article
stringlengths 515
334k
| abstract
stringlengths 112
2.3k
| section_names
stringlengths 5
818
|
|---|---|---|
perforation of the biliary system occurs
most frequently in the gallbladder , usually associated with ( and complicating
upto 10% cases of ) acute cholecystitis .
perforation of the extrahepatic biliary
tree is a rare entity , accounting for less than 10% of intraperitoneal biliary
rupture .
bile duct perforation is most commonly described in infants related to congenital
biliary system anomalies .
aetiology in the adult is commonly attributable to
intramural infection , necrosis of the wall of the bile duct secondary to
thrombosis , increased intraductal pressure secondary to obstruction , cirrhosis ,
and direct erosion by calculi .
overall , 70% of cases are related to calculi
. the incidence of biliary tract disease during pregnancy ranges from 0.050.3% . despite these apparent low
figures , complications from gallstones
represent the most common general surgical condition
requiring surgical intervention , second only to appendicitis .
indications for intervention of gallstones during pregnancy
include obstructive jaundice , acute cholecystitis , or pancreatitis failing
medical management .
we present the case of a young woman diagnosed with
gallstones in late pregnancy , complicated by acute gallstone pancreatitis and
subsequently spontaneous common bile duct perforation .
a twenty - year - old primigravida woman was
planned for elective caesarean section due to breech presentation .
the patient
had a past medical history of -thalassemia trait , but was not
normally on regular medication .
her mother had previously undergone a
cholecystectomy for gallstones . at 34 weeks gestation , she presented
acutely with a two - week history of worsening abdominal pain localised to the
epigastric region , associated with vomiting . on examination ,
blood results revealed raised inflammatory markers ( wbc 14.4 [ 4.011.0 ] ,
neutrophils 11.9 [ 2.07.5 ] , crp 60
[ 07.5 ] ) and evidence of pancreatitis ( amylase 1369
iu / l [ 36128 ] ) , mildly
raised bilirubin ( 24 mol / l [ 020 ] ) and raised alkaline phosphatase ( 183
an abdominal ultrasound revealed multiple small gallstones and a
thickened gallbladder wall , but no evidence of a dilated intra or extrahepatic
biliary system .
the patient was treated conservatively , rapidly improved , and liver function tests
normalised .
an emergency caesarean
section was performed and a term baby delivered , but no obvious cause was found
to explain her clinical condition .
the following day her clinical condition worsened , with progressive abdominal pain
and a metabolic acidosis .
she required aggressive resuscitation , inotropic , and ventilatory support and was ,
therefore , admitted to the intensive care unit .
a computed tomography ( ct )
revealed extensive free peritoneal fluid and gas of which the aetiology was not
apparent .
the patient underwent a prompt laparotomy and was found to have
generalised biliary peritonitis .
the gallbladder was intact but a 2 mm
perforation was found on the anterior surface of a dilated common
bile duct ( 12 mm ) . on table
cholangiography suggested obstruction of the distal
common bile duct caused by a 5 mm gallstone impacted within the distal common
bile duct .
the calculus was removed , and the duct was repaired over a t - tube .
a t - tube cholangiogram was performed
after 4 weeks , and the tube was uneventfully removed ( figure 1 ) .
although the pathogenesis of spontaneous biliary perforation is poorly understood ,
recognised mechanisms include the following : calculous perforation at the site
of impaction ; calculous erosion without impaction ; increased canalicular
pressure due to obstruction by tumour , stone , or spasm of the sphincter of
oddi ; intramural infection ; mural vessel infarction leading to mural necrosis ;
or rupture of a biliary tract anomaly such as cyst or diverticulum .
thus ,
because perforation of the biliary system is a recognised complication of
cholelithiasis , the diagnosis should be suspected if a perihepatic abscess or
peritonitis is combined with biliary stone disease . as early as 1882 , freeland reported the first case of extrahepatic biliary system rupture in an adult ( diagnosed at autopsy ) , an entity that was
subsequently first described in pregnancy by piotrowski et al . over a
century later . since this time ,
very few cases of spontaneous common bile duct
perforation in adults have been reported in the literature , with cases
occurring during pregnancy being even more scarce .
the importance of this clinical
scenario lies in the potential serious morbidity and not infrequent mortality
associated with missed biliary system perforation .
petrozza
et al . described two cases of gallbladder perforation due to cholelithiasis in
the early postpartum period .
both cases presented a diagnostic dilemma , and it
was concluded that a history of cholelithiasis in a patient with persistent
intra - abdominal symptoms in the postpartum period must alert to prompt
investigation and early management .
,
one patient was found to have suffered gallbladder rupture as a result of
cholecystitis , and in the second , a common bile duct perforation was found at
laparotomy with no obvious precipitating cause .
. also drew
attention to the similarity of symptoms of gallbladder disease in pregnancy to
mild pre - eclampsia , having in common hypertension , epigastric pain , and mildly
deranged liver function tests .
these cases highlight the importance of
recognising the possibility of delayed diagnosis of cholelithiasis as a result
of nonspecific abdominal symptoms during pregnancy and indicate early
investigation and treatment in order to reduce serious morbidity .
.
block and kelly reported the optimum time for surgical management of
gallstone pancreatitis to be in the second trimester or early postpartum
period , in order to minimise maternal / fetal mortality and recurrent
pancreatitis .
unfortunately , in those women presenting
late in pregnancy ( as in the case described ) , the balance of risk favours
watchful waiting until after delivery followed by elective cholecystectomy .
certainly , this risks early recurrence of acute pancreatitis , as well as rare but severe
consequences such as biliary peritonitis .
whether an early endoscopic retrograde cholangiopancreaticography ( ercp )
and sphincterotomy in those cases presenting with gallstone pancreatitis can be
an acceptable temporary preventive measure is unclear , but undertaking ercp is
not without risk , and the potential risks should be considered carefully in
individual cases . in this particular case ,
it is impossible to know whether the eroding calculus had been present during the
initial episode of pancreatitis .
magnetic resonance scanning is a commonly used
imaging modality in obstetrics , considered to be safe and avoiding the use of
ionising radiation .
therefore , magnetic resonance cholangiopancreatography
( mrcp ) would have been a reasonable next investigation during this patient s
initial presentation , and if a ductal stone had been revealed , then the indication
for ercp may have been clearer .
on the other hand , neonatal and postnatal
care of babies born early have progressed significantly , suggesting the
possibility of induction of labour perhaps at 3638 weeks
gestation in severely symptomatic or high - risk patients .
of course , every case
must be considered individually , taking into account maternal and fetal
history and health .
|
spontaneous perforation of the extrahepatic biliary system is a rare presentation of ductal stones .
we report the case of a twenty - year - old woman presenting at term with biliary peritonitis caused by common bile duct ( cbd ) perforation due to an impacted stone in the distal common bile duct .
the patient had suffered a single herald episode of acute gallstone pancreatitis during the third trimester .
the patient underwent an emergency laparotomy , bile duct exploration , and removal of the ductal stone .
the postoperative course was uneventful .
|
1. INTRODUCTION
2. CASE PRESENTATION
3. DISCUSSION
|
two male patients with type 2 diabetes ( t2d ) and coronary heart disease had plasma levels of brain natriuretic peptide ( bnp ) and n - terminal probrain natriuretic peptide ( nt - probnp ) measured as part of routine evaluation .
their glomerular filtration rate was normal ( > 75 ml / min per 1.73 m ) , hba1c was < 7% with metformin , and c - reactive protein ( crp ) was < 1 mg / dl in both .
patient a was 71 years old and had suffered an anterior myocardial infarction three years ago with st - segment elevation , which was initially treated with fibrinolytics and 24 h later using percutaneous coronary intervention with a drug eluting stent on the anterior coronary descending artery .
he presented with a > 70% stenosis on a diagonal artery , which was not treated and showed no significant stenosis in the remaining coronary arteries .
his bmi was 34.8 kg / m , waist circumference 116 cm and was stable without chest pain in nyha class i. the echocardiogram showed apical and lateral hypokinesia with left ventricular ejection fraction ( lvef ) of 58% .
plasma bnp and nt - probnp were 72 and 100 pg / ml , respectively ( reference values for age and sex : bnp median 28 pg / ml ( percentile 2575th , 1058 pg / ml ) ; nt - probnp median 45 pg / ml ( percentile 595th , 14140 pg / ml ) ) .
patient b , 58 years of age , had suffered a non - st - elevation myocardial infarction two years earlier , and had received standard treatment .
his bmi was 44.3 kg / m and his waist circumference 137 cm .
the echocardiogram showed mild hypokinesia of the basal segment of the inferior wall and mild dilation of left ventricle with eccentric hypertrophy .
lvef was 66% and plasma bnp and nt - probnp were 10 and < 20 pg / ml , respectively ( reference values for age and sex : bnp median 31 pg / ml , percentile 2575th , 14 - -49 pg / ml ; nt - probnp median 25 pg / ml , percentile 595th , 588 pg / ml ) .
these findings posed the question as to why it is that in some patients the plasma levels of cardiac natriuretic peptides ( cnp ) are unexpectedly low , and what is their value in the prognosis for these patients ?
the cnp act as a basic functional link between cardiovascular system homeostasis , inflammation and certain metabolic functions ( fig .
