malignancy is not a concern , specimen retrieval can be challenging , often resorting to morcellation of the specimen and at times enlarging a port site to facilitate removal of the specimen .
these practices increase the likelihood of contamination , implantation at the port site , port site hernias , tissue trauma , and increased operative time .
while the colpotomy incision is larger than a typical 10 mm port site , delivering a specimen through this vaginal incision is often difficult , time consuming , and can result into trauma to nearby structures .
improperly grasping the specimen can result in inadvertently incorporating nearby organs , such as the bowel or rectosigmoid epiploica . inadequately grasping the specimen often results in tearing or laceration of the specimen during each attempt at delivery through the colpotomy incision .
the importance of this matter is highlighted in women having surgery for gynecologic malignancy where an intact specimen is required for histopathological staging .
the benefits of minimally invasive surgery may not outweigh the risks of compromising the ability to adequately interpret the pathologic specimen .
for example , at our institution , patients diagnosed on final pathology with endometrial cancer after morcellation are offered pelvic radiation therapy that otherwise could have been avoided if delivery of an intact uterus and specimen was successful at the time osf the procedure .
the objective of this paper is to describe a novel technique to facilitate intact retrieval of large specimens during a minimally invasive hysterectomy .
a robotic - assisted hysterectomy and bilateral salpingooophorectomy were performed for a patient with a preoperative diagnosis of atypical endometrial hyperplasia .
the operation was uncomplicated until attempts were made to deliver the intact specimen through the vagina .
traction on the uterine manipulator during attempted delivery resulted in the specimen falling off the uterine manipulator .
a ringed forceps and then a single - tooth tenaculum were both used in attempt to retrieve the specimen . despite multiple attempts , none of the efforts were successful , and the specimen was lacerated in the process . in a final attempt
prior to undocking the robot and converting to a minilaparotomy , the specimen was grasped by the robotic arm and elevated off the pelvic floor . a 15 mm anchor tissue retrieval system no .
tr190sb2 retrievable bag along with a standard koh colpotomizer system pneumo - occluder balloon was used in a novel approach for specimen retrieval through the colpotomy incision ( figures 1 and 2 ) . first to ensure adequate pneumoperitoneum , the donut - shaped pneumo - occluding balloon was placed just over the shaft of the retrieval bag and inflated ( figure 3 ) .
the complete apparatus was then inserted into the vagina under direct visualization , and the pneumo - occluder balloon was inflated . after a pneumoperitoneum was obtained ,
the bag was deployed ( figure 4 ) and the uterus , fallopian tubes , and ovaries were easily placed inside the bag without difficulty using the robotic arm .
the bag was then closed , and the entire apparatus was effortlessly removed through the vagina with the complete specimen safely encapsulated and preserved .
the final pathology demonstrated figo stage i endometrial adenocarcinoma with mucinous features with 12% myometrial invasion and no lvi .
placement of the specimen within the retrieval bag is straightforward and easy to adapt to any minimally invasive gynecological surgery .
a koh colpotomizer system pneumo - occluder is slipped onto a 15 mm anchor retrieval system bag and inserted into the vagina .
after inflating the pneumo - occluder , a pneumoperitoneum is reestablished which is essential for adequate visualization . alternatively , for surgeons that use a mccartney tube ( gate healthcare ) rather that the koh for tlh or robotic hysterectomy , the device can be easily modified as illustrated in figures 5 , 6 , and 7 to accomplish the same ends .
this is also demonstrated in video clip 1 a ( see the supplementary material available online at doi:10.115/2012/454120 ) for removal of hysterectomy specimen and in video clip 2 a for removal of pelvic lymph node dissection . without the use of the mccartney tube
once the specimen is secured , the vaginal assistant applies downward traction onto the apparatus to extract the mass through the vaginal canal .
the force applied to the specimen within the bag squeezes the viscera smaller facilitating its delivery while simultaneously maintaining its architectural integrity .
we have successfully retrieved large and challenging specimens that could barely fit in the 15 mm anchor tissue retrieval system ( no . tr190sb2 ) .
the bag has a total volume capacity of 1860 ml accommodating very large specimens .
in addition , lubrication can be applied to the vagina or the outside of the bag without compromising the surgeon 's grip on the specimen .
our experience suggests that the anchor bag is superior to the endocatch bag , as more force can be exerted upon the anchor product prior to bag failure .
in addition , the anchor retrieval system can be used multiple times during a case .
in this paper we described a simple yet novel approach for specimen retrieval that promises to decrease operative time by facilitating safe and intact removal of large specimens following complex surgical procedures by minimally invasive approaches .
there is no additional cost associated with this technique , provided the surgeon was planning on using a retrieval system such as endocatch or the anchor product .
however , a prospective study would be imperative to compare the actual decrease in operative time , conversion to mini - laparotomy , and operative expenses associated with this new technique .
minimally invasive surgeries for gynecologic conditions are becoming more common due to the technical advantages of robotic surgery and the increasing comfort level and experience of advanced laparoscopic surgeons . as increasing complex procedures
becomes a more commonplace for gynecological surgeons , technological advancements will need to be made to overcome new challenges facing minimally invasive surgeons . facilitating retrieval of specimens , especially large or cancer - bearing organs , during minimally invasive surgery is paramount for the success of a minimally invasive procedure .
this is most apparent for women undergoing surgery for endometrial cancer ; however , this retrieval technique may have applications for even a wider range of minimally invasive surgical procedures . at our institution
this technique has also been used to remove an intact kidney and a segment of large intestine .
feasibility and safety of laparoscopic and robotic hysterectomies have been demonstrated in several studies [ 14 ] .
the benefits of this approach are readily apparent : more rapid recovery , shorter hospital stay , and less pain than conventional surgery [ 1 , 2 ] .
while these advantages are important , a minimally invasive approach is not warranted if it compromises the oncologic outcome .
adjuvant therapy is dictated by histologic grade , depth of myometrial invasion , and lymphovascular space invasion .
morcellating or fragmenting a hysterectomy specimen during retrieval not only limits the pathologic evaluation but it can also lead to seeding the abdominal and pelvic peritoneum . in cases where malignancy is not a primary concern , alternative methods of retrieval when
the uterine manipulator become dislodged such as using a tenaculum or ring forceps have been described .
although occurrences are rare , aggressive attempts to deliver a difficult specimen through the colpotomy incision can lead to unintended injury to the rectum or small bowel .
lastly , surgeons that perform minimally invasive hysterectomies on a routine basis know that precious time is wasted with fruitless attempts to deliver a uterus that is too large to fit through a small and narrow vagina as the case above demonstrates . since the routine adoption of this technique at our institution
, we have successfully used the technique in approximately 100 cases and have found specimen retrieval is less time consuming and less frustrating during minimally invasive hysterectomy .
in addition , the incidence of conversion to mini - laparotomy for specimen retrieval has been impacted . since adoption of this technique
, there has not been one instance where conversion was preformed solely for specimen retrieval .
data to support our observations are difficult to quantify as the time required to remove a specimen after completion of the vaginal colpotomy has not been routinely recorded at our institution .
nonetheless , over the last 30 cases preformed by one author , the average time to retrieve specimens that could not be spontaneously removed with the uterine manipulator was less than 2 minutes , ranging from 44 seconds to 3 minutes and 25 seconds .
since the introduction of this novel technique , we have found less time is required to remove large specimens .
total operative time is shorter which , in theory , can lead to a decrease of overall cost of robotic hysterectomy . despite numerous publications on the cost effectiveness of laparoscopic and robotic surgery , there is an equally valid argument that , in terms of dollars spent per case , conventional surgery is considerably less expensive .
multiple papers have addressed the higher cost for robotic hysterectomy and conventional laparoscopic hysterectomy [ 2 , 3 ] .
any new surgical technique that is cost effective and has the potential to decrease the overall cost of these procedures warrants further investigation . in conclusion ,
the technique described above is a simple adjunct to aid retrieval of large uteri and masses through a colpotomy incision .
we now use this technique almost exclusively when operating on women with endometrial cancer when the uterus does not deliver spontaneously with the uterine manipulator in an attempt to minimize exposure of cancer - bearing tissue to the pelvis . | introduction . with the widespread adoption of laparoscopic and robotic surgery ,
more and more women are undergoing minimally invasive surgery for complex gynecological procedures .
the rate - limiting step is often the delivery of an intact uterus or an unruptured adnexal mass . to avoid conversion to a minilaparotomy for specimen retrieval
, we describe a novel technique using an anchor tissue retrieval system bag in conjunction with a pneumo - occluder to easily retrieve large specimens through a colpotomy incision . surgical technique .
after completion of the robotic - assisted hysterectomy , the uterus , fallopian tubes , and ovaries were too large to be retrieved intact despite multiple attempts of delivery through the colpotomy incision . prior to resorting to a minilaparotomy or morcellation of the specimen , a 15 mm anchor retrieval bag with a pneumo - occluder
was placed through the vagina and the intact specimen was easily placed inside the bag under direct visualization and removed through the colpotomy incision intact . conclusion .
we routinely utilize this technique to retrieve hysterectomy specimens that are not readily delivered through the colpotomy incision and find this technique to be safe , highly efficient , and cost effective when there is a need to remove large intact specimens during minimally invasive surgery . |
in the past two decades ,
interest in porphyrins with aromatically
extended -systems has been steadily on the rise .
today areas of their application encompass optical limiting , organic light - emitting diodes ( oleds ) and other electronic
devices , upconversion by sensitized triplet
progress in all of these areas depends on the availability
of molecules with different functional groups , optimal solubility ,
and tailored photophysical properties , warranting continuous development
and optimization of synthetic methods . among -extended
porphyrins , fully symmetrical molecules
such as tetrabenzoporphyrins , tetranaphthporphyrins , and tetraanthraporphyrins captured the most attention , in part because
their synthetic chemistry has been most thoroughly developed .
these chromophores are characterized by strong and narrow absorption
bands in the red spectral region and form brightly phosphorescent complexes with pd and pt that are of interest for oled and imaging applications . in contrast ,
asymmetrically -extended porphyrins , which also have been known
for a long time , have been studied only occasionally .
structural asymmetry generally causes broadening of the porphyrin
optical spectra , which may be seen as a disadvantage from the applications
point of view .
, for technologies that rely on nonlinear
optical properties , asymmetrical -extension may bring about
unique possibilities , for example , as a method to increase two - photon
absorption ( 2pa ) cross sections .
a number of porphyrin - based systems with enhanced 2pa have been
designed in the past , and in some cases 2pa cross sections of several
thousand gm units have been reported . however , in only a few cases have triplet states of these systems
been evaluated in parallel .
triplet states , on
the other hand , are central to several key applications of porphyrins ,
including two - photon photodynamic therapy ( 2p pdt ) and two - photon
phosphorescence lifetime microscopy ( 2plm ) of oxygen . in those cases
when evaluation of the triplet states of 2p - absorbing
porphyrins has been attempted , measurements have been performed by
indirect methods , such as quantification of singlet oxygen ( a product
of the triplet quenching reaction ) or measuring downstream effects
of singlet oxygen itself .
such methods ,
however , can be subject to major errors due to a large number of interfering
parameters . at the same time
, it is quite
possible that modifications of porphyrins leading to an increase in
2pa can simultaneously cause severe triplet quenching effects , e.g. ,
via formation of low - lying charge transfer states and/or by influencing
the vibrational dynamics of the macrocycle , enhancing its nonradiative
triplet decay . as a result
, while gaining in
2pa , these porphyrins could lose their other critical property , i.e. ,
the ability to form long - lived triplet states .
our goal was
to develop 2p - absorbing porphyrins whose triplet states
can be monitored directly by emission .
the selection rules determining
linear one - photon ( 1p ) and 2p transition probabilities in centrosymmetrical
molecules , such as regular metalloporphyrins , are mutually exclusive .
consequently , strongly allowed 1p transitions
into b ( soret ) and q states , which are
states of ungerade symmetry , are not allowed for
2pa . on the other hand , gerade states , which can
be accessed via 2pa , lie at much higher energies , and their 2p spectra overlap with linear transitions ( e.g. ,
q bands ) , causing the 2p excitation pathway to be overshadowed by
1p absorption .
the above selection rules , however , should be relaxed
if there is no center - of - inversion symmetry in a porphyrin molecule ,
causing 1p states to become accessible by 2pa . given that pt
and pd complexes of -extended porphyrins exhibit
strong phosphorescence and that asymmetric -extension significantly
affects the electronic structure of porphyrins , we set out to examine whether asymmetrically -extended pt
and pd porphyrins can act as efficient triplet chromophores with intrinsically
enhanced 2pa . here
we present syntheses of nonfully symmetrical -extended syn - dibenzo- and syn - dinaphthoporphyrins
( dbps and dnps , respectively ; chart 1 ) and
their phosphorescent pt(ii ) and pd(ii ) complexes .
rational approaches
to a number of related tetrapyrroles have been explored previously . in particular
, there is an example of a 5,15-meso - diaryl dbp , reported by smith et al .
, that is very similar to one of our compounds ( porphyrin 20 ) but was obtained via different route , i.e. , from a sulfolenoporphyrin
precursor by the diels alder reaction .
the methodology explored
in our synthesis is based on the oxidative aromatization method and its 4,7-dihydroisoindole variant , developed by cheprakov and co - workers .
preliminary photophysical measurements showed that all of the synthesized
pt porphyrins exhibit very bright phosphorescence at ambient temperatures
and increased 2pa compared with nonextended porphyrins .
the 2pa is
especially enhanced in the case of porphyrins bearing alkoxycarbonyl
substituents on the fused benzo rings .
the developed methods should
facilitate the construction of nonfully symmetrical -extended
porphyrins for applications requiring enhanced 2pa / triplet action
cross sections , such as 2p pdt and 2plm .
two adjacent
pyrrole rings in each macrocycle are fused with external aromatic
fragments through the -carbon atoms , forming syn- ( or adj- ) dibenzo- and dinaphthoporphyrins .
if meso - aryl substituents are disregarded , these
macrocycles belong to the c2v symmetry group , unlike regular metalloporphyrins , which
have d4h - type symmetry
and thus possess a center of inversion .
previous studies have shown that planar meso - unsubstituted
-extended porphyrins are significantly more emissive than their
nonplanar meso - tetraarylated analogues .
however , meso - unsubstituted porphyrins are prone
to aggregation and often pose serious solubility problems . on the
other hand ,
nonplanar distortions in porphyrins are usually caused
by steric repulsion between -pyrrolic substituents ( e.g. , fused
benzo rings ) and meso - aryl groups .
placing only two meso - aryls opposite to each other allows the macrocycle
to evade the strain by undergoing in - plane as opposed to out - of - plane
deformation and thus preserve planarity .
moreover , in syn - dbps and syn - dnps
( chart 1 ) , aromatic rings are appended to pyrroles
only on
one side of the molecule , while the dipyrromethene
fragment on the opposite side remains strain - free .
mono - aryl - substituted dbp 21 lacking a meso - aryl group between the two benzo substituents was also synthesized .
to improve the solubility ,
the meso - aryl substituents
in the majority of the synthesized porphyrins were supplemented by meso - dialkoxycarbonyl groups . in all cases , dft ground - state
calculations ( b3lyp/6 - 31 g * ) confirmed planar macrocycle structures
( figure s1 in the supporting information ) .
the saturated precursor porphyrins for syn - dbps
and syn - dnps may be obtained from dipyrromethanes
bearing c1 synthons at the 1- and 9-positions and appropriate unsubstituted
counterparts ( scheme 1 ) . following the methods
developed by lindsey and co - workers , we chose to use bis(n , n - dimethylaminomethyl ) and bis(propyliminomethyl ) derivatives ( chart 2 ) , which have been shown to minimize scrambling
in the condensation reaction .
route a was selected because of easier
accessibility and higher stability of c1-dipyrromethanes derived from
unsubstituted pyrrole . generally , 1,9-bis(n , n - dimethylaminomethyl)dipyrromethanes
such as 1 ( chart 2 ) are simpler
to synthesize , and they lead to excellent results when condensations
are carried out in alcohols .
for example ,
in our case 1 was successfully employed in the synthesis
of porphyrin 13 in meoh ( see scheme 3 ) . however , in nonpolar solvents ( e.g. , toluene , benzene ) ,
which appeared to favor syntheses of the majority of our porphyrins
( see below ) , 1,9-bis(propyliminomethyl)dipyrromethane 2 allowed us to achieve significant improvements in the yield .
dipyrromethanes 1 and 2 ( chart 2 ) were
prepared in 5070% yield from pyrrole and the corresponding
aldehydes .
cyclohexadieno- ( 3 and 4 ) , cyclohexeno- ( 5 and 6 ) , and tetrahydronaphthaleno - fused ( 7 ) dipyrromethane
esters
were synthesized in 7090% yield from the respective pyrroles and aldehydes ( or dimethoxymethane ) following the previously developed
procedures ( scheme 2 ) . in the case of
sterically more hindered
structures 5 and 7 , longer times ( up to 72 h ) were required
to complete the condensations . generally , using acetic acid as the
solvent , as opposed to dichloromethane , was found to speed up the
reactions by factors of 2 .
the cyclohexadieno- ( 3 and 4 ) and
tetrahydronaphthaleno - fused ( 7 ) dipyrromethane esters were unstable
at room temperature and degraded rapidly during purification .