1 ) . increased secretion of cnp is a result of both mechanical and neurohumoral stimuli ( 1 , 2 ) . the main mechanical stimulus for the secretion of cnp is the increased pressure in the cardiac chambers leading to stretching of the myocardial fibers .
neurohumoral stimuli include endothelin-1 , angiotensin ii , adrenergic agonists and various cytokines ( 4 ) . increased left ventricular end - diastolic wall stress and left ventricular end - systolic wall stress correlate with an increase in plasma cnp in heart failure ( 5 , 6 ) .
however , studies on cardiac transplant patients have shown that plasma cnp levels remain high even after intra - cardiac pressures normalize following transplantation ( 7 ) . during an acute cardiac allograft rejection episode , bnp but not atrial natriuretic peptide ( anp )
plasma levels increase significantly above pre - rejection values independently of the surgical technique used ( 8) .
plasma bnp levels during acute rejection episodes do not correlate with hemodynamic variables but correlate with the levels of regulated on activation , normal t expressed and secreted , insulin growth factor binding protein-1 and neutrophil activating protein-2 ( 4 ) . in non - rejecting transplanted patients , despite the normalization of endothelin-1 plasma levels and diastolic and systolic functions , bnp remains high by comparison with control subjects ( 9 ) .
measuring cnp is useful as a diagnostic and prognostic tool ( 10 , 11 , 12 ) .
high plasma levels of cnp ( e.g. bnp > 63pg / ml or nt - probnp > 206 pg / ml ) can quite precisely predict which patients are at risk for events such as hospitalization due to systolic heart failure or cardiovascular death ( 13 , 14 , 15 , 16 , 17 , 18 ) .
obese patients may have strikingly low levels of plasma cnp ( 19 , 20 , 21 , 22 ) due to multiple mechanisms that might include increased neprilysin - neutral endopeptidase activity resulting in increased degradation of circulating cnp and increased adipose tissue expression of the np clearance receptor c ( nprc ) .
it has been proposed that suppression of probnp1 - 108 prohormone processing due to o - glycosylation at its cleavage site where furine or corin convertases act , prevents the formation of bnp77 - 108 ( functionally active ) and nt - probnp1 - 76 ( inactive ) fragments , an event that is purportedly more frequent in diabetics and in insulin - resistant states ( 23 , 24 , 25 ) . in heart failure patients ,
the precursor pro - hormone probnp1 - 108 is found circulating at lower than normal levels ( 26 , 27 ) .
recently , the paradigm - hf trial showed that angiotensin receptor - neprilysin inhibition using the compound lcz-696 , 200 mg twice daily , compared with enalapril at 10 mg twice daily significantly reduced the risk of cardiovascular death and hospitalization in patients with heart failure with an lvef 40% .
however , death rates from cardiovascular causes were similar among diabetics compared with non - diabetics ( 28 ) .
although this interesting finding could be an accidental one , other heart failure trials found that the benefit of this therapy in diabetics seems to be less than that in non - diabetics ( 29 , 30 , 31 ) .
this finding merits further research , since diabetics comprise 2535% of the subjects in most studies ( 29 , 30 , 31 , 32 ) .
the obese are differentiated from the metabolically healthy obese by an unhealthy metabolic profile ( 33 ) . the latter is defined by increased waist circumference ( > 94 cm in men and > 80 cm in women ) or bmi 30 kg / m accompanied by two or more of the following : hypertriglyceridemia ( 1.7 mmol / l ) , low hdl cholesterol ( < 1.03 mmol / l in men and < 1.29 mmol / l in women ) , hyperglycemia ( > 11.1 mmol / l or a diagnosis of diabetes ) , hypertension ( 130/85 mmhg ) or medication for high blood pressure ( 34 , 35 ) . in a 12-year follow - up of 61 299 subjects free from cardiovascular disease at baseline mrkedal et al . ( 36 ) observed that systolic heart failure development was similar among
metabolically healthy compared to non - metabolically healthy obese subjects , meaning that obesity per se creates a higher risk for developing systolic heart failure , especially if it is long - lasting and severe ( bmi > 40 kg / m ) .
recently , it was reported that subjects with high plasma levels of nt - probnp are at a greater risk of developing systolic heart failure whether they are obese or non - obese . however , unlike the non - obese who show a direct linear relationship between levels of nt - probnp and systolic heart failure , the obese show a u - shaped relationship ; i.e. those who have the lowest plasma levels are at just as much risk of developing systolic heart failure as those with the highest plasma levels ( 37 ) .
in contrast , the pathological weight loss caused by anorexia nervosa seems to be related to high plasma levels of anp ( 38 ) . in severe obesity ,
bariatric surgery reduces the bmi and improves control of diabetes but it is not yet known if it reduces the risk of cardiovascular events ( 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 ) . in patients with severe obesity ( bmi > 40 kg / m ) before gastric bypass surgery , the median level of nt - probnp was 54 pg / ml increasing by 125% after 1 year with no apparent relation to weight loss or glucometabolic parameters .
it was speculated that an improvement in secretory cardiac function following surgery was responsible for the increase in circulating nt - probnp ( 46 ) .
myocardial ischemia and cardiomyocyte stretch trigger the immediate release of cnp ( 47 , 48 ) even when the lvef is normal ( 49 , 50 ) . in a 9.2 year follow - up study of diabetic ( about 7% of the cohort ) and non - diabetic patients with chronic stable myocardial ischemia , high plasma levels of nt - probnp were significantly associated with mortality regardless of left ventricular function ( 51 ) .
recently , it was shown that in patients with stable coronary heart disease bnp and nt - probnp were strong predictors of long - term ( 6.53.3 years ) cardiovascular events and notably , when nt - probnp was added to the clinical predictors , it performed better than bnp in risk classification for adverse cardiovascular events ( 52 ) . serial measurement data for nt - probnp in patients with chronic stable angina have shown a wide intra - individual variation .
an increase by > 42% or a decrease by > 30% relative to baseline values is required to indicate a significant change ( 53 , 54 ) . in patients presenting with acute chest pain ,
cnp plasma levels help the diagnosis and prognosis when used together with a non - diagnostic ecg and a negative troponin .
cnp measured in these patients at rest predict future cardiac events at 30 and 180 days and at 1 year ( 55 , 56 , 57 , 58 ) .
if myocardial ischemia is evident during the stress test , an elevation of the plasma levels of nt - probnp detects ischemia and predicts cardiovascular events ( 59 ) .
likewise , in non - st - elevation acute coronary syndromes , cnp add prognostic information to the clinical indicators ( 60 , 61 , 62 , 63 ) ( table 1 ) . in a recent investigation ( 62 ) , bnp was shown to be an independent predictor of mortality on entering the study . at 1 year of
follow - up bnp plasma levels provided added value over the timi and grace scores .
cnp are stronger predictors of mortality than troponin in patients that will eventually develop myocardial infarction . in st - segment - elevation
acute coronary syndromes cnp plasma levels also add prognostic information independently of the lvef ( 50 , 64 , 65 ) and in addition , predict which patients with primary percutaneous coronary intervention ( pci ) may be discharged earlier thus saving health resources ( 66 ) .
it has been shown experimentally that anp is associated with salt - sensitive high blood pressure ( 67 ) . in humans
alleles associated with the highest levels of np were also associated with a lower risk of high blood pressure ( 68 , 69 ) . in keeping with these findings
, another study showed a relative deficiency of cnp at all the different stages of high blood pressure in humans consisting of low levels of probnp1 - 108 and anp99 - 126 .
low plasma levels of bnp76 - 108 and nt - probnp1 - 76 were observed in the first hypertensive stages together with a reduction of nt - anp1 - 98 in stage i ( 70 ) .
a possible therapeutic modality to supplement cnp in those cases in which circulating cnp are deemed deficient ( like in hypertension or heart failure ) is suggested by the development of a recombinant human serum albumin - atrial natriuretic factor ( anf ) ( 71 ) molecule .
this compound , when injected into mice increased circulating cgmp and decreased blood pressure demonstrated a half - life of > 80 min , which is considerably longer than the 510 min half - life for native anf ( 71 , 72 , 73 ) . from the preceding , it may be surmised that elevated cnp levels are strong markers of future cardiovascular events , especially cardiovascular death . on the other hand , cnp plasma levels lower than expected in obese and diabetic patients
the two patients discussed above had similar metabolic profiles , lvef > 50% but a different bmi and cnp profile .
patient a was not severely obese and had slight residual myocardial ischemia with bnp and nt - probnp within low to moderate values expected for systolic heart failure and cardiovascular mortality on follow - up .
patient b suffered from severe obesity , with very low plasma levels of cnp with a prognosis related to his bmi of > 40
kg / m and probably also to a deficit of cnp , placing him in the high - risk category for developing systolic heart failure and eventual cardiovascular death .
our patient a has markedly higher bnp and nt - probnp levels compared to patient b. this can largely be explained by the better ejection fraction of patient b , but there might also be other contributing factors .
for example , recent studies suggest an inverse relationship between circulating cnp levels and bodyweight as well as with insulin resistance and t2d ( 19 , 74 , 75 , 76 , 77 , 78 ) ( table 1 ) . this correlation can also be observed in patients with congestive heart failure , despite increased cnp levels , due to cardiac wall stress ( 79 ) .
cnp are cleared and degraded by neutral endopeptidase neprilysin and natriuretic peptide receptor c ( nprc ) ( 80 , 81 ) .
natriuretic peptide receptor a ( npra ) and nprc have been identified in human adipose tissue in abundance , implying that adipose tissue sustains a regulatory function on the np system ( 82 , 83 , 84 ) .
interestingly , compared to non - obese and normotensive individuals , nprc is increased in adipose tissue of obese hypertensive patients ( 85 ) .
insulin has been observed to induce nprc expression in human adipocytes ( 84 ) and monocytes ( 83 ) , and might , hence , link conditions associated with hyperinsulinemia ( e.g. obesity and insulin resistance ) , to a relative np deficit
. additionally , neprilysin , the np degrading endopeptidase , is expressed at increased levels in obesity ( 86 ) .
these data argue for obesity and insulin resistance being conditions in which cnp are degraded at an accelerated pace .
second , very recent experimental data also suggest that myocardial bnp expression is markedly decreased in mice fed a high fat diet ( 87 ) , an observation that warrants clinical confirmation .
a cnp deficit in patients with components of the metabolic syndrome might be of clinical relevance .
obese individuals have a higher prevalence of arterial hypertension compared to lean subjects ( 88 ) .
although obesity - related arterial hypertension has been intensively studied , not all mechanisms are well understood ( 89 , 90 ) . by reduced vasodilatory and sodium - excretion activity , as well as
aldosterone - system ( raas ) , a cnp deficit might contribute to obesity related hypertension .
interestingly , in lean subjects , application of a sodium load induces myocardial cnp secretion and stimulates natriuresis ( 91 , 92 , 93 , 94 ) , a response which is blunted in patients with obesity ( 95 ) .
together , these data argue that obesity promotes hypertension partly through reduced vascular and renal np responses as well as through impaired np - mediated raas inhibition .
second , cnp also have beneficial actions on cardiac remodelling in essential hypertension , reducing left ventricular hypertrophy ( 96 ) .
conversely , conditions with a cnp deficit are associated with cardiac hypertrophy in hypertensive patients . in this
regard , hypertensive patients with the metabolic syndrome present with lower anp and nt - probnp levels and increased left ventricular mass compared to hypertensive patients without the metabolic syndrome and insulin resistance ( 97 ) .
finally , the cnp deficit in patients with the metabolic syndrome might be part of a vicious circle which maintains metabolic disease .
for instance , cnp exert lipolytic properties mediated by a cgmp - dependent protein kinase g activating pathway .
cgmp - activated protein kinase g ( gk - i ) activates perilipin a and hormone sensitive lipase mediated triglyceride hydrolysis ( 98 , 99 , 100 , 101 ) . interestingly , these effects seem not to interact with the lipolytic effects of catecholamines ( 99 , 102 ) and the effect seems to be independent of the regulation of insulin ( 103 ) . besides a direct activating effect on lipolysis and lipid oxidation , cnp also control secretion of the insulin sensitizing adipokine adiponectin . in humans , anp acutely increases systemic levels of total and high molecular weight adiponectin ( 104 ) .
these findings are in line with some observational studies showing positive associations between systemic cnp and adiponectin concentrations , as for example in heart failure patients ( 105 , 106 ) .
this finding might also explain the adiponectin paradox in patients with congestive heart failure .
cnp also exert other relevant metabolic actions on adipose tissue . treating a human derived adipose cell line with anp results in the uncoupling of cellular respiration ( 107 ) as well as
the response seems to be mediated by p38 map kinase , which increases uncoupling protein 1 ( ucp1 ) transcription ( 107 ) . upon cold exposure , systemic np concentrations increase and nprc expression in
similarly , forced pkg expression in primary adipocytes leads to an increase in ucp1 expression and activates a thermogenic program .
administration of anp acutely increases lipid oxidation ( 102 , 109 ) and postprandial energy expenditure in healthy individuals ( 110 ) . circulating -hydroxybutyrate increases , indicating enhanced hepatic lipid oxidation .
apart from acute effects on lipid oxidation ( 110 ) , anp and bnp induce skeletal muscle mitochondrial biogenesis , respiration and lipid oxidation in human cells and in rodents , in vitro and in vivo
( 111 , 112 ) .
chronic overexpression of bnp and gk - i each led to increased muscle mitochondrial content , oxidative capacity and lipid oxidation in rodents ( 111 ) .
enhanced oxidative metabolism is associated with protection from diet induced obesity and insulin resistance ( 113 ) .
heterozygous npra knockout mice are prone to gaining weight and become insulin resistant ( 111 ) .
the mechanism linking np signalling to mitochondrial biogenesis and lipid oxidation in skeletal muscle includes activation of the co - transcriptional activator peroxisome proliferator activated receptor coactivator ( pgc)-1 and peroxisome proliferator activated receptor- , both of which are important factors of mitochondrial biogenesis in skeletal muscle ( 111 ) . in human myotubes , we observed that cnp stimulate pgc-1 , maximal mitochondrial respiratory capacity and lipid oxidation ( 112 ) .
moreover , npra expression positively correlates with pgc-1 expression in the skeletal muscle of individuals after a chronic physical training program .
similarly , cell culture studies show that cgmp restores glucose and insulin induced mitochondrial dysfunction in cultured c2c12 myotubes ( 114 ) , nitric oxide signalling via cgmp mediates activation of pgc-1 , and mitochondrial biogenesis in various murine tissues ( 115 ) .
hepatic and skeletal muscle lipid content has been associated with insulin resistance ( 113 ) . in liver and skeletal muscle ,
insulin resistance develops when bioactive lipid species accumulate , such as intracellular diacylglycerol ( 113 ) . in obesity and the metabolic syndrome , this lipid accumulation is primarily due to excessive caloric intake that exceeds the capacity of hepatocytes and myocytes to metabolize or export fatty acids , while refining mitochondrial respiration and enhancing lipid oxidation have been proved to improve lipid utilization and insulin sensitivity ( 116 , 117 , 118 ) . so far , there is no evidence that cnp directly interact with the insulin signalling cascade ( 109 , 119 ) .
it is tempting to speculate that cnp could ameliorate lipid - induced insulin resistance through improvements in hepatic ( 110 ) and muscular ( 112 ) lipid oxidation .
in line with this notion , cnp preserves mitochondrial function and insulin sensitivity in high fat diet mice ( 111 ) . cross sectional studies support the hypothesis that cnp protect from the development of obesity and t2d in patients ( 78 , 120 , 121,122 ) .
conversely , data from the framingham heart study and the malm diet and cancer study show that reduced cnp concentrations correlate with a higher probability of insulin resistance in lean and obese individuals ( 76 ) .
moreover , in the latter study low cnp concentrations are predictive of new - onset diabetes ( 123 ) .
together , these data suggest that a cnp deficit , as observed in obese patients with the metabolic syndrome , contributes and aggravates metabolic vascular disease , and thus , prognosis .
life style interventions as well as pharmacological approaches might be of benefit in this regard ( 28 , 124 ) .
while physical activity increases cnp levels acutely and augments npra expression in skeletal muscle cells ( 112 ) , physical activity coupled with a low calorie diet seems to increase cnp levels chronically ( 125 ) ; an observation that seems to depend on the amount of weight loss ( 126 , 127 ) .
experimental data further suggests that the incretin glucagon like peptide 1 ( glp1 ) might be of relevance in the regulation of np secretion .
the glp1 receptor agonist liraglutide , which is widely used for the treatment of t2d , has been shown to induce cardiac anp release in mice , leading to enhanced natriuresis and vasodilatation ( 128 ) without inducing congestive heart failure .
so far 25 clinical studies failed to demonstrate a similar effect of liraglutide when administered acutely ( 129 ) or sub - chronically to patients ( 130 ) .
chronic liraglutide treatment , accompanied by weight loss , however , seems to increase circulating anp as well as bnp concentrations in diabetic patients ( 131 ) .
clearly , future studies need to address the important question how a cnp deficit in obesity and t2d can be reversed most efficiently ( 132 ) .
the authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of this review .
this work was supported by a grant from the secretara de ciencia y tecnologa de la universidad nacional de crdoba ( grant number 69/08 ) to h r ramos , a grant from the german research foundation ( grant number bi1292/4 - 2 ) to a l birkenfeld and by funds from the university of ottawa to a j de bold .
|
since their discovery in 1981 , the cardiac natriuretic peptides ( cnp ) atrial natriuretic peptide ( also referred to as atrial natriuretic factor ) and brain natriuretic peptide have been well characterised in terms of their renal and cardiovascular actions .
in addition , it has been shown that cnp plasma levels are strong predictors of cardiovascular events and mortality in populations with no apparent heart disease as well as in patients with established cardiac pathology .
cnp secretion from the heart is increased by humoral and mechanical stimuli .
the clinical significance of cnp plasma levels has been shown to differ in obese and non - obese subjects .
recent lines of evidence suggest important metabolic effects of the cnp system , which has been shown to activate lipolysis , enhance lipid oxidation and mitochondrial respiration .
clinically , these properties lead to browning of white adipose tissue and to increased muscular oxidative capacity . in human association studies in patients without heart disease
higher cnp concentrations were observed in lean , insulin - sensitive subjects .
highly elevated cnp levels are generally observed in patients with systolic heart failure or high blood pressure , while obese and type-2 diabetics display reduced cnp levels .
together , these observations suggest that the cnp system plays a role in the pathophysiology of metabolic vascular disease . understanding
this role should help define novel principles in the treatment of cardiometabolic disease .
|
Introduction
The cardiologist's perspective
The endocrinologist's perspective
Declaration of interest
Funding
|
stress cardiomyopathy ( scm ) , a disease with many names like the broken heart syndrome ,
takotsubo cardiomyopathy , and apical ballooning syndrome , is characterized by regional myocardial dysfunction , typically occurring in the wake of a significant physical or emotional stressor .
the pathophysiology of the condition remains incompletely understood , yet the effects of catecholamines on select portions of myocardium are thought to play an integral role .
the degree of cardiac dysfunction in this condition is variable , and in the largest contemporary registry of patients with scm , only 9.9% developed cardiogenic shock .
although uncommon , this severe presentation of the disease is critical to appreciate , and we present a case of the disease at its extreme , with a patient in cardiogenic shock .
a 79-year - old woman with a history of an infratentorial meningioma was admitted to the coronary care unit in cardiogenic shock . the morning of admission
her past medical history was significant only for modest essential hypertension and no known coronary ischemia or dysrhythmias .
following routine induction of anesthesia with securement of the airway and institution of mechanical ventilation , sinus bradycardia associated with profound hypotension ( 70/40 mm hg ) ensued , and this hemodynamic perturbation subsequently progressed to cardiac standstill with absence of peripheral pulses .
cardiopulmonary resuscitation ( cpr ) was initiated and return of spontaneous circulation ( rosc ) was achieved after 90 s with one round of cpr and 1 mg of intravenous epinephrine .
after rosc , her vital signs were notable for sinus bradycardia with a systolic blood pressure initially between 140 and 170 mm hg , which quickly declined to less than 90 mm hg despite continuous infusions of high - dose norepinephrine ( 3 g / kg / min ) and dopamine ( 20 g / kg / min ) . the surface electrocardiogram post - rosc revealed new t - wave inversions in the precordial leads .
intra - operative transthoracic echocardiography shortly after cardiac arrest demonstrated a left ventricular ejection fraction ( lvef ) of 15 - 20% with severely hypokinetic mid and distal segments and a hyperkinetic left ventricular base consistent with scm ( fig . 1 and supplementary video 1 , www.cardiologyres.org ) .
in the setting of her continued hemodynamic instability , left heart catheterization was performed and an intra - aortic balloon pump was placed .
the coronary anatomy was notable for only mild , non - obstructive disease ( fig .
the right coronary artery has minimal disease , and supplies the posterior descending artery ( c ) . in the subsequent 6 h
serum troponin i levels peaked at 1.67 ng / ml and a newly prolonged qt interval developed ( fig .