( only compounds that were isolated and fully characterized
are identified in the text by bold numbers ) .
dipyrromethane esters
3 , 4 , and 5 were de - esterified and decarboxylated in a one - pot reaction
upon treatment with tfa , while benzyl esters 6 and 7
were first converted to carboxylic acids by reduction on pearlman s
catalyst ( pd(oh)2/c ) and then
introduced into the tfa - mediated decarboxylation without isolation .
de - esterification / decarboxylation was carried out immediately prior
to the porphyrin synthesis , since ,-unsubstituted
dipyrromethanes are even more unstable than their ester precursors
and degrade rapidly in air , presumably as a result of oxidation and/or
oligomerization .
dipyrromethane 12 was the least stable in the series ,
and it had to be handled especially promptly . on the basis of analyses
of the crude reaction mixtures , the yields of the dipyrromethanes
( starting with esters 37 ) ranged from 30 to 65% . reagents
and conditions : ( a ) p - tsoh , nbu4cl , ch2cl2 ,
r.t . , 24 h ( 3 ) , 48 h ( 4 ) , 72 h ( 5 ) .
( b ) p - tsoh , acoh , 24 h ( 6 ) , 48 h ( 7 ) .
( c ) h2/pd(oh)2/c , thf , r.t . , 2448 h.
( d ) ( i ) tfa / ch2cl2 1:1 , 20 c , 1 h ; ( ii )
nahco3 .
the [ 2 + 2 ] assembly leading
to the target porphyrins is shown
in scheme 3 . according
to the original procedures , condensation of 1,9-bis(n , n - dimethylaminomethyl)- or 1,9-bis(n - propyliminomethyl)dipyrromethanes ( chart 2 ) with 1,9-unsubstituted dipyrromethanes
is carried out optimally
in refluxing methanol in the presence of a 10-fold molar excess of
zn(oac)2 . in cases when the reactants bear functional groups sensitive to
solvolysis
, methanol may be substituted by toluene , also under reflux ,
in which case 1,9-bis(n - propyliminomethyl)dipyrromethanes
are the substrates of choice .
reagents and conditions : ( a )
( i ) zn(oac)22h2o ( 10 equiv ) , meoh , reflux ,
2 h ; ( ii ) ddq ( 3 equiv ) , r.t . , 2 h. ( b ) ( i ) zn(oac)22h2o ( 10 equiv ) , c6h6 , ar , reflux , 2 h ;
( ii ) air , reflux , 1218 h. ( c ) ddq ( 3 equiv ) , thf , reflux ,
3040 min .
( e )
pt insertion : ( method 1 ) pt(acac)2/benzoic acid , 130135
c , 26 h ; ( method 2 ) pt(acac)2/phcn , microwave ,
250 c ( 200 kpa , 105145 w ) , 40 min .
pd insertion :
pd(ac)2/phcn , microwave , 250 c ( 200 kpa ,
105145 w ) , 40 min .
( f ) ddq ( 5 equiv ) , toluene , reflux , 10
h. ( g ) ddq ( 3 equiv ) , thf , r.t .
, 10 min . following
the original method , reaction
of dipyrromethane 8 with 1 in meoh in the presence of
zn(oac)2 gave the zn complex of porphyrin 13 ( zn13 )
in 13% yield .
however , applying the same conditions directly to the
rest of our substrates proved inefficient .
for example , condensation
of dipyrromethanes 10 and 2 in meoh gave zn15 in a rather low 8% yield , and when the reaction was attempted
in toluene , the yield dropped below 5% .
other dipyrromethanes showed
similar results . on the basis of uv vis spectra , we concluded
that at least in part the low yields were caused by competing oxidation
of dipyrromethanes into dipyrromethenes ( dipyrrins ) , whose formation
could be easily detected by characteristic optical absorption .
dipyrrins are inert in the condensation . in order to prevent premature oxidation of dipyrromethanes and
establish overall milder conditions for the synthesis , the reaction
was attempted as a two - step process using dipyrromethane 2 as a reactant . in the first step ,
dipyrromethanes 912 were
condensed with 2 under an inert atmosphere ( ar ) in the
presence of zn(oac)2 in refluxing benzene .
the latter has
a lower boiling point than toluene , helping to avoid side reactions
and decomposition of the sensitive dypyrromethanes .
the formation
of the corresponding dehydroporphyrinogens and depletion of the starting
materials was monitored by maldi - tof analysis .
once the concentration
of the dehydroporphyrinogens stopped changing , usually after 23
h , the mixtures were flushed with air and left to react for an additional
816 h ( second step ) . at this stage
, the reaction progress
was monitored by uv vis spectroscopy , and the refluxing was
continued until the ratio of the intensity in the soret band region
( 350400 nm ) to the absorption by the side products
( dipyrrins ) near 500600 nm was at its maximum .
it should be
noted that not all of the dipyrromethanes were converted into dehydroporphyrinogens
in the first step , but the mixtures were simply allowed to reach their
respective steady states .
subsequent oxidation by air apparently was
mild enough to prevent fast oxidation of dipyrromethanes but effective
in converting dehydroporphyrinogens into porphyrins .
the dipyrromethanes
in the meantime continued to undergo condensation to generate new
dehydroporphyrinogens for oxidation .
these conditions allowed us to
obtain cyclohexadieno- ( 14 ) and cyclohexenoporphyrins ( 15 and 16 ) in 2030% yield and the least stable
porphyrin 17 in 13% yield . in all cases ,
aromatization of precursors 1317 into
the target dibenzo-
( 1821 ) and dinaphthoporphyrins ( 22 ) requires the removal of either two ( 13 , 14 , 17 ) or four
( 15 , 16 ) hydrogens from each exocyclic ring
annealed with the macrocycle , whereas dehydrogenation of less - saturated
rings generally occurs much more easily .
aromatizations may be inhibited
by the formation of porphyrin dications if the basicities of the corresponding
free bases are high .
the latter is typical
of highly nonplanar porphyrins , which must be converted into stable
metal complexes ( e.g. , with pd , pt , or cu ) prior to oxidation .
planar meso - unsubstituted and 5,15-diarylporphyrins may be oxidized directly as free bases , although metalation usually
facilitates the reaction .
the transformation
of zn17 into zn22 could be observed immediately
upon addition of ddq , even at room temperature , by the change of the
color of the mixture from red to deep green .
similarly , aromatization
of porphyrins zn13 and zn14 occurred when they were
treated with ddq in thf for just 3040 min , although heating
was required .
the zn complexes of 18 , 19 , and 22 were isolated in almost quantitative yields .
the aromatization of tetraalkoxycarbonylporphyrins 15 and 16 , however , proved to be much more difficult .
for example , treatment of 15 as either the free base
or the zn complex ( zn15 ) with excess ddq in refluxing
thf for 14 h did not show any sign of conversion , while the oxidation
in refluxing toluene produced mixtures of aggregated inseparable products .
our final targets in this study were phosphorescent pt and pd complexes ,
and therefore , we turned our attention to the methods developed earlier
for the synthesis of similar pt and pd tetraaryltetrabenzo- and tetranaphthoporphyrins . prior to oxidation ,
zn15 and zn16 were demetalated and then converted into pt and pd complexes
( see below ) .
the latter were smoothly oxidized using excess ddq in
toluene over 10 h , yielding the desired complexes m20 and m21 ( m = pt , pd ) in 7992%
yield .
insertion of pt(ii ) into the precursor porphyrins 15 and 16 and the target porphyrins 18 , 19 , and 22 was performed either in benzoic
acid
melt or using the microwave - assisted
method . in the former approach , the free - base
porphyrins were heated with a 34-fold molar excess of pt(acac)2 in benzoic acid at 130135 c for 26
h. subsequent methanol workup , chromatographic purification , and reprecipitation
from ch2cl2/meoh ( 1:50 ) afforded the pt(ii)porphyrin
complexes in 2056% yield . in spite of the moderate yields ,
the benzoic acid method in our hands
has proved to be general and frequently leads to success when other
methods , such as commonly used refluxing in benzonitrile , fail .
however ,
in this particular instance , the best results were achieved using
the microwave - assisted insertion , developed by bruckner and co - workers .
microwave treatment of mixtures of the free - base
porphyrins and pt(acac)2 ( 34 equiv ) in benzonitrile
for 40 min gave the corresponding pt complexes in 92% to quantitative
yield .
overall , the developed reaction sequences allowed us
to prepare
the target pt and pd porphyrins in yields of 515% relative
to the starting pyrrole esters ( scheme 2 ) .
the methods do not require expensive reagents and can be scaled to
gram quantities .
this section
provides a brief
overview of the photophysical properties of the newly synthesized
porphyrins , while leaving the detailed analysis for a separate account .
because of the presence of meso - aryl substituents
and alkoxycarbonyl groups , all of the porphyrins were found to be
well - soluble in a range of organic solvents ( e.g. , toluene , ch2cl2 , thf , dmf , and dma ) , where they showed no signs
of aggregation at the concentrations required for optical measurements
and above ( up to 10 m ) .
this solvent is especially convenient
because of its high boiling point ( 165 c ) , making it possible
to deoxygenate solutions by inert gas bubbling ( ar or n2 ) at room temperature without experiencing significant losses in
the solution volume .
the optical absorption features of the
pt and pd complexes of the synthesized porphyrins are very similar
to each other , with the bands of the pd porphyrins being red - shifted
by 515 nm relative to the pt complexes .
the triplet lifetimes
of the pd complexes pd19 through pd22 are 810 times longer than those of the respective
pt porphyrins , while their phosphorescence quantum yields are
about 2 times lower ( table 1 ) .
these trends
are common for pt and pd porphyrins , and
below we discuss only pt complexes pt18 through
pt22 , while the properties of the pd complexes
can be inferred by analogy .
the phosphorescence quantum yields
( ) and lifetimes ( ) were measured at 22 c in deoxygenated
solutions .
the fluorescence of rhodamine 6 g in etoh ( fl = 0.95 ) was used as a standard . for
comparison , under these conditions
the fluorescence quantum yield
of tetraphenylporphyrin ( h2tpp ) in deoxygenated c6h6 is 0.055 , and the phosphorescence quantum yield of
pt meso - tetraphenyltetrabenzoporphyrin ( ph4tbp ) in deoxygenated dmf is 0.085 .
the linear absorption and phosphorescence emission
spectra of porphyrins
pt18 through pt22 are shown
in figure 1 , and the relevant data are compiled
in table 1 .
similar graphs for the pd complexes
are shown in figure s2 in the supporting information .
it should be noted that the absorption features of zndbps zn18 and zn19 ( figure s3 in the supporting information ) correspond well with
the literature data on similar zn complexes .
( a )
absorption and ( b ) phosphorescence spectra of porphyrins pt18 through pt22 in dma .
the absorption
spectra were normalized by the corresponding q - band maxima ; the inset
shows the zoomed q - band region .
the phosphorescence spectra were normalized
by the respective phosphorescence quantum yields ( table 1 ) .
the absorption spectra of all
of the synthesized pt porphyrins
exhibit well - defined , narrow bands , resembling in that way the spectra
of the fully symmetrical pt tetrabenzoporphyrins ( pttbps ) ( d4h symmetry ) .
the soret ( or b ) ( s2 ) , q ( s1 ) , and phosphorescence
( t1 ) bands of porphyrins pt18 through
pt21 occupy somewhat intermediate positions between
the bands of regular nonextended pt porphyrins ( ptps ) and those of pttbps .
the q band of pt22 ( max =
593 nm ) is bathochromically shifted , approaching the q bands of pttbps
( 605615 nm ) , while its b band is
also red - shifted , but broadened similar to the b bands of tetranaphthoporphyrins .
these observations can be rationalized , at least
to a first approximation , by recalling that q and b bands in regular
symmetrical porphyrins are composed of orthogonally polarized transitions
( qx and qy , bx and by ) that are formed by configuration interaction involving single - electron
excitations between pairs of nearly degenerate homos and lumos . -extension lifts the degeneracy of one
of the homos ( a2u ) , leading to a spectral red shift as
well to an increase in the oscillator strength of the q band .
just as in fully symmetrical pttbps , the orthogonal transition dipoles
in pt syn - dbps and pt syn - dnps are
identical to each other .
consequently , the x and y bands are fully superimposed , giving rise to narrow spectral
lines , similar to those of pttbps .
in contrast , in anti - dbps , the x and y dipoles are
not equivalent , resulting in multiple spectral lines in both the q-
and b - band regions .
the transitions
in pt18 through pt21 are
polarized in the directions in which the syn - dbp
molecules have diameters larger than those of regular pt porphyrins
but smaller than those of pttbps . in syn - dnp pt22 ,
on the other hand , the macrocycle diameter along the polarization
axes is similar to that of pttbps .
the energies of the spectral bands
generally follow this simple relationship : the longer
the dipole , the lower the energy of the absorption band . of course ,
for accurate quantitative interpretation of the spectroscopic data ,
a detailed computational / photophysical study such as that performed
recently for zn benzoporphyrins will
be required . in the series of synthesized syn - dbps , porphyrins
pt20 and pt21 exhibit the
most bathochromically shifted q and b bands , while their phosphorescence
maxima are the most hypsochromically shifted . with the assumption
that the phosphorescent triplet states ( t1 ) in all of these
porphyrins are derived from the same electronic configurations as
the s1 ( or s2 ) states , the data for pt20 and pt21 appear to be consistent with
the expansion of the -conjugation onto the carbonyl groups
in the benzo rings in the macrocycles .
this expansion further destabilizes
one of the homos , causing a red shift in the absorption , but at the
same time decreases the exchange energy ( 2j ) because
of the increase in the size of the -system , narrowing the s1t1 gap and
raising the energy of the t1 state . between these two porphyrins ,
pt20 exhibits more bathochromically shifted bands
than pt21 , which is consistent with the presence
of only one meso - aryl substituent in the latter .
the meso - aryl groups in 5,15-diarylporphyrins ( see
the x - ray structure in figure 2 and the computed
structures in figure s1 in the supporting information ) are almost perpendicular to the porphyrin plane , and yet they participate in the conjugation with the macrocycle ,
thereby inducing small red shifts in the absorption spectra .
( top ) ortep view of the x - ray crystallographic structure
of porphyrin
pt19 ( 50% thermal ellipsoids ) with two stacked
nonidentical porphyrin molecules in the cell .
( bottom ) view of the plain porphyrin
skeleton of pt19 , showing the small nonplanar
distortion of the macrocycle and tilts of the two meso - aryl substituents .
the aryl group between the two benzo rings is
rotated by 83 relative to the porphyrin mean - square plane .
the phosphorescence quantum yields
of pt syn - dbps
( ph 0.35 ) were found to be among the highest
reported to date for phosphorescent tetrapyrroles .
the quantum yields
were measured against the fluorescence of rhodamine 6 g in etoh ( fl = 0.95 ) , and the emission spectra
were corrected for the wavelength dependences of the detector , excitation
source , and monochromators . it should be noted that under the same
conditions the phosphorescence quantum yield of pt meso - tetraphenyltetrabenzoporphyrin ( ph4tbp ) in deoxygenated
dmf was determined to be 0.085 , which is substantially lower than
reported previously .
the high emission yields of pt syn - dbps are in line with their relatively planar structures
( figure 2 and figure s1 in the supporting information ) , in accordance with the
previously observed correlation between nonplanarity of -extended
porphyrins and enhanced nonradiative triplet decay . on average only 60% of the triplet state in pt18 through pt21
decays via the nonradiative
channel ( table s1 in the supporting information ) , while in , e.g. , highly nonplanar ptph4tbps this fraction
is larger than 90% . in dnp pt22
, the proportion
of the t1 s0 intersystem crossing is
also increased to 90% because of the narrower t1s0 gap . in order to estimate the performance
of porphyrins pt18 through pt22 under 2p excitation
,
we compared their phosphorescence outputs to that of a regular symmetrical
pt porphyrin , i.e. , pt tetracyclohexenoporphyrin ( pttchp ) , which possesses d4h symmetry ( figure s4 in the supporting
information ) . the phosphorescence quantum yield of pttchp in
deoxygenated dma at 22 c is 0.42 .
our setup allows time - resolved
acquisition of phosphorescence upon excitation by trains of femtosecond
pulses from a tunable ti : sapphire laser ( see the supporting information for details ) . in pt porphyrins , because
of the ultrafast s1 t1 intersystem
crossing and therefore nearly quantitative formation of the triplet
state , the corrected
integrated intensity of the phosphorescence ( normalized by the phosphorescence
quantum yield and plotted against the excitation wavelength ) is directly
suitable for comparisons of 2pa cross sections of different porphyrins .
to ensure the 2p excitation regime ,
the signal in all cases was confirmed
to exhibit a strictly quadratic power dependence ( slope of the log
the results are summarized in figure 3 ( for the numerical data , see table s2 in the supporting information ) .
relative 2pa cross sections
( integrated intensities of 2p - excited
phosphorescence normalized by the phosphorescence quantum yield ) of
pttchp ( d4h ) and the
synthesized pt porphyrins .
the data are scaled in such a way that
the relative 2pa cross section of the most absorbing porphyrin , pt21 , equals 1.0 at 770 nm .
it can be seen that the apparent 2pa cross sections of pt syn - dbps and syn - dnps are indeed larger
than that of the reference pttchp . in the case of pt22
, measurements could not be conducted at wavelengths shorter
that 860 nm because of the interfering linear excitation into the
triplet state ( s0 t1 ) by the femtosecond
pulses .
( it should be kept in mind that in pt porphyrins , because
of the very strong spin orbit coupling , spin - forbidden s0
the most striking enhancement occurs in
the case of porphyrins pt20 and pt21 in which the benzo rings contain alkoxycarbonyl groups ,
as their apparent 2pa cross sections near 770 nm are 25-fold
larger than that of pttchp ( table s2 in the supporting
information ) . however , it is also clear that in all of the
porphyrins the 2pa continues to rise past 800810 nm , i.e. ,
twice the maximum of the soret band .
apparently , breaking the center - of - inversion
symmetry is not the dominant factor in the enhancement of the 2pa ,
and the most strongly absorbing 2pa states in these porphyrins are
still not the same as their linear 1p states .
these preliminary
findings raise the following questions : what
are the 2p - absorbing states in -extended pt porphyrins , and
why do alkoxycarbonyl groups cause such a pronounced enhancement of
the 2pa ?
one obvious notion is that the alkoxycarbonyl groups in porphyrins
pt20 and pt21 significantly
polarize these molecules .
for example , on the basis of dft calculations
( b3lyp/6 - 31 g * ) , the ground - state dipole moment of a zn porphyrin analogous
to zn20 is 6.12 d , whereas for the analogue of
zn19 it is only 0.85 d ( figure s1 in the supporting information ) .
such polarization could
in principle lead to an enhancement of 2pa , assuming that in the excited
state the dipole moment increases ( or changes sign ) .
these and other pertinent photophysical questions should
be addressed in a separate study , for which the present work sets
the necessary synthetic stage .
we have developed a synthesis of pt and pd complexes
of nonfully
symmetrical syn - dbps and syn - dnps
with solubilizing substituents .
these macrocycles possess nearly planar
structures and phosphoresce in solutions at ambient temperatures with
exceptionally high quantum yields .
preliminary evaluation showed that
structural asymmetry causes an increase in the 2pa into 1p - allowed
states ; however , much stronger 2p - absorbing states are positioned
at higher energies .
descriptions of materials , equipment , and general protocols are
provided in the supporting information .
hrms data are reported for the highest - intensity peak in the isotope
mass distribution and compared to the corresponding peak in the distribution
simulated by the mass spectrometer software .
the abbreviations used
in the h nmr peak assignments are shown on p s20 in the supporting information .
4-methoxycarbonylbenzaldehyde ( 2.40 g , 15
mmol ) and pyrrole ( 100 g , 1.5 mol ) were mixed together and purged
with ar for 10 min .
incl3 ( 0.3315 g , 1.5 mmol ) was added ,
and the reaction mixture was stirred under ar at room temperature
for 1.5 h. naoh ( 1.8 g , 43.5 mmol ) was added to the mixture , and stirring
was continued for additional 45 min .
the precipitates were removed
by filtration , and pyrrole was removed by distillation in vacuum ( 1
mmhg , 1820 c ) ; the remaining traces of pyrrole were
removed by washing the residue with hexane ( 3 30 ml ) .
5-(4-methoxycarbonylphenyl)dipyrromethane
( 1a ) precipitated from solution as pale - yellow crystals
upon cooling of the mixture to 0 c .
h nmr ( dmso - d6 ) ( ppm ) : 2.07 ( 12h , s , nch3 ) , 3.26 ( 4h , d , j = 2.51
hz , chn(ch3)2 ) , 3.81
( 3h , s , och3 ) , 5.37 ( 1h , s , ch ) , 5.53 ( 1h , d , j =
2.8 hz , pyr ) , 5.54 ( 1h , d , j = 2.8 hz ,
pyr ) , 5.72 ( 1h , d , j = 2.8 hz , pyr ) ,
5.73 ( 1h , d , j = 2.8 hz , pyr ) , 7.23 ( 2h ,
d , j = 8.3 hz , ar ) , 7.85 ( 2h , d , j = 8.3 hz , ar ) , 10.51 ( 2h , s , nh ) . to a solution of 1a ( 0.9468 g , 2.9
mmol ) in ch2cl2 ( 50 ml ) , n , n - dimethylethyleneiminium iodide ( eschenmoser s salt )
( 1.3871 g , 6.4 mmol ) was added .
the reaction mixture was stirred for
1 h at room temperature and then diluted with ch2cl2 ( 350 ml ) , and k2co3 ( 10% aq .
, 3 350 ml ) , dried over na2so4 , and concentrated in vacuum .
the title compound was
precipitated from ch2cl2 upon addition of hexanes ,
collected by filtration , and dried in vacuum .
h nmr ( dmso - d6 ) ( ppm ) : 2.07 ( 12h ,
s , nch3 ) , 3.26 ( 4h , d , j = 2.51 hz , chn(ch3)2 ) , 3.81 ( 3h , s , och3 ) , 5.37 ( 1h , s , ch ) , 5.53
( 1h , d , j = 2.8 hz , pyr ) , 5.54 ( 1h , d , j = 2.8 hz , pyr ) , 5.72 ( 1h , d , j = 2.8 hz , pyr ) , 5.73 ( 1h , d , j = 2.8 hz , pyr ) , 7.23 ( 2h , d , j = 8.3 hz , ar ) , 7.85 ( 2h , d , j = 8.3
hz , ar ) , 10.51 ( 2h , s , nh ) .
c nmr ( cdcl3 ) ( ppm ) : 44.2 , 44.9 , 52.0 , 56.6 , 107.1 ,
107.5 , 107.6 , 128.4 , 128.6 , 128.9 , 129.0 , 129.8 , 131.70 , 131.74 , 147.65 ,
147.67 , 167.0 . hrms
( esi - tof ) m / z : [ m + h ] calcd for c23h31n4o2 395.24413 , found 395.24368 .
3,5-dibutoxycarbonylbenzaldehyde ( 3.06 g ,
10 mmol ) and pyrrole ( 66 g , 1.0 mol ) were mixed together and purged
with ar for 10 min .
incl3 ( 0.221 g , 1.0 mmol ) was added ,
and the reaction mixture was stirred at room temperature under ar
for 1.5 h. naoh ( 1.2 g , 30 mmol ) was added to the mixture , and the
stirring was continued for 45 min .
the precipitates were removed by
filtration , and pyrrole was removed by distillation in vacuum ( 1
mmhg , 1820 c ) .
the residue was purified by chromatography
on a short silica gel column ( 10 cm , ch2cl2 )
to give crude 5-(3,5-dibutoxycarbonylphenyl)dipyrromethane ( 2a ) ( 3.6 g ) . to a solution of 2a ( 3.6 g ,
8.5 mmol ) in dry dmf ( 10 ml )
was added pocl3 ( 2.74 g , 17.9
mmol ) dropwise at 0 c under ar .
the reaction mixture was stirred
for 1 h at room temperature , poured into a saturated aqueous solution
of sodium acetate ( 250 ml ) , extracted with ch2cl2 ( 3 100 ml ) , and dried over na2so4 .
the solvent was removed in vacuum , and the residue was purified by
column chromatography ( silica gel , ch2cl2/etoac
= 4:1 ) to give crude 1,9-diformyl-5-(3,5-dibutoxycarbonylphenyl)dipyrromethane
( 2b ) ( 3.3 g ) .
n - propylamine ( 15
ml , 183 mmol ) was added to 2b ( 3.3 g , 6.9 mmol ) , and
the mixture was stirred for 1 h
at room temperature .
excess n - propylamine was removed
in vacuum to give the title compound 2 as a dark - orange
solid ( mp 112114 c ) .
h nmr ( cdcl3 ) ( ppm ) : 0.90 ( 6h , t , j = 7.4 hz , ch3 ) , 0.95 ( 6h , t , j = 7.4 hz , ch3 ) , 1.381.49 ( 4h , m , ch2 ) , 1.601.78 ( 8h , m , ch2 ) , 3.41 ( 4h , dd , j1 = 6.8 , j2 = 6.9 hz , nch2 ) ,
4.30 ( 4h , t , j = 6.7 hz , och2 ) , 5.59 ( 1h , s , broad , ch ) , 5.96 ( 2h , d , j =
3.6 hz , pyr ) , 6.50 ( 2h , d , j = 3.6 hz ,
pyr ) , 7.72 ( 2h , s , nh ) , 8.08 ( 2h ,
d , j = 1.4 hz , ar ) , 8.55 ( 1h , t , j = 1.4 hz , ar ) .
c nmr ( cdcl3 ) ( ppm ) : 11.7 , 13.7 , 19.2 , 24.2 , 30.6 , 44.1 , 62.3 ,
65.2 , 109.6 , 114.8 , 114.9 , 129.4 , 130.4 , 131.2 , 133.6 , 135.6 , 135.7 ,
142.2 , 151.4 , 165.7 .
maldi - tof ( m / z ) : calcd for c33h44n4o4 560.34 , found 561.24 [ m + h ] .
hrms ( esi - tof ) m / z : [ m + h ] calcd for c33h45n4o4 561.34349 , found
561.34371 .
p - toluenesulfonic acid ( 0.090 g , 0.48 mmol ) and tert - butylammonium chloride ( 0.135 g , 0.48 mmol ) were added to a solution
of 1-tert - butoxycarbonyl-5,6-dibutoxycarbonyl-4,5,6,7-dihydro-2h - isoindole ( 2 g , 4.75 mmol )
and 3,5-dibutoxycarbonylbenzaldehyde ( 0.727
g , 2.4 mmol ) in ch2cl2 ( 50 ml ) .
the reaction
mixture was stirred at room temperature under ar for 72 h. small amounts
of p - toluenesulfonic acid ( 0.045 g , 0.24 mmol ) and tert - butylammonium chloride ( 0.07 g , 0.25 mmol ) were added
to the reaction mixture after 24 h and then again after 48 h. the
reaction progress was monitored by tlc until the spot corresponding
to aldehyde disappeared .
the mixture was diluted with ch2cl2 ( 100 ml ) , washed with nahco3 ( 10% aq . ,
50 ml ) and brine ( 50 ml ) , and dried over na2so4 .
the solvent was removed in vacuum , and the residue was purified
by column chromatography ( silica gel , ch2cl2/thf = 50:1 ) to give the title compound 5 as an orange
oil .
h nmr ( cdcl3 )
( ppm ) ( mixture of conformers ) : 0.830.91 ( 12h , m , ch3 ) , 0.95 ( 6h , t , j = 7.3 hz , ch3 ) , 1.221.50
( 16h , m , ch2 ) , 1.501.53
( 18h , m , tbu ) , 1.481.60 ( 4h , m , ch2 ) , 1.681.78 ( 4h , m , ch2 ) , 2.242.47 ( 2h , m , chh ) , 2.552.80 ( 2h , m , chh ) , 2.993.27 ( 8h , m , chh
,
ch ) , 3.924.10 ( 8h , m , och2 ) , 4.30 ( 4h , t , j = 6.6 hz , och2 ) ,
5.505.54 ( 1h , m , broad , ch ) ,
7.887.95 ( 2h , m , ar ) , 8.398.44 ( 1h , m , ar ) , 8.528.50
( 2h , m , nh ) .