3 ) . inotropic and mechanical supports were rapidly weaned off over the following 6 h and she was successfully extubated .
repeat echocardiography 12 h after the initial study revealed a lvef of 75 - 80% , near cavity obliteration during systole , and no regional wall motion abnormalities ( supplementary video 2 , www.cardiologyres.org ) . at 6-month follow - up
, the patient denied any symptoms of heart failure and her ef normalized to 55% .
note the low voltages , inferior and lateral precordial lead st segment depressions , and the prolonged qt interval .
this case highlights many of the classic elements of scm : a post - menopausal female with a primary neurologic disease ; a temporal correlation with significant stress and exposure to catecholamines ; prolongation of the qt interval ; low - level cardiac enzyme elevations ; and reversible , often transient left ventricular dysfunction , which at the extreme , results in cardiogenic shock .
although reversal of left ventricular dysfunction is the norm in scm , the time course of this patient s improvement is remarkable .
most studies of scm cite echocardiographic and symptom improvement occurring within days to weeks of diagnosis , and to our knowledge , the earliest reported echocardiographic resolution occurred 5 days following diagnosis in these case series [ 2 , 3 ] .
scm has been associated with administration of catecholamines ( i.e. dobutamine during stress testing or epinephrine for treatment of anaphylaxis ) , and has occurred in the wake of cpr , which we believe occurred in this case . from a pathophysiological standpoint , a catecholamine surge , iatrogenic or otherwise , can impair myocyte contractility , particularly at the apex where the highest concentrations of adrenergic receptors are localized . in summary ,
we describe a case of transient , albeit severe scm that reversed rapidly . to our knowledge , this is the fastest recovery ever reported .
this case highlights the role of catecholaminergic excess , one of the proposed mechanisms underlying the disease s pathophysiology , and also reinforces the need of aggressive supportive therapy early in the disease .
the authors declare that there are no conflicts of interest regarding the publication of this paper .
|
we report the case of a 79-year - old woman who presented to our hospital for elective removal of an infratentorial meningioma and suffered a periprocedural cardiac arrest . shortly after uncomplicated induction of anesthesia prior to the surgery ,
the patient became hypotensive and bradycardic , culminating ultimately in a cardiac arrest with pulseless electrical activity .
return of spontaneous circulation occurred within 90 seconds of arrest , but the patient remained dependent on maximal doses of epinephrine and dopamine for hemodynamic support .
echocardiography performed on the day of cardiac arrest revealed a newly depressed left ventricular ejection fraction ( lvef ) of 15 - 20% with an apical ballooning pattern . left heart catheterization showed no obstructive coronary lesions to explain her depressed ejection fraction .
a diagnosis of stress cardiomyopathy ( scm ) was made given the echocardiographic findings and absence of concomitant coronary disease . within the next 24 hours
, the patient was liberated from inotropic support , and at 6-month follow - up , her lvef returned to 55% and she had no heart failure symptoms .
|
Introduction
Case Report
Discussion
Funding Sources
Conflicts of Interest
|
appropriate management of moderate postoperative pain is well achieved associating several analgesic drugs with adjuvant agents through synergistic interaction .
the potential advantage of this combined therapy is related to the minimization of the incidences of adverse effects of each drug and to the improvement of the outcome .
postoperative clinical situations , such as difficult patient extubation , respiratory depression due to opioids , and cardiovascular side effects , require a combined therapy to improve analgesia quality and decrease adverse effects of each drug when used alone .
this has led to administering pain drugs in continuous intravenous ( iv ) infusion with different mechanisms and sites of action , together with adjuvant agents .
tramadol hydrochloride ( hcl ) and ketorolac tromethamine are analgesic drugs commonly used in combination in postoperative pain management .
tramadol hcl is a synthetic , centrally acting analgesic with no anti - inflammatory activity and one of the most interesting and useful weak opioids for treatment of moderate to moderately severe pain with weak -receptor agonist properties and noradrenergic and serotonergic neurotransmission effects.[39 ] ketorolac tromethamine is a potent nonsteroidal anti - inflammatory drug ( nsaid ) with analgesic efficacy similar to opioids.[289 ] this drug is administered to treat moderate pain or , combined with reduced opioid doses , for severe pain . according to some studies , metoclopramide hcl as an adjuvant agent can improve analgesia and decrease the need for other pain drugs ; it is a dopamine and 5-ht receptor antagonist , commonly used as a prokinetic and antiemetic .
also , this has been recently investigated as an agent that can enhance the efficacy of analgesic drugs.[91113 ] some studies have also suggested a role of magnesium sulfate ( mgso4 ) as an adjuvant agent , an n - methyl - d - aspartate receptor antagonist , in the management of postoperative pain . the perioperative administration of iv mgso 4 is associated with smaller analgesic requirements , and magnesium sulfate could be of interest as an adjuvant to postoperative analgesia.[1416 ] the aim of this study was to investigate the chemical stability of tramadol hcl combined with ketorolac tromethamine and metoclopramide hcl in solution for 48 h ( 25c ) and 5 days ( 5c ) , with the presence of mgso4 .
milan , italy ) 2 mg / ml , tramadol hcl ( contramal ; grnenthal , gmbh , stolberg , germany ) 20 mg / ml , and ketorolac tromethamine ( lixidol ; roche s.p.a . , milan , italy ) 6 mg / ml stock solutions were prepared in high - performance liquid chromatographic ( hplc)-grade deionized water and stored at 4c during experimental assay .
well - defined volume samples were drawn from each stock solution , joined , and diluted in deionized water to produce 5 working standard solutions , with concentrations in the range of 0.51.5 g / ml for metoclopramide hcl , 2060 g / ml for tramadol hcl , and 39 g / ml for ketorolac tromethamine .
every 48 h the working standard solutions were prepared from stock solutions and stored at 4c .
limit of quantitation ( loq ) values were found to be 1 , 0.5 , and 0.1 g / ml for tramadol , ketorolac , and metoclopramide , respectively .
limit of detection ( lod ) values were 0.05 , 0.2 , and 0.1 g / ml for tramadol , ketorolac , and metoclopramide .
precision and accuracy were determined on spiked samples at 4 concentrations with respect to a calibration graph prepared every day ( n = 3 ) .
the precision of the method was evaluated as the intra- and interday relative standard deviation ( rsd ) of the measured peak areas by assaying spiked samples at 4 different concentrations .
all samples for these purposes were freshly prepared , including preparing the standard solution from the same stock solution .
six drug admixtures were prepared by transferring the contents of one ampule of metoclopramide hcl , 4 ampules of tramadol hcl , 2 ampules of ketorolac tromethamine , and 4 ampules of mgso4 ( 1 g/10 ml ampule , magnesio solfato monico ; monico s.p.a . , venezia mestre , italy ) to give each a final volume of 52 ml .
all the solutions were prepared on different days , stored at 25c and assessed over a period of 48 h. solutions were stored in amber colored glass bottles with air - tight caps to protect them from direct light exposure .
the nominal concentration of each drug in all prepared solutions was 0.19 mg / ml for metoclopramide hcl , 7.69 mg / ml for tramadol hcl , and 1.15 mg / ml for ketorolac tromethamine .
all the drug admixtures were visually inspected immediately after preparation ( 0 h ) and at 24 and 48 h against a black and white background to ensure any physical changes ( eg , color , phase separation , precipitation ) .
a 100 l sample was drawn from each solution at intervals of 0 ( initial ) , 24 , and 48 h and diluted to 20 ml with deionized water ; we prepared solutions in triplicate at each time interval to have 54 samples .
twenty microliters of each sample was injected into the hplc system to determine concentration of each drug .
as previously described , 6 drug admixtures were prepared on different days , stored at 5c ( refrigerated ) , assessed , and visually inspected at intervals of 0 ( initial ) , 1 , 3 , and 5 days .
a 100 l of sample was drawn from each solution and diluted to 20 ml with deionized water ; we prepared solutions in triplicate at each time interval to have 72 samples .
twenty microliters of each sample was injected into the hplc system to determine concentration of each drug . the hplc method developed by kk et al
was modified for use in this study.[1718 ] the instrumentation included a binary pump ( model g1312a ; agilent technologies ; hewlett - packard , waldbronn , germany ) and an ultraviolet ( uv)variable wavelength detector ( model g1314a ; agilent ) . at the beginning of our study
, we looked for the maximum uv absorption wavelength for each drug solution on the basis of uv spectra for each solution . as we used uv
variable wavelength detector , and metoclopramide and tramadol retention times were very close reciprocally , we found that 271 nm was the best wavelength for both drugs to have good sensitivity and maximum signal / noise ratio . for ketorolac drug solutions
separation was achieved using a reversed - phase c18 5 m particle size column ( luna c18 ( 2 ) 100a , 150 4.6 mm ; chemtek analytica srl , anzola emilia ( bo ) , italy ) , equipped with a 2-cm precolumn , which was maintained at 25c with a column temperature controller ( thermosphere ts-130 ; phenomenex , torrance , california ) .
hplc - gradient analysis were performed using the following mobile phase : 0.01 m phosphate buffer , potassium dihydrogen phosphate [ kh2po4 ] ultra for molecular biology ( fluka and riedel - de han , buchs sg , switzerland ) and acetonitrile ( sigma - aldrich supelco , bellefonte , pennsylvania ) ( 75:25 , v / v ) with the addition of 0.1% triethylamine , adjusted to ph 3 with phosphoric acid ( solvent a ) and h2o acetonitrile ( 50:50 , v / v , solvent b ) .