c nmr ( cdcl3 ) ( ppm ) ( mixture of conformers ) : 13.62 , 13.64 , 13.71 ,
19.01 , 19.04 , 19.1 , 19.2 , 22.2 , 22.3 , 22.4 , 22.5 , 23.5 , 23.6 , 23.7 ,
23.8 , 28.39 , 28.42 , 30.3 , 30.47 , 30.52 , 30.54 , 30.6 , 40.56 , 40.58 ,
40.7 , 40.8 , 40.90 , 40.94 , 40.96 , 41.00 , 41.1 , 64.45 , 64.49 , 64.55 ,
64.60 , 64.63 , 65.3 , 80.63 , 80.65 , 80.69 , 116.9 , 117.17 , 117.23 , 117.3 ,
118.76 , 118.84 , 118.9 , 119.0 , 125.3 , 125.4 , 125.5 , 128.1 , 128.3 , 128.4 ,
128.5 , 129.77 , 129.8 , 129.9 , 131.85 , 131.90 , 133.2 , 133.3 , 139.5 ,
139.7 , 139.8 , 160.68 , 160.70 , 160.9 , 165.25 , 165.33 , 172.6 , 172.7 ,
172.76 , 172.79 , 173.01 , 173.02 .
maldi - tof ( m / z ) : calcd for c63h90n2o16 1130.63 , found 1153.42 [ m + na ] , 1169.39 [ m
+ k ] . hrms ( esi - tof ) m / z : [ m + h ] calcd for c63h91n2o16 1131.63623 , found 1131.63613 .
p - toluenesulfonic acid ( 0.033 g , 0.175 mmol ) was added to a solution
of 1-benzyloxycarbonyl-5,6-dimethoxycarbonyl-4,5,6,7-dihydro-2h - isoindole ( 1.3 g , 3.5 mmol )
and dimethoxymethane ( 0.133 g , 1.75 mmol ) in acetic acid ( 25 ml ) ,
and the reaction mixture was stirred at room temperature under ar
for 24 h. the mixture was poured into cold water ( 80 ml ) , and the
formed precipitate was collected by filtration and dried in vacuum .
the resulting solid was dissolved in meoh ( 20 ml ) and precipitated
upon addition of water ( 1015 ml ) at 0 c .
the precipitate
was collected by centrifugation , washed with water ( 3 20 ml ) ,
and dried in vacuum to give the title compound as a white solid ( mp
104106 c ) .
h nmr ( cdcl3 ) ( ppm ) ( mixture of conformers ) : 2.652.78
( 2h , m , chh ) , 2.903.03 ( 2h ,
m , chh ) , 3.043.24 ( 6h , m ,
chh , ch ) ,
3.263.38 ( 2h , m , ch ) , 3.623.69
( 12h , m , och3 ) , 3.693.81
( 2h , m , ch2 ) , 5.175.33
( 4h , m , ch2ph ) , 7.257.34
( 10h , m , ar ) , 9.309.48 ( 2h , m , nh ) .
c nmr ( cdcl3 ) ( ppm ) ( mixture
of conformers ) : 21.80 , 21.87 , 21.92 , 21.98 , 22.4 , 22.5 , 22.6 , 23.7 ,
23.76 , 23.83 , 23.9 , 24.0 , 40.7 , 40.8 , 51.4 , 51.95 , 52.00 , 65.7 , 65.8 ,
116.42 , 116.48 , 116.54 , 116.58 , 126.2 , 126.5 , 126.6 , 127.66 , 127.68 ,
127.71 , 127.90 , 127.92 , 128.0 , 128.4 , 128.5 , 129.15 , 129.24 , 136.2 ,
161.4 , 162.3 , 173.33 , 173.35 , 173.36 , 173.39 , 173.45 , 173.49 , 173.52 ,
173.55 . maldi - tof ( m / z ) : calcd for
c41h42n2o12 754.27 , found
752.96 [ m h ] , 776.95 [ m + na ] , 792.92
[ m + k ] . hrms ( esi - tof ) m / z : [ m + h ] calcd for c41h43n2o12 755.28099 , found 755.27822 .
dipyrromethane
ester 5 ( 1.98 g , 1.75 mmol ) was dissolved in ch2cl2 ( 15 ml ) , and the solution was cooled to 0 c
on an ice bath .
the ice bath was removed , and the reaction mixture was stirred
at room temperature for 1.5 h. the mixture was diluted with ch2cl2 ( 100 ml ) , washed with nahco3 ( 10%
aq . , 50 ml ) and brine ( 50 ml ) , and dried over na2so4 . the solvent was removed in vacuum , and the residue was purified
by column chromatography ( silica gel , ch2cl2/thf , gradient from 15:1 to 2:1 ) to give crude dipyrromethane 10
as a dark - brown oil ( 0.92 g ) .
the thus - obtained crude dipyrromethane
10 was used immediately in the porphyrin synthesis ( see below ) .
dipyrromethane
ester 6 ( 1.23 g , 1.63 mmol ) was dissolved in thf ( 50
ml ) , and the solution was purged with ar for 10 min .
catalyst ( 0.2 g ) was added , and the reaction
mixture was purged with ar again for 10 min and then with h2 for 1520 min .
the mixture was kept under vigorous stirring
overnight under h2 ( 1 atm ) .
the mixture was purged with
ar and then passed through celite to remove the catalyst .
the resulting
solution was concentrated in vacuum to give crude ,-bis(carboxy)methylenebis(1,1-(5,6-dibutoxycarbonyl-4,5,6,7-tetrahydro-2h - isoindole ) ) ( 6a ) ( 0.936 g ) as a slightly
pink viscous oil .
maldi - tof ( m / z ) : calcd for c27h30n2o12 574.18 , found 596.90 [ m + na ] , 612.88 [ m + k ] .
crude 6a ( 0.936 g , 1.63 mmol ) was dissolved
in thf ( 5 ml)/ch2cl2 ( 20 ml ) , and the solution
was cooled to 0 c on an ice bath .
the ice bath was removed , and the reaction
mixture was stirred at room temperature for 1 h. the mixture was diluted
with ch2cl2 ( 100 ml ) , washed with nahco3 ( 10% aq .
, 2 50 ml ) and brine ( 50 ml ) , and dried over
na2so4 . the solvent was removed in vacuum , and
the residue was purified on a short column ( silica gel , 10 cm , ch2cl2/thf , 25:1 ) to give crude dipyrromethane 11
( 0.42 g ) as a dark - brown oil .
maldi - tof ( m / z ) : calcd for c25h30n2o8 486.20 , found 487.01 [ m + h ] .
dipyrromethane
3 was prepared from 2-tert - butoxycarbonyl-4,7-dihydro-2h - isoindole ( 1.18 g ,
5.4 mol ) and benzaldehyde ( 0.285 g , 2.7 mmol ) following the procedure
described above for dipyrromethane 5 .
dipyrromethane
4 was prepared from 1-tert - butoxycarbonyl-4,7-dihydro-2h - isoindole ( 0.8672 g , 4 mmol ) and 3,5-dibutoxycarbonylbenzaldehyde
( 0.606 g , 2 mmol ) following the procedure described above for dipyrromethane 5 .
dipyrromethane 7 was prepared from 1-benzyloxycarbonyl-4,9-dihydro-2h - benzo[f]isoindole ( 1.175 d , 3.88 mmol )
and 3,5-dibutoxycarbonylbenzaldehyde ( 0.593 g , 1.94 mmol ) following
the procedure described above for dipyrromethane 6 .
maldi - tof ( m / z ) : calcd for c41h42n2o4 626.31 , found 627.13
[ m + h ] .
to avoid degradation , dipyrromethanes 3 ,
4 , 7 , 8 , 9 , and 12 were
immediately introduced into their respective subsequent syntheses
without purification . 1 ( 0.5664 g , 1.44 mmol ) and 8 ( 0.47 g ,
1.44 mmol ) were dissolved in methanol ( 145 ml ) , and the reaction mixture
was purged with ar .
an excess of zn(oac)22h2o ( 3.1409 g , 14.4 mmol ) was added , and the reaction mixture was refluxed
for 2 h , during which time the solution color turned deep red .
after
2 h , the reaction mixture was cooled to room temperature , and ddq
( 0.9820 g , 4.3 mmol ) was added .
the methanol was evaporated under reduced pressure , and the product
was purified by column chromatograpy ( silica gel , ch2cl2 ) .
the fraction containing the target porphyrin ( as monitored
by uv vis spectroscopy ) was collected and evaporated to dryness
to give crude zn13 .
zn13 ( 0.14 g , 0.2 mmol )
was treated with ddq ( 0.145 g , 0.64 mmol ) in refluxing thf ( 200 ml )
during 30 min .
the
reaction mixture was concentrated in vacuum and subjected to column
chromatography ( silica gel , ch2cl2 ) .
after solvent
removal and drying in vacuum , zn18 was isolated
as a purple crystalline powder .
uv vis ( thf ) max ( nm ) : 427 , 561 , 599 .
h nmr ( cdcl3 ) ( ppm ) : 4.11 ( 3h , s , och3 ) , 7.09 ( 2h , d , j = 8.0 hz , bn ) , 7.59 ( 2h , dd , j1 = j2 = 7.5 hz , bn ) , 7.877.95 ( 4h , m , bn ,
ph ) , 8.018.09 ( 3h , m , ph ) , 8.19 ( 2h , d , j = 8.3 hz , ar ) , 8.40 ( 2h , d , j = 8.3 hz , ar ) , 8.82 ( 2h , d , j = 4.3
hz , pyr ) , 9.07 ( 2h , d , j = 4.3 hz , pyr ) ,
9.25 ( 2h , d , j = 7.5 hz , bn ) ,
10.20 ( 2h , s , meso - h ) .
maldi - tof
( m / z ) : calcd for c42h26n4o2zn 684.07 , found 684.36 [ m ] .
zn18 ( 0.020 g , 0.029 mmol ) was
dissolved in
ch2cl2 ( 35 ml ) , and the mixture was shaken with
hcl ( 20% aq . , 2 35 ml ) in a separatory funnel .
the organic
layer was washed with water ( 30 ml ) and dried over na2so4 .
the solvent was removed in vacuum , and the solid was subjected
to column chromatography ( silica gel , ch2cl2 ) .
target porphyrin 18 was isolated as a purple crystalline
powder ( mp > 300 c ) .
vis ( thf )
max ( nm ) : 420 , 527 , 560 , 585 , 641 .
h
nmr ( cdcl3 ) ( ppm ) : 2.00 to 1.91
( 1h , m , broad ) , 1.71 to 1.58 ( 1h , m , broad ) , 4.14
( 3h , s , och3 ) , 7.33 ( 2h , d , j = 8.1 hz , ph ) , 7.71 ( 2h , dd , j1 = 7.9 , j2 = 7.3 hz , bn ) , 7.95 ( 2h , dd , j1 = 7.4 , j2 = 7.3 hz , bn ) , 8.018.08 ( 3h ,
m , ph ) , 8.178.23 ( 2h , m , bn ) , 8.328.38
( 2h , m , ar ) , 8.468.51 ( 2h , m , ar ) , 8.898.95 ( 2h , m ,
pyr ) ,
9.249.29 ( 2h , m , pyr ) , 9.429.49 ( 2h , m , bn ) ,
10.4710.53 ( 2h , m , meso - h ) .
c nmr ( cdcl3 ) ( ppm ) : 52.5 , 99.1 ,
114.7 , 120.4 , 120.9 , 125.5 , 127.6 , 127.6 , 128.3 , 129.2 , 129.3 , 129.6 ,
129.9 , 130.0 , 132.8 , 134.5 , 137.5 , 139.9 , 140.3 , 141.3 , 144.6 , 144.8 ,
144.9 , 145.8 , 167.4 .
maldi - tof ( m / z ) : calcd for c42h28n4o2 620.22 , found 621.69 [ m + h ] .
hrms ( esi - tof ) m / z : [ m + h ] calcd for c42h29n4o2 621.22848 , found
621.22687 . 2
( 0.373 g , 0.67 mmol ) and
9 ( 0.350 g , 0.67 mmol ) were dissolved in benzene ( 65 ml ) , and the
solution was purged with ar . zn(oac)22h2o
was added in excess ( 1.45 g , 6.7 mmol ) , and the mixture was heated
under reflux with a dean stark trap for 2 h under ar .
the reaction
mixture was purged with air , and the refluxing was continued under
air for 12 h. the reaction progress was monitored by uv
the solvent was removed in vacuum , and the remaining
material was subjected to column chromatography ( silica gel , ch2cl2/etoac = 20:1 ) .
the band containing the target
porphyrin was collected , and the solvent was removed in vacuum to
give crude zn14 . zn14 ( 0.205 g , 0.2 mmol ) was
dissolved in thf ( 50 ml ) , and ddq ( 0.095 g , 0.42 mmol ) was added .
the reaction mixture was heated under reflux for 40 min , and the reaction
progress was monitored by uv vis spectroscopy .
the solvent
was removed under vacuum , and the residue was diluted with ch2cl2 ( 100 ml ) , washed with na2so3 ( 2 50 ml ) and water , and dried over na2so4 .
the solution was concentrated , and the product was
purified by column chromatography ( silica gel , ch2cl2/etoac = 20:1 ) to give porphyrin zn19 as a purple crystalline powder .
uv vis ( thf ) max ( nm ) : 430 , 561 , 597
. maldi - tof
( m / z ) : calcd for c60h56n4o8zn 1024.34 , found 1024.35 [ m ] .
zn19 ( 0.200 g , 0.195 mmol ) was
dissolved in
ch2cl2 ( 150 ml ) .
( 20% , 2 100 ml ) and then water ( 100 ml )
and dried over na2so4 .
the solvent was removed
in vacuum , and the product was purified by column chromatography ( silica
gel , ch2cl2/etoac = 20:1 ) and further by reprecipitation
from ch2cl2 upon addition of meoh / h2o ( 50:0.5 v / v ) .
the precipitate was isolated by centrifugation ,
washed with meoh , and dried in vacuum to give the title compound as
a purple crystalline powder ( mp 274275 c ) .
vis ( thf ) max ( nm ) : 421 , 529 ,
562 , 584 , 640 .
h nmr ( cdcl3 ) ( ppm ) :
2.04 to 1.52 ( 2h , m , broad ) , 0.89 ( 6h , t , j = 7.4 hz , ch3 ) , 0.96 ( 6h , t , j = 7.4 hz , ch3 ) , 1.361.54 ( 8h , m , ch2 ) , 1.701.85 ( 8h , m , ch2 ) , 4.42 ( 4h , t , j = 6.7 hz , och2 ) ,
4.48 ( 4h , t , j = 6.7 hz , och2 ) , 7.23 ( 2h , d , j = 8.1 hz , bn ) , 7.74 ( 2h , dd , j1 = j2 = 7.3 hz , bn ) , 8.07 ( 2h , dd , j1 = j2 = 7.3 hz , bn ) , 8.87 ( 2h , d , j = 4.4 hz , pyr ) , 9.099.13 ( 4h , m , ar ) , 9.159.18
( 1h , m , ar ) , 9.30 ( 2h , d , j = 4.4 hz ,
pyr ) , 9.429.45 ( 1h , m , ar ) , 9.48 ( 2h , d , j = 7.9 hz , bn ) , 10.53 ( 2h , s , meso - h ) .
c nmr ( cdcl3 )
( ppm ) : 13.69 , 13.74 , 19.2 , 19.3 , 30.6 , 30.8 , 65.6 , 65.8 , 99.4 , 112.2 ,
119.5 , 121.1 , 124.9 , 127.7 , 127.9 , 130.0 , 130.06 , 130.14 , 130.3 , 131.6 ,
131.9 , 137.1 , 137.5 , 138.4 , 139.9 , 140.3 , 141.6 , 142.3 , 144.5 , 144.9 ,
145.1 , 165.8 , 166.1 .
maldi - tof ( m / z ) : calcd for c60h58n4o8 962.43 , found 963.11 [ m + h ] .
hrms ( esi - tof ) m / z : [ m + h ] calcd for c60h59n4o8 963.43269 , found
963.43134 .
zn17 was prepared similarly to
zn14 ( see the synthesis of zn19 above )
from 2 ( 0.316 g , 0.56 mmol ) and 12 ( 0.353 g , 0.56 mmol ) .
the reaction was complete in 18 h. zn17 was purified by column
chromatography ( silica gel , ch2cl2/thf = 70:1 ) .
maldi - tof ( m / z ) : calcd for c68h64n4o8zn 1128.40 , found
1128.52 [ m ] .
zn17 ( 0.09 g , 0.08 mmol ) was
dissolved in ch2cl2 ( 30 ml ) , and ddq ( 0.038
g , 0.168 mmol ) was added .
the reaction mixture was stirred for 10
min at room temperature and was monitored by uv vis spectroscopy .
the solvent was removed in vacuum , and the remaining material was
dissolved in ch2cl2 ( 100 ml ) , washed with na2so3 ( 2 50 ml ) and water ( 50 ml ) , and dried
over na2so4 .
the mixture was chromatographed
( silica gel , ch2cl2/thf = 70:1 ) , and zn22 was isolated as a green crystalline powder .
vis ( thf ) max ( nm ) :
452 , 583 , 623 .
maldi - tof ( m / z ) :
calcd for c68h60n4o8zn
1124.37 , found 1124.05 [ m ] .
zn22 ( 0.080 g , 0.071 mmol ) was dissolved in
ch2cl2 ( 100 ml ) , and the solution was shaken
with hcl aq .