0% b , 610 min 80% b , 1015 min 0% b [ table 1 ] .
the elution - gradient program each mobile phase was prepared fresh daily , filtered through a 0.45 m 47 mm nylon membrane filter ( supelco , bellefonte , pennsylvania nylon 66 filter membranes pore size 0.20 m , diameter 47 mm ) , and degassed ultrasonically for 20 min before use .
the flow rate was 1 ml / min , and the injected volume was 20 l .
the run time was 15 min and the approximate retention times for metoclopramide , tramadol , and ketorolac were 2.8 , 3.4 , and 13.4 min , respectively [ figure 1 ] .
uv chromatogram of metoclopramide hydrochloride ( a ) , tramadol hydrochloride ( b ) , and ketorolac tromethamine ( c ) , eluted at 2.8 , 3.4 , and 13.4 min , respectively .
calibration curves were produced using linear regression of the peak area against concentration of each drug [ table 2 ] .
we obtained 5 calibration curves for each drug and all the curves were linear over the concentration ranges considered for each drug : metoclopramide hcl r = 0.991 ; tramadol hcl r = 0.995 ; ketorolac tromethamine r = 0.961 .
the results were averaged and analyzed by linear simple regression model of y = mx + q by the least - squares method .
the t test was used to examine the concentration difference at each time , and the significance level of error was less than 0.001 .
linear simple regression model ( y = mx + q ) by the least - squares method all the data reported in tables 3 and 4 were submitted to anova and differences analyzed by tukey 's honestly significant differences test .
no significant admixture and time - dependent effect interactions were observed ( p 0.05 ) .
percentage % sd of metoclopramide hydrochloride , tramadol hydrochloride , and ketorolac tromethamine remaining in the admixtures with magnesium sulfate after storage at 25c for 48 h percentage % sd of metoclopramide hydrochloride , tramadol hydrochloride , and ketorolac tromethamine remaining in the admixtures with magnesium sulfate stored at 5c for 5 days
milan , italy ) 2 mg / ml , tramadol hcl ( contramal ; grnenthal , gmbh , stolberg , germany ) 20 mg / ml , and ketorolac tromethamine ( lixidol ; roche s.p.a . , milan , italy ) 6 mg / ml stock solutions were prepared in high - performance liquid chromatographic ( hplc)-grade deionized water and stored at 4c during experimental assay .
well - defined volume samples were drawn from each stock solution , joined , and diluted in deionized water to produce 5 working standard solutions , with concentrations in the range of 0.51.5 g / ml for metoclopramide hcl , 2060 g / ml for tramadol hcl , and 39 g / ml for ketorolac tromethamine .
every 48 h the working standard solutions were prepared from stock solutions and stored at 4c .
limit of quantitation ( loq ) values were found to be 1 , 0.5 , and 0.1 g / ml for tramadol , ketorolac , and metoclopramide , respectively .
limit of detection ( lod ) values were 0.05 , 0.2 , and 0.1 g / ml for tramadol , ketorolac , and metoclopramide .
precision and accuracy were determined on spiked samples at 4 concentrations with respect to a calibration graph prepared every day ( n = 3 ) .
the precision of the method was evaluated as the intra- and interday relative standard deviation ( rsd ) of the measured peak areas by assaying spiked samples at 4 different concentrations .
all samples for these purposes were freshly prepared , including preparing the standard solution from the same stock solution .
six drug admixtures were prepared by transferring the contents of one ampule of metoclopramide hcl , 4 ampules of tramadol hcl , 2 ampules of ketorolac tromethamine , and 4 ampules of mgso4 ( 1 g/10 ml ampule , magnesio solfato monico ; monico s.p.a . , venezia mestre , italy ) to give each a final volume of 52 ml .
all the solutions were prepared on different days , stored at 25c and assessed over a period of 48 h. solutions were stored in amber colored glass bottles with air - tight caps to protect them from direct light exposure .
the nominal concentration of each drug in all prepared solutions was 0.19 mg / ml for metoclopramide hcl , 7.69 mg / ml for tramadol hcl , and 1.15 mg / ml for ketorolac tromethamine .
all the drug admixtures were visually inspected immediately after preparation ( 0 h ) and at 24 and 48 h against a black and white background to ensure any physical changes ( eg , color , phase separation , precipitation ) .
a 100 l sample was drawn from each solution at intervals of 0 ( initial ) , 24 , and 48 h and diluted to 20 ml with deionized water ; we prepared solutions in triplicate at each time interval to have 54 samples .
twenty microliters of each sample was injected into the hplc system to determine concentration of each drug .
as previously described , 6 drug admixtures were prepared on different days , stored at 5c ( refrigerated ) , assessed , and visually inspected at intervals of 0 ( initial ) , 1 , 3 , and 5 days .
a 100 l of sample was drawn from each solution and diluted to 20 ml with deionized water ; we prepared solutions in triplicate at each time interval to have 72 samples .
twenty microliters of each sample was injected into the hplc system to determine concentration of each drug .
the hplc method developed by kk et al was modified for use in this study.[1718 ] the instrumentation included a binary pump ( model g1312a ; agilent technologies ; hewlett - packard , waldbronn , germany ) and an ultraviolet ( uv)variable wavelength detector ( model g1314a ; agilent ) . at the beginning of our study
, we looked for the maximum uv absorption wavelength for each drug solution on the basis of uv spectra for each solution . as we used uv
variable wavelength detector , and metoclopramide and tramadol retention times were very close reciprocally , we found that 271 nm was the best wavelength for both drugs to have good sensitivity and maximum signal / noise ratio . for ketorolac drug solutions , we found that 323 nm was the best absorption wavelength .
separation was achieved using a reversed - phase c18 5 m particle size column ( luna c18 ( 2 ) 100a , 150 4.6 mm ; chemtek analytica srl , anzola emilia ( bo ) , italy ) , equipped with a 2-cm precolumn , which was maintained at 25c with a column temperature controller ( thermosphere ts-130 ; phenomenex , torrance , california ) .
hplc - gradient analysis were performed using the following mobile phase : 0.01 m phosphate buffer , potassium dihydrogen phosphate [ kh2po4 ] ultra for molecular biology ( fluka and riedel - de han , buchs sg , switzerland ) and acetonitrile ( sigma - aldrich supelco , bellefonte , pennsylvania ) ( 75:25 , v / v ) with the addition of 0.1% triethylamine , adjusted to ph 3 with phosphoric acid ( solvent a ) and h2o acetonitrile ( 50:50 , v / v , solvent b ) .
the elution - gradient program was 06 min 0% b , 610 min 80% b , 1015 min 0% b [ table 1 ] .
the elution - gradient program each mobile phase was prepared fresh daily , filtered through a 0.45 m 47 mm nylon membrane filter ( supelco , bellefonte , pennsylvania nylon 66 filter membranes pore size 0.20 m , diameter 47 mm ) , and degassed ultrasonically for 20 min before use .
the flow rate was 1 ml / min , and the injected volume was 20 l .
the run time was 15 min and the approximate retention times for metoclopramide , tramadol , and ketorolac were 2.8 , 3.4 , and 13.4 min , respectively [ figure 1 ] .
uv chromatogram of metoclopramide hydrochloride ( a ) , tramadol hydrochloride ( b ) , and ketorolac tromethamine ( c ) , eluted at 2.8 , 3.4 , and 13.4 min , respectively .
calibration curves were produced using linear regression of the peak area against concentration of each drug [ table 2 ] .
we obtained 5 calibration curves for each drug and all the curves were linear over the concentration ranges considered for each drug : metoclopramide hcl r = 0.991 ; tramadol hcl r = 0.995 ; ketorolac tromethamine r = 0.961 .
the results were averaged and analyzed by linear simple regression model of y = mx + q by the least - squares method .
the t test was used to examine the concentration difference at each time , and the significance level of error was less than 0.001 . linear simple regression model ( y = mx + q ) by the least - squares method all the data reported in tables 3 and 4
no significant admixture and time - dependent effect interactions were observed ( p 0.05 ) .
percentage % sd of metoclopramide hydrochloride , tramadol hydrochloride , and ketorolac tromethamine remaining in the admixtures with magnesium sulfate after storage at 25c for 48 h percentage % sd of metoclopramide hydrochloride , tramadol hydrochloride , and ketorolac tromethamine remaining in the admixtures with magnesium sulfate stored at 5c for 5 days
at the mediterranean institute for transplantation and highly specialized therapies ( ismett , palermo , italy ) is already using an experimental protocol for the treatment of moderate to severe pain , which involves the administration of intravenous mixture of tramadol ( 7.69 mg / ml ) , metoclopramide ( 0.19 mg / ml ) , ketorolac ( 1.15 mg / ml ) , and magnesium sulfate ( 77 mg / ml ) . to use this drug combination
is necessary that the drugs are compatible with each other and the mixture is stable over time .
table 3 shows drugs solution concentration after storage period up to 48 h. the data obtained are in agreement with data reported in the literature regarding a mixture of tramadol , ketorolac , metoclopramide , and ranitidine in a solution for intravenous perfusion .
data reported in table 4 show the remaining percentage of drug concentrations in all admixtures prepared and assessed over a period of 5 days .
it is possible to observe constant percentage concentrations remaining for all the 3 drugs at 1 , 3 , and 5 days when stored at 5c .
we believe that the information has clinical utility in facilitating the preparation and dispensing of the tramadol , metoclopramide , ketorolac , and magnesium sulfate mixture in postoperative pain management . on the basis of our results ,
the mixture may be considered for a possible commercial formulation , at the concentrations listed .
in this study , precise and accurate hplc method suitable for stability evaluation of tramadol hcl combined with ketorolac tromethamine and metoclopramide hcl is described .
the physical appearance of the solutions remained constant during the study period , without the formation of any visible discoloration , cloudiness , or precipitation . in admixtures containing mgso4 and stored at 25c , each drug , at the same time , reached at least 98% of the initial concentration during 48 h. solutions prepared adding mgso4 assessed over a period of 5 days at 5c show the lower confidence limit of the estimated regression line of the concentration at 98% of the initial concentration . within these limits , for the purpose of pre - preparing drug admixtures to use with confidence , tramadol hcl infusions may be prepared in advance and then thawed before use in clinical units .
moreover , information about the chemical stability of the drugs could be utilized in further investigations focused on iv infusion solutions .
|
background : tramadol hydrochloride ( hcl ) and ketorolac tromethamine are analgesic drugs , which are commonly used in combination in postoperative pain management . according to some studies , metoclopramide and magnesium sulfate ( mgso4 ) as adjuvant agents can improve analgesia and decrease the need for other pain drugs.materials and methods : the chemical stability of tramadol hcl combined with ketorolac tromethamine and metoclopramide hcl has been studied using a stability - indicating high - performance liquid chromatographic assay method .
calibration curves were produced using linear regression of the peak area against concentration of each drug , with an r2 value 0.96 .
our aim was to investigate the stability of admixture solution of tramadol hcl combined with ketorolac tromethamine and metoclopramide hcl for 48 h ( 25c ) and 5 days ( 5c ) , with mgso4 , which has never been assessed.results:data obtained for admixtures prepared and stored at temperatures of 25c and 5c , show that all drugs have reached at least 98% of the initial concentration.conclusions:for the purpose of pre - preparing drug admixtures to use with confidence , tramadol hcl infusions may be prepared in advance and then thawed before use in clinical units . on the basis of our results , the intravenous mixture of tramadol ( 7.69 mg / ml ) , metoclopramide (
0.19 mg / ml ) , ketorolac ( 1.15 mg / ml ) , and magnesium sulfate ( 77 mg / ml ) may be considered for a possible commercial formulation .
|
INTRODUCTION
MATERIALS AND METHODS
Preparation of stock solutions
Admixture preparation and analysis
High-performance liquid chromatography
Data analysis
RESULTS AND DISCUSSION
CONCLUSIONS
|
antiphospholipid syndrome ( aps ) may predispose the patient to deep vein thrombosis ( dvt ) , pulmonary thromboembolism ( pte ) , pulmonary hypertension and pulmonary infarction , and occasionally progress to pulmonary cavitations .
corticosteroid and immunosuppressive therapy directed at reducing antibody are not advised routinely . in a special situation ,
recognized as catastrophic apl syndrome with recurrent pte along with anticoagulant therapy , the recommendations is to start therapy with immunosuppressive agents .
however , treatment with corticosteroid and other immunosuppressive agents predispose the patients to diabetes mellitus and opportunistic infections such as fungal infection .
opportunistic fungal infection such as mucormycosis and invasive aspergilosis are almost always reported in patients with major risk factors such as diabetic ketoacidosis , long term neutropenia , post transplantation , and high dose long term corticosteroid treatment .
these infections are aggressive , rapidly progressive , angioinvasive , and life threatening diseases .
pulmonary mucormycosis has a rapid progressive course and result in lung cavitations with high a mortality rate .
some less common condition resulting in catastrophic condition are surgical procedures , anticoagulation withdrawal , medications , obstetric complications , neoplasia and systemic lopus erythmatosus ( sle ) flares .
the present case is one of the unique presentations of apl syndrome complicated with catastrophic flare up in a young woman , who died after a possible of fungal infection and immunosuppressive withdrawal .
a 35-year - old woman presented to respiratory clinic because of acute onset of fever , dyspnea exacerbation , hemoptesis , and aggravated bilateral lower limbs edema .
she had two significant episodes of dvt 12 and 10 years earlier after each childbearing .
her first infant suffered from congenital heart disease , and died at the age of 5 months .
afterwards , her condition progressed to pulmonary hypertension and right sided heart failure gradually in the last years . in spite of conventional treatment for dvt and pulmonary thromboembolism
, her condition had developed to severe pulmonary hypertension , severe dyspnea and severe lower limb edema .
she had high serum concentration of antiphospholipid antibodies ( igg : 22 gpl , igm : 17 mpl ) and anticardiolipine antibody ( igg : 25gpl , igm : 21mpl ) , but normal rheumatologic tests including antinuclear antibody ( ana ) , rheumatoid factor ( rf ) , and anti - neutrophil cytoplasmic antibodies ( anca ) .
perfusion lung scanning demonstrated perfusion defects , which was interpreted as high probability of pulmonary thromboembolism ( figure 1 ) .
perfusion lung scan : multiple segmental perfusion defects compatible with the presence of pulmonary thromboembolism .
she started receiving warfarin aiming at an international normalized ratio ( inr ) of 3 to 4 .
the measurement of serum levels of antiphospholipid antibodies was repeated on the occasion of deciding about immunosuppressive therapy . assuming the presence of multiple deep vein thrombosis , pulmonary thromboembolism , progressive pulmonary hypertension and positive antiphospholipid antibodies , prednisolone ( 60 mg / day ) and azathioprine ( 50 mg twice a day ) started hoping to prevent more catastrophic events .
she was on treatment with warfarine , azathioprine and prednisolon for 12 months , after which corticosteroid was tapered and discontinued because of hyperglycemia .
the patient then continued to receive azathioprine , warfarine and oral glucose lowering agents , and felt well until recently .
last year , she suffered from several attacks of paroxsismal atrial tachycardia ; therefore , she was admitted to the hospital . because of poor compliance , glycemic control was poor .
there was no history of any serious infection during the last four years , and serial assays for complete blood count was normal . at the last presentation ,
the laboratory findings were as follows : urea ; 55 mg / dl , creatinine ; 1.1 mg / dl , glucose ; 350 mg / dl , prothromobine time ; 25 seconds , inr ; 4.4 , wbc ; 10000/l / with 75% segment and 20% lymphocyte .
microscopic examination of urine showed : wbc ; 4 - 5/high power field , rbc ; 4 - 5/high power field and negative for bacteria .
chest computer tomography ( ct ) scan showed cavity in the medial segment of left lower lobe and a cavity in the apical segment of right lower lobe ( figure 3 ) .
blood culture for bacterial infections , and sputum smear for acid fast bacillus ( afb ) were negative .
the patient was in poor condition , and semi invasive or invasive procedures were not performed .
liposomal amphotricine was not available ; therefore , amphotricin b started empirically for possible invasive fungal infection of lung , and azathioprine was discontinued .
blood glucose remained in an acceptable range by regular insulin therapy . at the end of the first week
, the patient continued to be much better , and blood sugar , urea and creatinine were remained in acceptable range .
after two weeks of treatment , the patient was discharged and amphotricine was replaced by oral itraconasol . because of recurrent paroxysmal atrial tachycardia , the patient was readmitted on day 7 after discharge . at this admission ,
the patient succumbed to severe dyspnea , increased urea ( 192 mg / dl ) and creatinine ( 4.4 mg / dl ) , state of confusion , and was transferred to icu on the day 5 of the admission .
the portable chest radiography was not remarkable for complications such as pneumothorax or new infection .
the patient failed to improve , and died on the second day of icu care with multi organ failure and homodynamic instability .
chest computed tomography showing a cavity in the medial segment of left lower lobe and a cavity in the apical segment of right lower lobe ( arrows ) .
the patients did have the criteria for apl syndrome such as recurrent venous thrombosis , pte , pulmonary hypertension , and pregnancy morbidity as well as the presence of high serum levels of antiphospholipid antibody and anticardiolipine . as it occurred in the present case , pulmonary thromboembolism accompanied by dvt
anticoagulant therapies with heparin followed by life - long warfarine is the optimal prophylactic treatment .
since apl syndrome was not suspected in the present patient , anticoagulant was discontinued at the end of 6 month ; therefore , subsequent multiple thromboembolic events resulted in pulmonary hypertension .
as this case illustrates , apl syndrome can be associated with chronic thromboembolic pulmonary hypertension .
the prevalence of pulmonary hypertension in patient with apl syndrome is estimated to be 2 to 4% .
on the other hand , the prevalence of apl in patients with chronic thromboembolic pulmonary hypertension is around 10 to 20% .
primary non - thromboembolic pulmonary hypertension was also reported in patients with primary apl syndrome .
however , the prevalence of apl has been reported more frequently in thrombembolic type than in primary non - thromboembolic type of pulmonary hypertension .
there is also evidence that apl may contribute to the pathogenesis of pulmonary hypertension in patients with connective tissue diseases .
some cases of apl syndrome fall into a catastrophic situation , which is characterized by overwhelming small vessel occlusive disease simultaneously affecting many organs in a short period of time .
the catastrophic situation represents less than 1% of all patients with apl syndrome , and is usually life - threatening with a 50% mortality rate .
cerebral and cardiac involvements are the main causes of death in catastrophic variant of the syndrome , followed by bacterial and fungal infections . because of highly - suspected fungal infection in the present case , azathioprine was discontinued .
this might have predisposed the patient to flare up of catastrophic syndrome , which was presented as stupor , hemodynamic instability and renal failure after an initial improvement in the condition of patient due to amphotricine therapy .
infections ( 22% ) and surgical procedures ( 10% ) are the most common precipitating factors of catastrophic syndrome reported in catastrophic antiphospholipid syndrome registry followed by anticoagulation withdrawal or low inr ( 8% ) , medications ( 7% ) , obstetric complications ( 7% ) , neoplasia ( 5% ) and sle flare up ( 3% ) . in the present case ,
pulmonary cavitations in patients with apl syndrome are rare , and there are only few case reports of the condition caused by pulmonary embolism and infarction followed by cavitations
. it might be important to mention that the lung cavitations in the present case could not be due to microthrombosis , which is one of the major features of catastrophic syndrome .