( 10% , 4 100 ml ) in a separatory funnel , washed
with water ( 100 ml ) , and dried over na2so4 .
the solvent was removed in vacuum , and the product was purified by
column chromatography ( silica gel , ch2cl2/thf
= 70:1 ) .
the title porphyrin 22 was precipitated from
ch2cl2/thf solution upon addition of meoh ( 1:100
v / v ) and isolated as a green crystalline powder ( mp 286288
c ) .
vis ( thf ) max ( nm ) : 422 , 454 , 521 , 560 , 594 , 661 .
h nmr ( cdcl3 ) ( ppm ) : 0.72 ( 1h , broad ) , 0.25 ( 1h ,
broad ) , 0.83 ( 6h , t , j = 7.4 hz , ch3 ) , 0.96 ( 6h , t , j = 7.4 hz , ch3 ) , 1.311.43
( 4h , m , ch2 ) , 1.441.55
( 4h , m , ch2 ) , 1.651.76
( 4h , m , ch2 ) , 1.761.86
( 4h , m , ch2 ) , 4.41 ( 4h ,
t , j = 6.7 hz , och2 ) , 4.47 ( 4h , t , j = 6.7 hz , och2 ) , 7.59
( 2h , s ) , 7.67 ( 2h , dd , j1 =
7.4 , j2 = 7.0 hz ) , 7.76 ( 2h ,
dd , j1 = 7.4 , j2 = 6.9 hz ) , 7.88 ( 2h , d , j = 8.1 hz ) , 8.44 ( 2h , d , j = 8.1 hz ) , 8.66 ( 2h , d , j = 4.3 hz ,
pyr ) , 9.05 ( 2h , d , j = 1.4 hz , ar ) , 9.07
( 2h , d , j = 4.3 hz , pyr ) , 9.12 ( 1h , dd , j1 = j2 = 1.4 hz , ar ) , 9.17 ( 2h , d , j = 1.4 hz , ar ) , 9.59 ( 1h , dd , j1 = j2 = 1.5 hz , ar ) ,
9.82 ( 2h , s ) , 10.33 ( 2h , s , meso - h ) .
c nmr ( cdcl3 ) ( ppm ) : 13.7 , 13.8 ,
19.2 , 19.3 , 30.6 , 30.8 , 65.6 , 65.8 , 98.3 , 110.2 , 119.7 , 121.0 , 125.3 ,
126.2 , 127.1 , 128.6 , 129.0 , 129.7 , 130.1 , 130.2 , 130.3 , 131.5 , 132.5 ,
132.57 , 132.63 , 135.1 , 137.7 , 138.1 , 138.2 , 141.3 , 141.5 , 142.6 , 144.0 ,
145.5 , 146.5 .
maldi - tof ( m / z ) : calcd
for c68h62n4o8 1062.46 ,
found 1063.21 [ m + h ] .
hrms ( esi - tof ) m / z : [ m + h ] calcd for c68h63n4o8 1063.46399 , found 1063.46533 .
zn15 was synthesized
similarly to porphyrin zn14 ( see the synthesis of zn19 above ) from 2 ( 0.554 g , 1 mmol ) and 10 ( 0.92
g , 1 mmol ) .
the reaction was complete in 16 h. the product was purified
by column chromatography ( silica gel , ch2cl2/etoac = 20:1 ) to give the title compound zn15 as a purple solid .
maldi - tof ( m / z ) : calcd for c80h96n4o16zn 1432.61 , found 1432.87 [ m ] .
zn15 ( 0.300 g , 0.21 mmol ) was dissolved in ch2cl2 ( 200 ml ) , and the solution was shaken with
hcl aq .
( 20% , 2 100 ml ) in a separatory funnel , washed with
water ( 100 ml ) , and dried over na2so4 .
the solvent
was removed in vacuum , and the product was purified by column chromatography
( silica gel , ch2cl2/etoac = 20:1 ) and further
by reprecipitation from ch2cl2 upon addition
of meoh .
the precipitate was isolated by centrifugation , washed with
meoh , and dried in vacuum to give the title compound as a purple crystalline
powder ( mp 140142 c ) .
vis
( thf ) max ( nm ) : 409 , 504 , 535 , 575 , 629 .
h nmr ( cdcl3 ) ( ppm ) ( mixture of conformers ) : 3.02
to 2.58 ( 2h , m , broad ) , 0.581.01 ( 24h , m , ch3 ) , 1.041.19 and 1.331.86 ( 32h ,
m , ch2 ) , 2.973.27
( 4h , m , chh ) , 3.553.71 ( 4h ,
m , chh ) , 3.834.02 ( 4h , m ,
och2 ) , 4.174.27
( 4h , m , och2 ) , 4.404.49
( 8h , m , och2 ) , 4.494.72
( 4h , m , ch ) , 8.94 ( 2h , d , j = 4.5 hz , pyr ) , 8.948.96 ( 1h , m , ar ) ,
8.979.00 ( 1h , m , ar ) , 9.079.12 ( 2h , m , ar ) , 9.139.16
( 1h , m , ar ) , 9.209.24 ( 1h , m , ar ) , 9.38 ( 2h , d , j = 4.5 hz , pyr ) , 10.2310.24 ( 2h , m , meso - h ) .
c nmr ( cdcl3 ) ( ppm ) ( mixture of conformers ) : 13.3 , 13.4 , 13.67 , 13.70 ,
13.74 , 13.77 , 13.77 , 13.78 , 18.75 , 18.84 , 19.14 , 19.17 , 19.21 , 19.27 ,
19.31 , 22.7 , 24.0 , 24.3 , 28.1 , 28.5 , 29.7 , 30.27 , 30.34 , 30.63 , 30.65 ,
30.74 , 40.7 , 40.8 , 41.8 , 41.9 , 64.6 , 64.7 , 64.9 , 65.56 , 65.66 , 65.72 ,
101.6 , 115.0 , 115.1 , 117.3 , 129.1 , 129.2 , 130.03 , 130.04 , 130.06 ,
130.5 , 130.6 , 130.7 , 131.1 , 131.8 , 136.6 , 136.8 , 137.0 , 137.1 , 138.59 ,
138.60 , 140.0 , 140.1 , 141.8 , 142.0 , 142.3 , 144.8 , 144.9 , 146.3 , 165.5 ,
165.8 , 166.0 , 166.1 , 172.5 , 172.6 , 173.1 , 173.3 .
maldi - tof ( m / z ) : calcd for c80h98n4o16 1370.70 , found 1371.14 [ m + h ] .
hrms ( esi - tof ) m / z : [ m + h ] calcd for c80h99n4o16 1371.70497 , found 1371.70702 .
zn16 was synthesized
similarly to porphyrin zn14 ( see the synthesis of zn19 above ) from 2 ( 0.484 g , 0.86 mmol ) and 11
( 0.42 g , 0.86 mmol ) .
the reaction was complete in 12 h. the product
was purified by column chromatography ( silica gel , ch2cl2/thf , gradient from 50:1 to 25:1 ) .
maldi - tof ( m / z ) : calcd for c52h52n4o12zn 988.29 , found
987.93 [ m ] .
zn16 ( 0.200 g ,
0.2 mmol ) was dissolved in ch2cl2 ( 200 ml ) ,
and the solution was shaken with hcl aq .
( 20% , 2 100 ml ) in
a separatory funnel , washed with water ( 100 ml ) , and dried over na2so4 .
the solvent was removed in vacuum , and the
product was purified by column chromatography ( silica gel , ch2cl2/thf = 5:1 ) and further by reprecipitation from
ch2cl2/thf ( 1:1 ) upon addition of meoh .
the
precipitate was isolated by centrifugation , washed with meoh , and
dried in vacuum to give the title compound as a brown crystalline
powder ( mp 287288 c ) . yield 0.182 g ( 98% ) .
vis
( dma ) max ( nm ) : 404 , 499 , 531 , 568 , 623 .
h nmr ( cdcl3 ) ( ppm ) ( mixture of conformers ) : 3.75
to 3.17 ( 2h , m , broad ) , 0.901.04 ( 6h , m , ch3 ) , 1.421.53 ( 4h , m , ch2 ) , 1.741.85 ( 4h , m , ch2 ) , 3.623.99 ( 4h , m , chh ) , 3.76 ( 3h , s , och3 ) , 3.77 ( 3h , s , och3 ) ,
3.80 ( 3h , s , och3 ) , 3.82 ( 3h ,
s , och3 ) , 4.074.28 ( 4h ,
m , chh ) , 4.394.49 ( 4h , m ,
och2 ) , 4.504.65
( 4h , m , ch ) , 8.89 ( 1h , d , j = 4.5 hz , pyr ) , 8.90 ( 1h , d , j = 4.5 hz , pyr ) , 9.069.12 ( 2h , m , ar ) , 9.139.17
( 1h , m , ar ) , 9.33 ( 1h , d , j = 4.5 hz ,
pyr ) , 9.34 ( 1h , d , j = 4.5 hz , pyr ) ,
9.649.79 ( 1h , m , meso - h ) ,
10.05 ( 2h , s , meso - h ) .
c nmr ( cdcl3 ) ( ppm ) ( mixture of conformers ) : 13.7 ,
13.8 , 19.25 , 19.27 , 23.7 , 23.9 , 24.0 , 30.7 , 30.8 , 40.87 , 40.90 , 41.05 ,
41.07 , 52.19 , 52.23 , 65.6 , 95.59 , 95.63 , 101.1 , 101.2 , 117.82 , 117.83 ,
129.8 , 130.1 , 130.5 , 130.8 , 136.6 , 136.7 , 138.39 , 138.42 , 142.56 ,
142.59 , 166.1 , 166.2 , 173.36 , 173.38 , 173.44 .
maldi - tof ( m / z ) : calcd for c52h54n4o12 926.37 , found 927.13 [ m + h ] , 965.07
[ m + k ] .
hrms ( esi - tof ) m / z : [ m + h ] calcd for c52h55n4o12 927.38102 , found 927.38357 . a free - base porphyrin , platinum(ii )
acetylacetonate , and benzoic acid were mixed together and heated to
130140 c under ar .
the reaction progress was monitored
by uv vis spectroscopy ; samples were analyzed every 15 min .
the reaction was stopped after the q00 band maximum of
the free - base porphyrin disappeared .
the mixture was dispersed in
meoh / h2o ( 2:1 , 50 ml ) , and the precipitate , which
contained the target pt porphyrin , was isolated by centrifugation .
it was redispersed in meoh / h2o ( 2:1 , 20 ml ) and
centrifuged again .
this washing procedure was repeated three times ,
after which the final solid was dried in vacuum .
the product was purified
by column chromatography , and the fraction containing the target compound
was collected .
the solvent was removed in vacuum , and the target metalloporphyrin
was reprecipitated from ch2cl2 by addition of
meoh .
a free - base porphyrin was dissolved
in dry benzonitrile
( 24 ml ) , and the solution was placed into a thick - walled
microwave vial ( biotage , 5 ml ) .
platinum(ii ) acetylacetonate or palladium(ii )
acetate was added , and the vessel was sealed , after which the mixture
was subjected to microwave irradiation at 250 c ( 2 bar ,
105145 w ) for 40 min under stirring .
the reaction progress
was monitored by uv vis spectroscopy ; samples were analyzed
every 20 min .
the reaction was stopped after the q00 band
maximum of the free - base porphyrin disappeared .
after the mixture
was cooled , benzonitrile was removed in vacuum , and the product was
purified by column chromatography on silica gel .
the fraction containing
the target compound was collected , and the solvent was removed in
vacuum .
the target metalloporphyrin was reprecipitated from ch2cl2 upon addition of meoh , and the precipitate
was collected by centrifugation and dried in vacuum .
pt15 was synthesized
from 15 ( 0.153 g , 0.108 mmol ) and pt(acac)2 ( 0.170 g , 0.433 mmol ) in benzoic acid ( 3 g ) .
the reaction was complete
in 6 h. chromatography was performed using ch2cl2/thf ( 50:1 ) .
the target compound was isolated as an orange solid
( mp 157160 c ) .
vis ( thf )
max ( nm ) : 391 , 503 , 535 .
h nmr ( cdcl3 ) ( ppm ) ( mixture of conformers ) : 0.591.01
( 24h , m , ch3 ) , 1.041.19
and 1.341.86 ( 32h , m , ch2 ) , 2.873.12 ( 4h , m , chh ) ,
3.483.63 ( 4h , m , chh ) , 3.864.02
( 4h , m , och2 ) , 4.174.59
( 16h , m , och2 , ch ) , 8.79 ( 2h , d , j =
4.9 hz , pyr ) , 8.908.94 ( 2h , m , ar ) , 8.989.06 ( 2h ,
m , ar ) , 9.129.16 ( 1h , m , ar ) , 9.199.25 ( 3h , m , ar ,
pyr ) , 10.0310.08 ( 2h , m , meso - h ) .
c nmr ( cdcl3 ) ( ppm ) ( mixture of
conformers ) : 13.37 , 13.44 , 13.68 , 13.70 , 13.73 , 13.76 , 13.78 , 18.76 ,
18.83 , 19.15 , 19.18 , 19.20 , 19.26 , 19.31 , 23.68 , 23.71 , 23.99 , 28.82 ,
29.16 , 29.69 , 30.30 , 30.36 , 30.63 , 30.65 , 30.73 , 40.36 , 40.41 , 41.86 ,
41.94 , 64.6 , 64.7 , 64.9 , 65.6 , 65.7 , 65.8 , 104.0 , 117.3 , 177.4 , 119.4 ,
129.8 , 129.9 , 130.3 , 130.5 , 130.6 , 130.7 , 131.2 , 131.3 , 136.6 , 136.7 ,
137.0 , 137.1 , 137.2 , 137.8 , 138.3 , 138.4 , 138.6 , 138.7 , 139.1 , 139.2 ,
139.4 , 140.5 , 141.70 , 141.79 , 141.83 , 165.5 , 165.7 , 166.0 , 166.1 ,
172.5 , 172.6 , 173.1 , 173.2 .