however constellation of long term uncontrolled hyperglycemia state , immunosuppressive therapy , and severely decompensated pulmonary circulation , could be predisposing the patient into opportunistic angioinvasive fungal infection such as mucurmycosis .
pulmonary mucormycosis is most often encountered in patients with diabetic ketoacidosis , uncontrolled diabetes , hematological malignancy , severe burn , and after solid organ transplantation .
the definite diagnosis of pulmonary mucormycosis is usually difficult and ante - mortem diagnosis has been made infrequently . because of ill and decompensated condition in the present case , invasive diagnostic procedures such as bronchoscopy either percutaneous or open lung biopsy , were not possible .
the signs and symptoms of the present case might suggest that physicians should be aware of flare up of a catastrophic situation in patients with apl syndrome , if they decide to taper or discontinue the immunosuppressive or corticosteroid regimens .
besides , as the infection may be a possible cause of flare up or relapse , close observation of any infectious condition must be considered .
|
antiphospholipid syndrome ( aps ) is a systemic disease that causes venous and arterial thrombosis in virtually any organ .
sometimes it is complicated into pulmonary infarction and cavitation , pulmonary hypertension , and catastrophic course with high morbidity and mortality .
the present case is a 35-year - old woman with one episode of postpartum deep veins thrombosis ( dvt ) 12 years earlier and the second one after the second labor two years later . in spite of usual therapy for each episode of dvt
, the condition had progressed into severe pulmonary hypertension . the diagnosis of primary apl syndrome was confirmed four years ago .
she had been on warfarin , low dose of steroid , and azathioprine since the diagnosis of apl syndrome .
after one year treatment with steroid and azathiprine the patient showed progressive well being ; however , because of hyperglycemia the steroid tapered and discontinued .
she had several attacks of paroxismal atrial tachycardia in the last year .
on the last time , she presented with severe dyspnea , hemoptesis , and lower limbs edema .
chest radiography and lung ct scan demonsterated the presence of lung cavitations .
because of high suspicious for fungal pulmonary infection , azathioprine was also discontinued . however , constellation renal failure , hemodynamic instability , and confusion caused the patient to succumb to death .
the definite diagnosis of lung cavitations was not obtained .
|
Introduction
Case Description
Discussion
Conclusion
None
|
the patients in the african - heritage ( n = 9 ) and caucasian - heritage ( n = 11 ) groups had similar distributions of sex , age , and duration of diabetes ( male 75 vs. 70% , p = 0.89 ; mean sd age 53.3 7.2 vs. 55.2 4.6 years , p = 0.50 ; and duration 10.3 10.7 vs. 6.8 6.4 years , p = 0.37 , respectively ) .
systolic blood pressure and diastolic blood pressure were 124.4 vs. 122.1 mmhg ( p = 0.75 ) and 77.0 vs. 76.1 mmhg ( p = 0.81 ) , respectively .
the patients were nave to antihypertensive therapy , and equal numbers in each group received metformin ( n = 6 ) and insulin ( n = 2 ) .
a1c and urinary albumin were measured by high - pressure liquid chromatography ( ha 8 - 121 ; biomen , berkshire , u.k . ) and immunoturbidimetry , respectively .
microalbuminuria was excluded on the basis of three consecutive albumin - to - creatinine ratios <3 mg / mmol in sterile , early - morning urine samples and a urinary albumin excretion rate < 30 mg / day .
renal plasma flow ( rpf ) was measured by the constant infusion method ( 6,7 ) .
a bolus dose of 8 mg / kg paraminohippurate ( merck , sharp & dohme , hoddesdon , u.k . ) was given with a 20 mg / min infusion .
after a 90-min equilibration period , the concentration of the infusate was multiplied by the infusion flow rate and divided by the mean of duplicate plasma samples at this and subsequent time points .
plasma paraminohippurate was assayed after deproteinizing the samples with 6% trichloroacetic acid for 10 min at 70c and sequentially adding sodium nitrite , ammonium sulfamate , and n-1-naphthylethylenediamine using a cobas mira ( roche , lewes , u.k . ) .
after initial equilibration , an amino acid mixture ( vamin ; pharamcia & upjohn , milton keynes , u.k . ) was infused ( 0.043 ml kg min ) .
rpf was assessed 80 min later , and then l - nmma ( clinalfa , laufelfingen , switzerland ) was begun at the nonpressor dose of 20 g kg min .
both infusions were continued for a further 20 min , after which a final rpf measurement was made . during the studies ,
blood pressure was monitored automatically ( dinamap ; critikon , basingstoke , u.k . ) , and whole blood was sampled from a venflon in a hand vein to measure glucose by the oxidase method ( one touch ; lifescan , high wycombe , u.k . ) every 10 min .
mean arterial pressure ( map ) was calculated as the diastolic blood pressure plus one - third of the pulse pressure .
renal blood flow ( rbf ) was calculated by dividing the rpf by 1 hematocrit and renal vascular resistance ( rvr ) by dividing map by rbf .
the study was approved by the ethics committee of the whittington hospital national health service trust .
analyses between or within the groups were performed using spss for windows ( version 10 ; spss , chicago , il ) .
continuous variables were compared with parametric or nonparametric tests and associations tested with spearman 's rank correlation test or pearson 's x test according to their distribution .
categorical variables were compared using a test with continuity correction or fisher 's exact test .
clearance and rpf measurements were corrected for a body surface area of 1.73 m. data are expressed as means sd unless otherwise stated .
analyses between or within the groups were performed using spss for windows ( version 10 ; spss , chicago , il ) .
continuous variables were compared with parametric or nonparametric tests and associations tested with spearman 's rank correlation test or pearson 's x test according to their distribution .
categorical variables were compared using a test with continuity correction or fisher 's exact test .
clearance and rpf measurements were corrected for a body surface area of 1.73 m. data are expressed as means sd unless otherwise stated .
comparative baseline measurements of rpf and systolic and diastolic blood pressures were similar between the african - heritage and caucasian - heritage groups ( rpf 533.7 174.7 vs. 565.3 260.8 ml / min per 1.73 m , p = 0.78 ; systolic 124.9 23.7 vs. 121.6 12.3 mmhg , p = 0.29 ; and diastolic 77.1 9.5 vs. 76.3 5.7 mmhg , p = 0.81 , respectively ) .
there were no differences in creatinine clearance or median urinary albumin excretion rate ( 93.7 19.9 vs. 98.9 19.5 ml / min per 1.73 m , p = 0.57 , and 12.6 [ 4.125.0 ] vs. 14.0 [ interquartile range 8.524.1 ] mg / day , p = 0.79 ) .
averaged blood glucose was similar ( 6.7 0.9 vs. 7.4 0.9 mmol / l ; p = 0.14 ) .
a1c was lower in the african - heritage than in the caucasian - heritage group ( 6.8 0.69 vs. 8.0 0.94% ; p = 0.005 ) .
the l - nmma infusion was associated with significant changes in systolic blood pressure in the african - heritage group ( fig .
relative to the baseline and post amino acid measurements , there was a mean rise of 10.0 mmhg ( 95% ci 2.317.9 ; p = 0.017 ) and 7.3 mmhg ( 1.013.7 ; p = 0.03 ) , respectively , in the african - heritage group and 4.3 mmhg ( 1.8 to 10.4 ; p = 0.23 ) and 2.4 mmhg ( 3.5 to 8.3 ; p = 0.38 ) in the caucasian - heritage group .
final blood pressure was higher in the african - heritage group ( 137.5 9.0 vs. 123.4 14.2 mmhg ; p < 0.05 ) and was associated with a fall in rbf ( 46.0 ml / min per 1.73 m ; p < 0.05 ) and a rise in rvr ( from 0.12 0.06 to 0.14 0.04 mmhg ml / min per 1.73 m ; p = 0.036 ) .
the changes in rvr correlated with map ( r = 0.77 ; p = 0.004 ) .
in this study , patients without hypertension or renal disease of african heritage had an increased sensitivity to the renal vasoconstrictive effect of no synthase ( nos ) inhibition .
these data suggest that a reduction in no bioavailability may adversely affect autoregulatory processes that could potentially increase vulnerability to renal damage ( 8) .
we used the amino acid infusion to optimize renal blood flow and suppress tubuloglomerular feedback as a contributor to vasoconstriction .
therefore , the reduction in renal blood flow that we observed was probably due to an effect of nos inhibition on the renovascular smooth muscle .
early in the course of diabetes , no production is necessary to forestall a rise in blood pressure .
hypertension is associated with the generation of no - quenching free radicals and is a prerequisite for the development of renal disease ( 1012 ) .
furthermore , the renal expression of nos in patients with diabetes is related to the degree of vasculopathy ( 13 ) .
it could therefore be considered that upregulation of no production in patients of african heritage is related to a mechanism that opposes an enhanced vasoconstrictor tendency .
although consistent with experimental studies , these outcomes require caution before being generalized . confirmatory studies in patients with and without diabetes with greater power and the evaluation of the role of vasoconstrictive cytokines , angiotensin
|
objective we compared the renal and systemic vascular ( renovascular ) response to a reduction of bioavailable nitric oxide ( no ) in type 2 diabetic patients without nephropathy and of african and caucasian heritage.research design and methods under euglycemic conditions , renal blood flow was determined by a constant infusion of paraminohippurate and changes in blood pressure and renal vascular resistance estimated before and after an infusion of l-ng-monomethyl-l-arginine.resultsin the african - heritage group , there was a significant fall in renal blood flow ( 46.0 ml / min per 1.73 m2 ; p < 0.05 ) and rise in systolic blood pressure ( 10.0 mmhg [ 95% ci 2.317.9 ] ; p = 0.017 ) , which correlated with an increase in renal vascular resistance ( r2 = 0.77 ; p = 0.004).conclusions the renal vasoconstrictive response associated with no synthase inhibition in this study may be of relevance to the observed vulnerability to renal injury in patients of african heritage .