hrms ( esi - tof ) m / z : [ m + h ] calcd for c80h97n4o16pt 1565.65650 , found 1565.65826 .
pt15 was synthesized
from 15 ( 0.065 g , 0.0475 mmol ) and pt(acac)2 ( 0.056 g , 0.143 mmol ) .
pd15 was synthesized
from 15 ( 0.017 g , 0.0124 mmol ) and pd(oac)2 ( 0.011 g , 0.05 mmol ) .
chromatography
was performed using ch2cl2/thf ( 50:1 ) to give
crude pd15 ( 0.018 g ) , which was used in the subsequent
aromatization reaction without further purification .
pt16 was synthesized
from 16 ( 0.05 g , 0.054 mmol ) and pt(acac)2 ( 0.085 g , 0.22 mmol ) .
pt16 was isolated as an orange
crystalline solid ( mp > 300 c ) .
vis
( dma ) max ( nm ) : 384 , 500 , 531 .
h nmr
( cdcl3 ) ( ppm ) ( mixture of conformers ) : 0.881.00
( 6h , m , ch3 ) , 1.421.54
( 4h , m , ch2 ) , 1.711.87
( 4h , m , ch2 ) , 2.993.52
( 8h , m , chh ) , 3.71 ( 3h , s , och3 ) , 3.75 ( 3h , s , och3 ) , 3.76 ( 3h , s , och3 ) , 3.79 ( 3h , s , och3 ) , 4.064.34
( 4h , m , ch ) , 4.404.53 ( 4h ,
m , och2 ) , 8.78 ( 1h , d , j = 4.8 hz , pyr ) , 8.79 ( 1h , d , j = 4.9 hz , pyr ) , 8.848.91 and 8.969.00
( 2h , m , ar ) , 9.079.11 ( 1h , m , ar ) , 9.129.20 ( 3h , m ,
ar , pyr ) , 9.219.29 and 9.489.63 ( 2h , m , meso - h ) , 9.71 ( 1h , s , broad , meso - h ) .
the c nmr spectrum was not recorded because
of the low solubility . maldi - tof ( m / z ) : calcd for c52h52n4o12pt 1119.32 , found 1119.73 [ m ] .
hrms ( esi - tof ) m / z : [ m + h ] calcd for c52h53n4o12pt 1120.33057 , found
1120.33205 .
pd16 was synthesized
from 16 ( 0.015 g , 0.0162 mmol ) and pd(oac)2 ( 0.0145 g , 0.065 mmol ) .
chromatography
was performed using ch2cl2/thf ( 25:1 ) to give
crude pd16 ( 0.0165 g ) , which was used in the
aromatization step without further purification ( see below ) .
pt18 was synthesized
from 18 ( 0.015 g , 0.026 mmol ) and pt(acac)2 ( 0.041 g , 0.1 mmol ) in benzoic acid ( 1.5 g ) .
the target compound was isolated as a pink crystalline powder ( mp
> 300 c ) .
uv vis ( dma ) max / nm ( log ) : 401 ( 5.33 ) , 519 ( 4.19 ) , 556 ( 4.91 ) .
h nmr ( cdcl3 ) ( ppm ) : 4.13 ( 3h , s , och3 ) , 7.04 ( 2h , d , j = 8.3 hz , bn ) , 7.58 ( 2h , dd , j1 = 8.0 , j2 = 7.8 hz , bn ) , 7.897.96 ( 4h , m , bn ,
ph ) , 8.028.12 ( 3h , m , ph ) , 8.26 ( 2h , d , j = 8.2 hz , ar ) , 8.46 ( 2h , d , j = 8.2 hz , ar ) , 8.82 ( 2h , d , j = 4.7
hz , pyr ) , 9.16 ( 2h , d , j = 4.7 hz , pyr ) ,
9.25 ( 2h , d , j = 7.8 hz , bn ) ,
10.39 ( 2h , s , meso - h ) .
c nmr ( cdcl3 ) ( ppm ) : 52.5 , 101.4 , 114.5 , 119.9 ,
122.1 , 125.9 , 127.2 , 127.3 , 128.2 , 128.6 , 129.37 , 129.43 , 129.8 , 130.4 ,
130.5 , 132.5 , 133.8 , 136.3 , 137.5 , 139.2 , 139.4 , 140.2 , 141.5 , 145.9 ,
167.3 .
maldi - tof ( m / z ) : calcd for
c42h26n4o2pt 813.17 , found
813.41 [ m ] . hrms ( esi - tof ) m / z : [ m ] calcd for c42h26n4o2pt 813.17012 , found 813.16860 . pt19 was synthesized
from 19 ( 0.12 g , 0.132 mmol ) and pt(acac)2 ( 0.21 g , 0.53 mmol ) in benzoic acid ( 2.00 g , 16.4 mmol ) .
the reaction
was complete in 6 h. chromatography was performed using ch2cl2/thf = 50:1 .
the target compound was isolated as a
dark - red crystalline powder ( mp > 300 c ) .
uv vis ( dma ) max / nm ( log ) : 402
( 5.34 ) , 521 ( 4.16 ) , 559 ( 4.92 ) .
h nmr ( cdcl3 ) ( ppm ) : 0.87 ( 6h , t , j = 7.4
hz , ch3 ) , 0.94 ( 6h , t , j = 7.4 hz , ch3 ) , 1.351.51 ( 8h , m , ch2 ) , 1.681.83 ( 8h , m , ch2 ) , 4.41 ( 4h , t , j = 6.7
hz , och2 ) , 4.45 ( 4h , t , j = 6.7 hz , och2 ) , 6.96 ( 2h , d , j = 8.0
hz , bn ) , 7.60 ( 2h , dd , j1 = j2 = 7.5 hz ,
bn ) , 7.96 ( 2h , dd , j1 = j2 = 7.2 hz , bn ) ,
8.78 ( 2h , d , j = 3.5 hz , pyr ) , 9.03 ( 2h ,
d , j = 1.6 hz , ar ) , 9.07 ( 2h , d , j = 1.6 hz , ar ) , 9.15 ( 1h , dd , j1 = j2 = 1.6 hz , ar ) , 9.22 ( 2h , d , j = 3.6 hz , pyr ) , 9.31 ( 2h , d , j = 7.5
hz , bn ) , 9.44 ( 1h , dd , j1 = j2 = 1.6 hz ,
ar ) , 10.50 ( 2h , s , meso - h ) .
c nmr ( cdcl3 ) ( ppm ) : 13.7 , 13.8 , 19.2 ,
19.3 , 30.6 , 30.8 , 65.6 , 65.8 , 101.4 , 114.5 , 120.1 , 120.5 , 125.2 , 127.4 ,
127.5 , 128.7 , 129.8 , 130.1 , 130.3 , 131.7 , 132.1 , 136.1 , 137.1 , 137.1 ,
137.4 , 137.7 , 138.1 , 139.1 , 140.1 , 141.7 , 142.4 , 165.8 , 166.1 . maldi - tof
( m / z ) : calcd for c60h56n4o8pt 1155.37 , found 1155.01 [ m ] .
hrms ( esi - tof ) m / z : [ m ] calcd for c60h56n4o8pt 1155.37445 , found 1155.37554 .
pt19 was synthesized
from 19 ( 0.02 g , 0.021 mmol ) and pt(acac)2 ( 0.024 g , 0.062 mmol ) and purified as in method 1 above .
pd19 was synthesized
from 19 ( 0.01 g , 0.01 mmol ) and pd(oac)2 ( 0.0093
g , 0.042 mmol ) .
pd19 was isolated as a dark - purple
crystalline solid ( mp 296297 c ) .
uv vis ( dma ) max ( nm ) : 417 , 534 , 570 .
h nmr ( cdcl3 ) ( ppm ) : 0.88 ( 6h , t , j = 7.4 hz , ch3 ) , 0.95 ( 6h , t , j = 7.4 hz , ch3 ) , 1.351.54 ( 8h , m , ch2 ) , 1.681.84 ( 8h , m , ch2 ) , 4.41 ( 4h , t , j = 6.8 hz , och2 ) ,
4.46 ( 4h , t , j = 6.7 hz , och2 ) , 7.01 ( 2h , d , j = 8.1 hz , bn ) , 7.62 ( 2h , dd , j1 = 7.8 , j2 = 7.4 hz , bn ) , 7.95 ( 2h , dd , j1 = j2 = 7.4 hz , bn ) , 8.76 ( 2h , d , j = 4.6 hz , pyr ) , 9.03 ( 2h , d , j = 1.6 hz , ar ) , 9.07 ( 2h , d , j = 1.6
hz , ar ) , 9.139.19 ( 3h , m , pyr , ar ) , 9.29 ( 2h , d , j = 7.8 hz , bn ) , 9.44 ( 1h , dd , j1 = j2 = 1.6 hz , ar ) , 10.42 ( 2h , s , meso - h ) .
c nmr ( cdcl3 ) ( ppm ) :
13.69 , 13.74 , 19.2 , 19.3 , 30.6 , 30.8 , 65.6 , 65.8 , 101.2 , 114.6 , 120.0 ,
120.3 , 125.1 , 127.3 , 127.4 , 129.4 , 129.8 , 130.2 , 130.6 , 131.7 , 132.0 ,
136.9 , 137.5 , 137.7 , 137.8 , 138.6 , 138.7 , 139.4 , 140.8 , 142.1 , 142.8 ,
165.8 , 166.1 .
maldi - tof ( m / z ) : calcd
for c60h56n4o8pd 1066.31 ,
found 1065.90 [ m ] .
hrms ( esi - tof ) m / z : [ m ] calcd for c60h56n4o8pd 1066.31478 , found 1066.31410 .
pt22 was synthesized
from 22 ( 0.032 g , 0.03 mmol ) and pt(acac)2 ( 0.05 g , 0.12 mmol ) in benzoic acid ( 2 g ) .
the target compound was isolated as a dark - green crystalline powder
( mp > 300 c ) .
uv vis ( dma ) max / nm ( log ) : 423 ( 5.02 ) , 549 ( 4.16 ) , 593 ( 4.93 ) .
h nmr ( cdcl3 ) ( ppm ) : 0.81 ( 6h , t , j = 7.3 hz , ch3 ) , 0.95 ( 6h , t , j = 7.3 hz , ch3 ) , 1.301.41 ( 4h , m , ch2 ) , 1.411.53 ( 4h , m , ch2 ) , 1.651.74 ( 4h , m , ch2 ) , 1.751.84 ( 4h , m , ch2 ) , 4.40 ( 4h , t , j = 6.7 hz , och2 ) ,
4.46 ( 4h , t , j = 6.7 hz , och2 ) , 7.357.43 ( 2h , m , broad ) ,
7.607.67 ( 2h , m , broad ) , 7.697.76 ( 2h , m , broad ) ,
7.767.83 ( 2h , m , broad ) , 8.368.40 ( 2h , m , broad ) ,
8.67 ( 2h , m , broad ) , 9.02 ( 2h , d , j =
1.5 hz , ar ) , 9.11 ( 2h , m , broad ) , 9.15 ( 1h , dd , j1 = j2 = 1.6 hz , ar ) , 9.17 ( 2h , d , j = 1.5
hz , ar ) , 9.63 ( 1h , dd , j1 = j2 = 1.5 hz , ar ) , 9.72 ( 2h , s ,
broad ) , 10.46 ( 2h , s , broad , meso - h ) .
hrms ( esi - tof ) m / z : [ m ] calcd for c68h60n4o8pt 1256.40747 , found 1256.40717 .
pd22 was synthesized
from 22 ( 0.004 g , 0.0038 mmol ) and pd(oac)2 ( 0.0034 g , 0.015 mmol ) .
pd22 was isolated as a dark - green crystalline powder ( mp > 300
c ) .
vis ( dma ) max ( nm ) : 435 , 561 , 605 .
h nmr ( cdcl3 )
( ppm ) : 0.81 ( 6h , t , j = 7.4 hz , ch3 ) , 0.95 ( 6h , t , j = 7.4 hz , ch3 ) , 1.281.41
( 4h , m , ch2 ) , 1.411.54
( 4h , m , ch2 ) , 1.641.74
( 4h , m , ch2 ) , 1.741.84
( 4h , m , ch2 ) , 4.40 ( 4h ,
t , j = 6.5 hz , och2 ) , 4.46 ( 4h , t , j = 6.6 hz , och2 ) , 7.427.49
( 2h , m , broad ) , 7.607.69 ( 2h , m , broad ) ,
7.707.78
( 2h , m , broad ) , 7.807.87 ( 2h , m , broad ) , 8.388.47
( 2h , m , broad ) , 8.668.74 ( 2h , m , broad ) , 9.009.05
( 2h , m , broad ) , 9.109.20 ( 5h , m , broad ) , 9.609.66
( 1h , m , broad ) , 9.739.82 ( 2h , m , broad ) , 10.4610.53
( 2h , m , broad ) .
the c nmr spectrum was not recorded because
of the low solubility . maldi - tof ( m / z ) : calcd for c68h60n4o8pd 1166.34 , found 1165.88 [ m ] .
hrms ( esi - tof ) m / z : [ m ] calcd for c68h60n4o8pd 1166.34628 , found 1166.34671 .
the porphyrin was dissolved
in toluene ( 50 ml ) , and ddq was added .
the reaction mixture
was refluxed , and the reaction progress was monitored by uv
the reaction was stopped when no further changes could
be observed in the spectra .
the reaction mixture was diluted with
ch2cl2 ( 100 ml ) , washed with na2so3 ( 10% aqueous solution , 2 50 ml ) and water ( 50 ml ) ,
and dried over na2so4 .
the solvents were removed
in vacuum , and the product was purified by column chromatography on
silica gel .
the fraction containing the target compound was collected ,
and the solvent was removed in vacuum .
the target metalloporphyrin
was reprecipitated from ch2cl2 upon addition
of meoh , and the precipitate was collected by centrifugation and dried
in vacuum .
pt20 was synthesized from
pt15 ( 0.05 g , 0.033 mmol ) using ddq ( 0.03 g ,
0.132 mmol ) .
the reaction was complete in 10 h. chromatography was
performed using ch2cl2/thf = 50:1 .
pt20 was isolated as a bright - purple crystalline solid ( mp 256258
c ) .
uv vis ( dma ) max / nm ( log ) : 412 ( 5.43 ) , 527 ( 4.29 ) , 568 ( 5.01 ) .
h nmr ( cdcl3 ) ( ppm ) : 0.87 ( 6h , t , j = 7.4 hz , ch3 ) , 0.95 ( 6h , t , j = 7.4 hz , ch3 ) , 1.05 ( 6h , t , j = 7.4 hz , ch3 ) , 1.10 ( 6h , t , j = 7.4 hz , ch3 ) , 1.341.62 ( 16h , m , ch2 ) , 1.691.92 ( 16h , m , ch2 ) , 4.35 ( 4h , t , j = 7.1 hz , och2 ) , 4.42
( 4h , t , j = 6.8 hz , och2 ) , 4.46 ( 4h , t , j = 6.7 hz , och2 ) ,
4.54 ( 4h , t , j = 6.8 hz , och2 ) , 7.55 ( 2h , s , bn ) ,
8.85 ( 2h , d , j = 4.9 hz , pyr ) , 9.03 ( 2h ,
d , j = 1.6 hz , ar ) , 9.07 ( 2h , d , j = 1.6 hz , ar ) , 9.17 ( 1h , dd , j1 = j2 = 1.6 hz , ar ) , 9.31 ( 2h , d , j = 4.9 hz , pyr ) , 9.46 ( 1h , dd , j1 = j2 = 1.6 hz , ar ) ,
9.61 ( 2h , s , bn ) , 10.63 ( 2h , s , meso - h ) .
c nmr ( cdcl3 )
( ppm ) : 13.66 , 13.73 , 13.79 , 13.83 , 19.16 , 19.17 , 19.26 , 19.32 , 30.63 ,
30.68 , 30.73 , 65.65 , 65.68 , 65.8 , 66.1 , 103.3 , 116.0 , 120.8 , 121.4 ,
126.9 , 129.4 , 130.1 , 130.4 , 130.6 , 131.4 , 132.2 , 132.3 , 132.7 , 135.4 ,
136.7 , 137.1 , 137.5 , 137.6 , 139.1 , 140.3 , 140.9 , 141.1 , 141.2 , 165.2 ,
165.9 , 166.7 , 168.8 . hrms ( esi - tof ) m / z : [ m + h ] calcd for c80h89n4o16pt 1557.59389 , found 1557.5965 .
pd-20 was prepared
from pd15 ( 0.018 g , 0.012 mmol ) using ddq ( 0.011
g , 0.049 mmol ) .
the reaction was complete in 8 h. chromatography was performed using
ch2cl2/thf = 50:1 .
pd20 was isolated as a dark - purple crystalline solid ( mp 243245
c ) .
vis ( dma ) max ( nm ) : 427 , 541 , 581 .
h nmr ( cdcl3 ) ( ppm ) : 0.88 ( 6h , t , j = 7.4 hz , ch3 ) , 0.95 ( 6h , t , j = 7.4 hz , ch3 ) , 1.05 ( 6h , t , j = 7.4 hz , ch3 ) , 1.10 ( 6h , t , j = 7.4 hz , ch3 ) , 1.351.62 ( 16h , m , ch2 ) , 1.701.92 ( 16h , m , ch2 ) , 4.36 ( 4h , t , j = 7.1 hz , och2 ) , 4.42
( 4h , t , j = 6.8 hz , och2 ) , 4.47 ( 4h , t , j = 6.7 hz , och2 ) ,
4.54 ( 4h , t , j = 6.8 hz , och2 ) , 7.61 ( 2h , s , bn ) ,
8.86 ( 2h , d , j = 4.8 hz , pyr ) , 9.04 ( 2h ,
d , j = 1.5 hz , ar ) , 9.08 ( 2h , d , j = 1.5 hz , ar ) , 9.17 ( 1h , dd , j1 = j2 = 1.6 hz , ar ) , 9.32 ( 2h , d , j = 4.8 hz , pyr ) , 9.46 ( 1h , dd , j1 = j2 = 1.6 hz , ar ) ,
9.66 ( 2h , s , bn ) , 10.67 ( 2h , s , meso - h ) .
c nmr ( cdcl3 )
( ppm ) : 13.67 , 13.74 , 13.80 , 13.84 , 19.16 , 19.18 , 19.27 , 19.32 , 30.63 ,
30.69 , 30.74 , 65.65 , 65.66 , 65.8 , 66.2 , 102.7 , 115.7 , 120.7 , 121.0 ,
126.9 , 129.3 , 130.0 , 130.5 , 130.9 , 131.7 , 132.09 , 132.12 , 132.4 , 136.0 ,
136.7 , 137.2 , 137.3 , 137.8 , 137.9 , 140.3 , 141.50 , 141.52 , 165.2 , 166.0 ,
166.8 , 168.8 .
maldi - tof ( m / z ) : calcd
for c80h88n4o16pd 1466.52 ,
found 1465.90 [ m ] .
hrms ( esi - tof ) m / z : [ m + h ] calcd for c80h89n4o16pd 1467.53279 , found 1467.53562 .
pt21 was
synthesized from
pt16 ( 0.04 g , 0.036 mmol ) using ddq ( 0.032 g ,
0.143 mmol ) .
the reaction was complete in 12 h. chromatography was
performed using ch2cl2/thf = 5:1 .
pt21 was isolated as a pink crystalline powder ( mp > 300 c ) .
vis ( dma ) max ( nm ) :
405 ( 5.40 ) , 522 ( 4.27 ) , 562 ( 5.03 ) .
h nmr ( dmso - d6 , 80 c ) ( ppm ) : 0.99 ( 6h , t , j = 7.3 hz , ch3 ) , 1.491.61 ( 4h , m , ch2 ) , 1.791.90 ( 4h , m , ch2 ) , 4.20 ( 6h , s , och3 ) , 4.21 ( 6h , s , och3 ) ,
4.51 ( 4h , t , j = 6.5 hz , och2 ) , 7.918.03 ( 2h , m , broad ) ,
8.098.27 ( 2h , m , broad ) , 8.658.99 ( 10h , m , broad ) .
the c nmr spectrum was not recorded because of the low
solubility . maldi - tof ( m / z ) : calcd
for c52h44n4o12pt 1111.26 ,
found 1111.72 [ m ] .
hrms ( esi - tof ) m / z : [ m ] calcd for c52h44n4o12pt 1111.26014 , found 1111.26239 .
pd21 was
synthesized from
pd16 ( 0.0165 g , 0.016 mmol ) using ddq ( 0.0145
g , 0.064 mmol ) .
the target porphyrin was purified by column chromatography
( silica gel , ch2cl2/thf = 5:1 ) and then by reprecipitation
from thf upon addition of meoh .
pd21 was isolated
as a dark - purple crystalline powder ( mp > 300 c ) .
vis ( dma )
max ( nm ) : 421 , 534 , 574 .
h nmr ( dmso - d6 , 80 c ) ( ppm ) : 1.00 ( 6h , t , j = 7.3 hz , ch3 ) , 1.501.61 ( 4h , m , ch2 ) , 1.801.89 ( 4h , m , ch2 ) , 4.124.22 ( 12h , m , och3 ) , 4.50 ( 4h , t , j = 6.5 hz , och2 ) , 7.627.72
( 2h , m , broad ) , 7.737.86 ( 2h , m , broad ) ,
8.008.26
( 3h , m , broad ) , 8.298.50 ( 4h , m , broad ) , 8.508.59
( 2h , m , broad ) , 8.858.92 ( 1h , m , broad ) .
maldi - tof ( m / z ) : calcd for c52h44n4o12pd 1022.20 , found 1021.71 [ m ] .
hrms ( esi - tof ) m / z : [ m ] calcd for c52h44n4o12pd 1022.20031 , found 1022.20218 . | significant effort has been directed
in recent years toward porphyrins
with enhanced two - photon absorption ( 2pa ) .
however , the properties
of their triplet states , which are central to many applications , have
rarely been examined in parallel . here
we report the synthesis of
asymmetrically -extended platinum(ii ) and palladium(ii ) porphyrins ,
whose 2pa into single - photon - absorbing states is enhanced as a result
of the broken center - of - inversion symmetry and whose triplet states
can be monitored by room - temperature phosphorescence .
5,15-diaryl - syn - dibenzoporphyrins ( dbps ) and syn - dinaphthoporphyrins
( dnps ) were synthesized by [ 2 + 2 ] condensation of the corresponding
dipyrromethanes and subsequent oxidative aromatization .
butoxycarbonyl
groups on the meso - aryl rings render these porphyrins
well - soluble in a range of organic solvents , while 5,15-meso - aryl substitution causes minimal nonplanar distortion of the macrocycle ,
ensuring high triplet emissivity .
a syn - dbp bearing
four alkoxycarbonyl groups in the benzo rings and possessing a large
static dipole moment was also synthesized .
photophysical properties
( 2pa brightness and phosphorescence quantum yields and lifetimes )
of the new porphyrins were measured , and their ground - state structures
were determined by dft calculations and/or x - ray analysis .
the developed
synthetic methods should facilitate the construction of -extended
porphyrins for applications requiring high two - photon triplet action
cross sections . |
despite progression in the development of pharmacological therapy , treatment of the alpha synucleinopathy parkinson s disease ( pd ) as well as treatment of atypical parkinsonism proteinopathies , including a range of synucleinopathies and nonsynucleinopathic neurodegenerative disorders , is still challenging .
early diagnosis and differentiation of pd from atypical parkinsonism syndromes are clinically important as it allows timely symptomatic treatment and improvement of quality of life.1 to date , our knowledge of the mechanisms whereby the pathological form of alpha - synuclein causes structural and functional damage to the nervous system is limited .
consequently , there is a lack of specific diagnostic tools to allow early differentiation between idiopathic and atypical parkinsonism syndromes and optimization of individualized therapy .
therefore , there is an urgent need for novel disease markers . during the ongoing search for such markers , analysis of intraneural alpha - synuclein depositions in small autonomic nerve fibers in the skin
has recently emerged as promising diagnostic target in patients with pd.2 skin samples are obtained in vivo via superficial punch biopsies and are immunohistochemically stained for alpha - synuclein and nerve fiber specific markers .
the procedure was shown to be safe and quantitative results showed high sensitivity and specificity in the detection of pd.3,4 however , challenges include the need for standardizing immunohistochemical analysis techniques and identifying distinct patterns of alpha - synuclein deposition among synucleinopathies in order to allow diagnostic discrimination . in this perspective , we reflect on the scientific and clinical opportunities linked to alpha - synuclein assessment in skin biopsies .
potentialities include elucidation of the peripheral nervous system pathology in synucleinopathies , such as pd , identification of novel targets to study response to neuroprotective treatment , and improvement of clinical management in patients with neurodegenerative diseases .
moreover , we reflect on future challenges in exploring the diagnostic value of skin biopsy assessment for alpha - synuclein deposition and implementing the technique in clinical practice .
the skin is innervated by afferent somatic nerves , unmyelinated c - fibers , and lightly myelinated a - delta fibers that originate in the dorsal nerve root ganglia ( small fibers ) . in skin biopsy studies of pd ,
alpha - synuclein accumulation in cutaneous autonomic small fibers has been recently linked to autonomic nervous system disturbances even in the early stages of the disease , suggesting that quantification of this protein via immunohistochemical staining might be a valid biomarker of the disease and could help improve the accuracy of diagnosis and treatment.2 this study also demonstrated that alpha - synuclein deposition normalized to intraepidermal nerve fiber density is increased in cutaneous sudomotor ( sweat gland innervating ) and pilomotor ( pilomotor muscle innervating ) nerve fibers in patients with pd and that more severe alpha - synuclein deposition relates to greater autonomic dysfunction and more advanced motor symptoms .
importantly , a clear increase in pathology was observed in the early stages of pd with substantially higher alpha synuclein depositions in patients at hoehn and yahr stage 2 compared to stage 1 .
this early increase of small fiber pathology indicates the possible utility of skin biopsy assessment in early diagnostic work - up and short - term clinical studies in patients with pd .
interestingly , another investigation found morphological similarities between alpha - synuclein deposition - related fiber damage in cutaneous small fibers and those present in cerebral neurites , which led the authors to postulate that the skin constitutes a window into brain pathology in patients with pd.3 additional research confirmed and further advanced these observations .
a study in 28 patients with pd and 23 control subjects found > 90% sensitivity and > 90% specificity to distinguish pd from control participants with quantification of alpha - synuclein deposition in either pilomotor or sudomotor nerve fibers.4 additional class iii evidence was provided in a study of 21 patients with pd , 30 control subjects , and 20 patients with parkinsonism syndromes assumed not to have alpha - synuclein deposits ( ie , vascular parkinsonism , tauopathies , parkinsonism with evidence of parkin mutations ) . in this study ,
alpha - synuclein depositions were detected in all patients with pd but were not found in any skin sample in control subjects or patients with presumably nonsynucleinopathic parkinsonism syndromes.5 research indicated that alpha - synuclein deposition in cutaneous small fibers might facilitate early differentiation of pd from other synucleinopathies . in a disease comparison study , alpha - synuclein deposition in nerve fibers innervating pilosebaceous units ( including pilomotor fibers ) was found in 62% of pd patients but only in 7% of patients with atypical parkinsonism syndromes.6 however , sample sizes were relatively small in this study ( 34 patients with pd , 33 patients with atypical parkinsonism syndromes , 20 control subjects ) and the group of atypical parkinsonism syndromes included both synucleinopathic syndromes , such as dementia with lewy bodies and multiple system atrophy ( msa ) , and tauopathies , such as progressive supranuclear palsy and alzheimer s disease , potentially limiting interpretability of the observed diagnostic discrimination . within the group of synucleinopathic neurodegenerative disorders , distinct distributions of alpha - synuclein deposition between the central and the peripheral nervous system have been assumed .
particularly , in contrast to pd , msa has been traditionally believed to be a pure central disorder with deposition of the presumably pathological form of alpha - synuclein ( phosphorilyzed alpha - synuclein ) being limited to the brain and preganglionic nerve fibers.7 consistent with this assumption , a previous investigation in skin biopsies of patients with msa found no accumulation of the presumably pathogenic form of alpha - synuclein ( phosphorylated alpha - synuclein ) in cutaneous autonomic adrenergic nerve fibers.8 however , unexpectedly , another study also included assessment of phosphorylated alpha - synuclein in peripheral somatic nerve fibers in both msa and pd patients and found pathological alpha - synuclein accumulation in sensory fibers in 67% of msa patients , whereas a similar fraction of pd patients showed alpha - synuclein pathology in autonomic fibers.9 this important observation does not only question the common conception of msa being a pure or predominant central nervous system disorder but also highlights the necessity of intraneural alpha - synuclein quantification beyond autonomic fibers in future research .
similar to the pd phenotype , patients with pure autonomic failure ( paf ) showed length - dependent somatic and autonomic small fiber loss , which increased with alpha - synuclein load.10 notably , in the comparison between paf and msa , differences in skin denervation patterns seem not to relate to patterns of autonomic function tests , including cardiovascular function assessed via head - up tilt test and sympathetic nerve activity assessed via microneurography.11 by contrast , in pd patients , alpha - synuclein deposition in cutaneous small fibers strongly correlates with autonomic functions and severity of autonomic symptoms.2 the discrepancy observed between peripheral alpha - synuclein pathology patterns in msa and pd , as well as the differences between structural nerve fiber damage and autonomic functions in patients with msa and paf but not pd do not only highlight our limited understanding of the underlying mechanisms but also indicate the unique opportunity of the technique to identify disease - specific patterns . defining these patterns might facilitate early diagnosis , disease monitoring , and investigation of response to neuroprotective treatment .
however , an encompassing characterization of alpha - synuclein deposition among autonomic and nonautonomic small nerve fibers in large populations of patients with synucleinopathies and longitudinal assessment of pathology related to disease progression are still lacking .
furthermore , it appears necessary to elucidate to what degree structural nerve fiber damage relates to loss of functional integrity of the affected nerve fiber types in order to improve our understanding of the functional relevance of alpha - synuclein deposition in cutaneous small fibers .
the skin is innervated by afferent somatic nerves , unmyelinated c - fibers , and lightly myelinated a - delta fibers that originate in the dorsal nerve root ganglia ( small fibers ) . in skin biopsy studies of pd ,
alpha - synuclein accumulation in cutaneous autonomic small fibers has been recently linked to autonomic nervous system disturbances even in the early stages of the disease , suggesting that quantification of this protein via immunohistochemical staining might be a valid biomarker of the disease and could help improve the accuracy of diagnosis and treatment.2 this study also demonstrated that alpha - synuclein deposition normalized to intraepidermal nerve fiber density is increased in cutaneous sudomotor ( sweat gland innervating ) and pilomotor ( pilomotor muscle innervating ) nerve fibers in patients with pd and that more severe alpha - synuclein deposition relates to greater autonomic dysfunction and more advanced motor symptoms .
importantly , a clear increase in pathology was observed in the early stages of pd with substantially higher alpha synuclein depositions in patients at hoehn and yahr stage 2 compared to stage 1 .
this early increase of small fiber pathology indicates the possible utility of skin biopsy assessment in early diagnostic work - up and short - term clinical studies in patients with pd .
interestingly , another investigation found morphological similarities between alpha - synuclein deposition - related fiber damage in cutaneous small fibers and those present in cerebral neurites , which led the authors to postulate that the skin constitutes a window into brain pathology in patients with pd.3 additional research confirmed and further advanced these observations .
a study in 28 patients with pd and 23 control subjects found > 90% sensitivity and > 90% specificity to distinguish pd from control participants with quantification of alpha - synuclein deposition in either pilomotor or sudomotor nerve fibers.4 additional class iii evidence was provided in a study of 21 patients with pd , 30 control subjects , and 20 patients with parkinsonism syndromes assumed not to have alpha - synuclein deposits ( ie , vascular parkinsonism , tauopathies , parkinsonism with evidence of parkin mutations ) . in this study ,
alpha - synuclein depositions were detected in all patients with pd but were not found in any skin sample in control subjects or patients with presumably nonsynucleinopathic parkinsonism syndromes.5
research indicated that alpha - synuclein deposition in cutaneous small fibers might facilitate early differentiation of pd from other synucleinopathies . in a disease comparison study ,
alpha - synuclein deposition in nerve fibers innervating pilosebaceous units ( including pilomotor fibers ) was found in 62% of pd patients but only in 7% of patients with atypical parkinsonism syndromes.6 however , sample sizes were relatively small in this study ( 34 patients with pd , 33 patients with atypical parkinsonism syndromes , 20 control subjects ) and the group of atypical parkinsonism syndromes included both synucleinopathic syndromes , such as dementia with lewy bodies and multiple system atrophy ( msa ) , and tauopathies , such as progressive supranuclear palsy and alzheimer s disease , potentially limiting interpretability of the observed diagnostic discrimination . within the group of synucleinopathic neurodegenerative disorders , distinct distributions of alpha - synuclein deposition between the central and the peripheral nervous system have been assumed . particularly , in contrast to pd , msa has been traditionally believed to be a pure central disorder with deposition of the presumably pathological form of alpha - synuclein ( phosphorilyzed alpha - synuclein ) being limited to the brain and preganglionic nerve fibers.7 consistent with this assumption , a previous investigation in skin biopsies of patients with msa found no accumulation of the presumably pathogenic form of alpha - synuclein ( phosphorylated alpha - synuclein ) in cutaneous autonomic adrenergic nerve fibers.8 however , unexpectedly , another study also included assessment of phosphorylated alpha - synuclein in peripheral somatic nerve fibers in both msa and pd patients and found pathological alpha - synuclein accumulation in sensory fibers in 67% of msa patients , whereas a similar fraction of pd patients showed alpha - synuclein pathology in autonomic fibers.9 this important observation does not only question the common conception of msa being a pure or predominant central nervous system disorder but also highlights the necessity of intraneural alpha - synuclein quantification beyond autonomic fibers in future research .
similar to the pd phenotype , patients with pure autonomic failure ( paf ) showed length - dependent somatic and autonomic small fiber loss , which increased with alpha - synuclein load.10 notably , in the comparison between paf and msa , differences in skin denervation patterns seem not to relate to patterns of autonomic function tests , including cardiovascular function assessed via head - up tilt test and sympathetic nerve activity assessed via microneurography.11 by contrast , in pd patients , alpha - synuclein deposition in cutaneous small fibers strongly correlates with autonomic functions and severity of autonomic symptoms.2 the discrepancy observed between peripheral alpha - synuclein pathology patterns in msa and pd , as well as the differences between structural nerve fiber damage and autonomic functions in patients with msa and paf but not pd do not only highlight our limited understanding of the underlying mechanisms but also indicate the unique opportunity of the technique to identify disease - specific patterns .
defining these patterns might facilitate early diagnosis , disease monitoring , and investigation of response to neuroprotective treatment .
however , an encompassing characterization of alpha - synuclein deposition among autonomic and nonautonomic small nerve fibers in large populations of patients with synucleinopathies and longitudinal assessment of pathology related to disease progression are still lacking .
furthermore , it appears necessary to elucidate to what degree structural nerve fiber damage relates to loss of functional integrity of the affected nerve fiber types in order to improve our understanding of the functional relevance of alpha - synuclein deposition in cutaneous small fibers .
while quantification of alpha - synuclein deposition in pilomotor muscles might be a valid biomarker for pd , several current limitations would have to be overcome to enable broad clinical implication of the technique .
first , and probably most important , consensus on the optimal technique of immunohistochemical staining methods and analysis appears to be necessary to generate comparable data and define clinical standards .
several techniques have been proposed including : 1 ) normalizing total alpha - synuclein burden to loss of intraepidermal nerve fibers to control for damage not related to alpha - synuclein ; 2 ) using an antibody selective for the presumably neuropathy inducing phosphorylated form of alpha - synuclein ; and 3 ) quantifying burden of native alpha - synuclein.2,4,9 while normalization to loss of intraepidermal nerve fibers and use of phosphorylated alpha - synuclein - specific antibodies seem superior over native alpha - synuclein quantification , the optimal analysis technique remains uncertain .
second , accumulations of alpha - synuclein were found in different types of autonomic cutaneous small fibers , including sudomotor ( sweat gland innervating ) , vasomotor ( blood vessel innervating ) , and pilomotor ( pilomotor muscle innervating ) fibers but it remains to be determined which fiber type ( or which combination of types ) is the optimal diagnostic target . however , two investigations assessing alpha - synuclein burden normalized to loss of intraepidermal nerve fibers found most severe pathology in pilomotor nerve fibers , potentially indicating high diagnostic value of this type of autonomic small fibers.2,4 the uncertainty of the optimal neural target structure ( or set of target structures ) as well as the optimal skin site where the biopsy should be obtained from might , inter alia , explain why in previous studies positive staining for phosphorylated alpha - synuclein ranged between 0% and 70% in patients with pd.1214 third , longitudinal studies in large populations of patients with pd and other synucleinopathies to relate cumulative nerve fiber damage over time to progression of clinical symptoms and confirm external validity of the technique are still lacking .
finally , immunohistochemical quantification of alpha - synuclein burden in small fibers captures structural pathology but may not reflect the functional integrity that results from structural damage . viewed in conjunction , these limitations and challenges underscore that consensus on standardized testing protocols as well as further assessment of sensitivity and specificity of cutaneous nerve fiber evaluation for both structural and functional damage related to alpha - synuclein deposition are needed.1517 while autonomic skin nerve fibers can be assessed for structural integrity using immunohistochemical quantification of nerve fiber densities in skin biopsies , several techniques have been designed to assess functional integrity of these fibers .
in fact , functional assessment of autonomic skin nerves fibers has taken on increasing importance in the evaluation of autonomic neuropathy.18 cutaneous small fibers are responsive to a range of physical , chemical , and mechanical stimuli that can evoke an axon - reflex - mediated response .
controlled activation of cutaneous vasomotor ( blood vessel innervating ) fibers by acetylcholine administered via iontophoresis leads to orthodromic conduction of an action potential to an axon branch point and , consecutively , antidromic conduction to a neighboring population of cutaneous blood vessels . on activation ,
both c - fibers and a - delta fibers release substance p and calcitonin gene - related peptide , leading to vasodilation and plasma extravasation in an indirect skin area , surrounding the area of acetylcholine application.19 this neurally mediated response can be evoked by chemical ( eg , acetylcholine , histamine , and capsaicin ) and electrical stimulation and can be quantified via laser doppler assessment as a measure of functional integrity of vasomotor fibers.20 the most widely used application includes iontophoresis of acetylcholine using a predefined current and dosage . similarly , axon - reflex responsivity can be assessed in sudomotor nerve fibers via cholinergic and in pilomotor fibers via adrenergic stimulation.21,22 research has shown that sudomotor axon - reflex function is impaired in pd and impairment increases in severity with increasing disease stage.23 however , to date , it is unknown whether axon - reflex responsiveness relates to alpha - synuclein load in pilomotor and vasomotor nerve fibers .
therefore , it remains to be defined to what degree structural nerve fiber damage related to alpha - synuclein deposition translates into dysfunction of the affected fiber .
while autonomic skin nerve fibers can be assessed for structural integrity using immunohistochemical quantification of nerve fiber densities in skin biopsies , several techniques have been designed to assess functional integrity of these fibers .
in fact , functional assessment of autonomic skin nerves fibers has taken on increasing importance in the evaluation of autonomic neuropathy.18 cutaneous small fibers are responsive to a range of physical , chemical , and mechanical stimuli that can evoke an axon - reflex - mediated response .
controlled activation of cutaneous vasomotor ( blood vessel innervating ) fibers by acetylcholine administered via iontophoresis leads to orthodromic conduction of an action potential to an axon branch point and , consecutively , antidromic conduction to a neighboring population of cutaneous blood vessels . on activation ,
both c - fibers and a - delta fibers release substance p and calcitonin gene - related peptide , leading to vasodilation and plasma extravasation in an indirect skin area , surrounding the area of acetylcholine application.19 this neurally mediated response can be evoked by chemical ( eg , acetylcholine , histamine , and capsaicin ) and electrical stimulation and can be quantified via laser doppler assessment as a measure of functional integrity of vasomotor fibers.20 the most widely used application includes iontophoresis of acetylcholine using a predefined current and dosage .
similarly , axon - reflex responsivity can be assessed in sudomotor nerve fibers via cholinergic and in pilomotor fibers via adrenergic stimulation.21,22 research has shown that sudomotor axon - reflex function is impaired in pd and impairment increases in severity with increasing disease stage.23 however , to date , it is unknown whether axon - reflex responsiveness relates to alpha - synuclein load in pilomotor and vasomotor nerve fibers .
therefore , it remains to be defined to what degree structural nerve fiber damage related to alpha - synuclein deposition translates into dysfunction of the affected fiber .
there is emerging evidence on an important pathogenic role of deposition of alpha - synuclein in cutaneous small nerve fibers , quantitative analysis of which might facilitate differentiation between synucleinopathies , early diagnosis , disease monitoring , and evaluation of response to neuroprotective treatment .
further research is warranted to standardize analysis techniques , elucidate progression of pathology over time , and define the association between structural and functional impairment of nerve fibers affected by alpha - synuclein deposition . | despite progression in the development of pharmacological therapy , treatment of alpha synucleinopathies , such as parkinson s disease ( pd ) and some atypical parkinsonism syndromes , is still challenging . to date
, our knowledge of the mechanisms whereby the pathological form of alpha - synuclein causes structural and functional damage to the nervous system is limited and , consequently , there is a lack of specific diagnostic tools to evaluate pathology in these patients and differentiate pd from other neurodegenerative proteinopathies .
recent studies indicated that alpha - synuclein deposition in cutaneous small nerve fibers assessed by skin biopsies might be a valid disease marker of pd and facilitate early differentiation of pd from atypical parkinsonism syndromes .
this observation is relevant since early diagnosis may enable timely treatment and improve quality of life .
however , challenges include the necessity of standardizing immunohistochemical analysis techniques and the identification of potential distinct patterns of intraneural alpha - synuclein deposition among synucleinopathies . in this perspective , we explore the scientific and clinical opportunities arising from alpha - synuclein assessment using skin biopsies . these include elucidation of the peripheral nervous system pathology of pd and other synucleinopathies , identification of novel targets to study response to neuroprotective treatment , and improvement of clinical management . furthermore , we discuss future challenges in exploring the diagnostic value of skin biopsy assessment for alpha - synuclein deposition and implementing the technique in clinical practice . |
the abdominal organ herniation trough the right diaphragm is even rarer due to the liver protective function .
the case reported concerns about a patient suffering from massive delayed right diaphragmatic hernia with right liver and bowel dislocation .
a 41-year - old patient was referred to our emergency department with complaints of wheezing and cough since 1 month . during a previous admission , a diagnosis of right basal pneumonia
his medical history was significant for motorcycle accident ~20 years before involving and bladder rupture .
a chest x - ray revealed right basal consolidation with inhomogenous opacity at the medium and lower chest area .
a computed tomography ( ct ) scan demonstrated massive right diaphragmatic hernia with dislocation of the liver that appeared overturned ~180 , transverse and ascending colon and part of small bowel ( fig .
1 ) .
figure 1:thorax and abdominal ct showing the dislocation of right upper abdominal viscera thorax and abdominal ct showing the dislocation of right upper abdominal viscera he was then admitted to the surgery department for a laparoscopic exploration that confirmed the radiological finding of inveterate right diaphragmatic hernia with an 8 cm defect . because of the impossibility to reduce the liver in abdomen , due to the thoracic adhesions , a right anterolateral thoracotomy was then performed .
the liver was uneventfully reinstated and colon and small bowel were replaced in anatomical position .
postoperative oxygen saturation was normal and a chest x - ray was performed before discharge and revealed complete re - expansion of the lung .
the diaphragmatic hernia is the herniation of abdominal organs into the chest through a diaphragmatic defect .
these can be congenital or acquired . acquired diaphragmatic hernia occurs , in most of the cases , as a result of blunt or penetrating thoraco - abdominal trauma .
left hemidiphragmatic hernia is more common because liver exerts a protective function against the herniation of the viscera .
herniation of the abdominal organs may be completely asymptomatic ; due to this reason , ~66% of diaphragmatic rupture are not recognized at the time of trauma .
the chest negative pressure causes the gradual migration of abdominal contents leading to the onset of symptoms .
we can classify this clinical condition in two types : type i ( early ) and type ii ( delayed ) .
clinical presentation includes gastrointestinal symptoms ( abdominal pain , nausea , vomiting and sub - occlusion ) , respiratory ( dyspnea , orthopnea and chest pain ) or cardiocirculatory ( hemodynamic compromission ) [ 14 ] .
the initial diagnostic tool is chest or abdominal x - ray but ct scan is the best modality to assess the extent of dislocation , the size of diaphragmatic defect and the belt - like constriction of abdominal contents , referred to as the collar sign .
surgery is always necessary for the treatment ; the approach could be laparotomic / thoracotomic or minimal invasive . in our case ,
minimally invasive approach was used as a diagnostic tool in order to evaluate the diaphragmatic defect and choose the best approach ( thoracotomic or laparotomic ) . in delayed case
, diagnosis may be a compulsory thoraco - abdominal approach in order to lyse adhesions between abdominal organs and thoracic structure .
diagnosis of diaphragmatic hernia should always be considered in patient with chest or abdominal trauma because the mortality rate can reach 31% in the first 24 hours following the trauma .
it should also be considered many years after trauma in case of onset of typical symptoms .
| abstractright diaphragmatic hernia is an uncommon injury following abdominal trauma .
a case of delayed right post - traumatic diaphragmatic hernia is presented .
the patient referred us with wheezing and cough since 1 month .
a chest - abdominal computed tomography scan demonstrated a large diaphragmatic defect with liver and intestinal dislocation .
the patient underwent surgical intervention with diaphragmatic repair .
no complications were observed during admission and follow - up is actually negative for recurrence . |
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.