|
RESEARCH DESIGN AND METHODS
Statistical analysis
RESULTS
CONCLUSIONS
|
following exposure of a population to radiation , concern about the risk of radiation - induced cancer can be a major source of anxiety,1,2 particularly for parents concerned about the future health of their children.3 despite the obvious importance of preventing accidental radiation exposure and reducing exposure levels , it is not currently known whether there is a practical way to specifically mitigate radiation - induced cancer risk once exposure has occurred.4,5 post - exposure measures to increase survival from acute radiation are available for individuals needing intensive care after high - dose exposure ; however , for large populations exposed to low radiation doses , there are no medical interventions which are currently advised to reduce the probability of a radiation - induced malignancy later in life .
research into chemical and biologic radio - protectors has been ongoing for many decades,6 and while some agents have been found to confer a certain degree of protection against acute effects when delivered within a short period after irradiation , many need to be administered prior to exposure in order to be effective . in the absence of a specific medical countermeasure , a consensus regarding other strategies proven to be effective in minimizing radiation - induced cancer risk and/or cancer risk more generally , would likely be a valuable public health tool .
in addition to direct effects on cancer burden , empowering individuals in exposed populations by providing safe , proven methods to lower their cancer risk could assist in decreasing anxiety and improving coping skills.7 one candidate for such an approach is caloric restriction ( cr ) .
controlled dietary intake has long been studied in terms of its effects on increasing longevity and reducing cancer incidence.8 cr has also been investigated for its potential as an adjuvant cancer treatment , to slow the growth of existing tumors.9,10 although many specific mechanisms of cr have been documented , it has effects on a wide range of physiological and cellular systems , not all of which are understood .
in fact , the levels of cr required to produce the greatest longevity effects in experimental animals are perhaps beyond what could be reasonably maintained by most people , and certainly would not be advised for children or young people during their developmental years.11 yet , understanding the kinetics of how cancer preventative / suppressive approaches can be instigated long after the time of irradiation affects the risk of radiation - induced cancer can help us to determine the utility of such approaches , and whether they can specifically prevent or merely offset radiation - induced cancer risk .
seven weeks after 1-week - old mice were subjected to 3.8 gy irradiation , they were fed either a diet equivalent to their ad libitum calorie intake ( 95 kcal / mouse / week ) or switched to a nutritionally - balanced diet limited to approximately 1/3 fewer calories ( 65 kcal / mouse / week).12 the results showed that irradiation alone decreased tumor - free lifespan , cr alone increased tumor - free lifespan , and initiating cr after irradiation was able to partially mitigate radiation - induced cancer . separating the data by cause of death revealed that different tumor types / sites responded differently to cr , with radiation - induced lymphomas showing little to no response , while a clear effect could be seen for late - occurring solid tumors .
here , we analysed the pathology results further to identify cases of lethal solid tumors and applied a multistage mathematical model of carcinogenesis to the data13 in order to gain mechanistic insight into the protective effect of cr , specifically as it relates to radiation - induced cancers .
we used lifespan data from male b6c3f1 mice published by shang et al.12 briefly , mice were either irradiated with 3.8 gy of x - rays at 1 week of age , or were sham - irradiated .
this radiation dose was chosen because it is known to efficiently induce both haematopoietic and various solid tumors in the b6c3f1 mouse strain , providing the opportunity to examine whether cr would have differential effects by tumor type . at 7 weeks of age ,
irradiated and unirradiated mice were switched from an ad libitum diet to either a diet limited to 95 kcal / week / mouse ( equivalent to ad libitum ) or to a nutrient - balanced but calorically restricted diet of 65 kcal / week / mouse ( a caloric reduction of approximately one - third ) . at this age , mice are fully developed adults but have not yet reached their peak body weight , allowing the mice to physiologically adapt to long - term cr without interrupting normal development or inducing a sharp decline in body weight .
the four groups were monitored over their natural lifespan , with detailed autopsy and pathology analysis for each mouse . tumor spectrum and latency , and their effects on overall lifespan , were used as measures of the interaction between radiation - induced carcinogenesis and cr . in the original study ,
lifespans were compared based on tumors that the mice harboured at the time of autopsy . however , as such tumors could be either incidental or lethal , the age of the mouse at the time of death may not have been directly related to any one of the tumors discovered at autopsy.14 thus , we elected to use data only from mice with tumor(s ) that were diagnosed as lethal , with the autopsy and pathology records re - evaluated by a veterinary pathologist .
this approach allowed us to compare the age - specific tumor mortality in a manner analogous to that used in human cancer epidemiology .
since cr was not observed to have a significant effect on leukaemia / lymphoma , we limited our analyses to deaths due to any solid tumor ( except sarcoma , which was rare ) , and then further analysed deaths due to lethal hepatocellular carcinoma ( hcc ) , lethal lung tumor , or lethal hemangioma .
we used a simple implementation of the armitage - doll model,13 which postulates that a normal single cell must undergo several critical steps , such as mutations or other rate - limiting events , to become a malignant cell .
armitage and doll used this model to explain the temporal variation in the death rate for solid cancer , and it has since been used across a large number of epidemiological studies to estimate the stages of cancers at many sites.15,16 the mortality rate of cancer at age t , i(t ) ( tumor deaths / mouse - day ) , is related to the probabilities of the transition between each step , p ( transitions per unit time ) . because the transitions must proceed in a unique , sequential order , i(t ) is expressed as shown in equation ( 1 ) , below , where k is the number of critical stages in the course of carcinogenesis a cell needs to pass through before becoming fully malignant , t is the age of the mice , and pi is the probability of a transition from the ( i1 ) to the i
change ( i k ) .
( 1)i(t)=p1p2pk(k-1)tk-1the logarithm of i(t ) is directly proportional to the logarithm of age , as shown in equation ( 2 ) , where a is a constant representing the product of the transition ( pk ) rates divided by ( k1 ) ! , and t
( in our analysis ) is the mid - point of age divided into 200-day increments .
( 2)log i(t)=a+(k-1)logtby plotting the log of age - specific mortality rate , i(t ) , against the log of age ( t ) obtained from the lifespan and pathology data , we used a linear fit ( estimated using the non - linear least squares method with r software version 3.1.3 ) to calculate the intercept ( a ) and slope ( k1 ) for each of the three site - specific tumors and all solid tumors to derive the model parameters pk and k. an estimate of the per - stage transition rate , pk , was calculated from the product of the transition rates , pk .
figure 1 shows the model fit for all solid tumors for the four treatment groups , with the model parameters for all solid tumor data and for the three site - specific analyses shown in table 1 .
the data clearly show a more rapid increase in the mortality rate with age in the two 65 kcal groups ( cr ) compared to the mortality rate of mice on the 95 kcal diet .
this is consistent with the increased lifespan associated with cr reported in the original data , since , although there is a stronger effect of age on the increase in the mortality rate , the mortality rate starts at a lower point and remains lower for most of the animals lifespans , exceeding that of the standard diet only at the most advanced ages , by which time few animals remain alive .
the increase in the slope and thus in the model parameter k with cr is significant for all solid tumors , hcc , and lung tumor deaths ( table 2 ) , but is not significant for hemangioma . for
all solid tumors and hcc , the increase in k associated with cr was equivalent in irradiated and unirradiated mice . on the contrary
, irradiation did not change the estimate of k , with no significant change in the slopes of the graphs of solid tumor deaths or any of the site - specific data . for each of the four treatment groups ,
the age - specific mortality data for all solid tumors shows an increase with age that is consistent with the power function characteristic of the simple armitage - doll model ( r > 0.98 ) .
hcc represented between 27% to 56% of the diagnoses for solid tumor death in each group , and the model for the hcc data ( r > 0.93 ) was very consistent with the all - solid tumor model .
although the model fits were reasonable ( r > 0.88 ) , there were too few cases of lung cancer to provide a model fit for each group , and the model fit was poor for hemangioma ( r > 0.38 ) .
both the pooled solid tumor and hcc models showed a significant increase in k with cr .
an increase in k of around 3 reflects a more rapid increase in the mortality rate in aging mice on the lower calorie diet , albeit starting from a lower baseline mortality rate . according to the tenets of the model
the carcinogenic process which delay the onset of tumors but also allow for the accumulation of cells in the penultimate stages of tumor formation .
the reduction in the product of the transition rates with cr could be the result of an overarching effect common to all of the steps ( such as a decrease in the spontaneous mutation rate ) , or the result of a specific effect which alters the probability of one particular transition .
the results presented here confirm that solid tumor mortality is indeed lower when mice are subjected to cr for most of their lifespan , and excludes artefacts due to variations in tumor stage or accelerated discovery of incidental tumors due to earlier death caused by another tumor type .
the similarity in the effect of cr on both parameters between the irradiated and unirradiated groups may suggest that there is no or little interaction between the detrimental effects of irradiation and the beneficial effects of the dietary regimen .
it is thus possible that cr offsets both spontaneous and radiation - induced carcinogenesis in a similar fashion .
this could mean that existing data on the optimal levels , duration , and timing of cr may be applicable to individuals who have previously been exposed to radiation .
interestingly , although the products of the transition rates did increase , the modelled values of k were not significantly lower in the irradiated mice , consistent with the notion that the probability of mutations was altered without concomitant alterations in the oncogenic pathway .
it may be that a radiation - induced dna mutation , such as inactivation of a tumor suppressor gene , might instantaneously move cells one or more steps forward through a multistage progression towards carcinogenesis , effectively removing the step(s ) .
although not significant , the effect of irradiation on the values of k were estimated to be in the range of a loss of a single step , and , thus , such an explanation is not excluded by our data . since radiation may act on the carcinogenic process by inducing genomic instability , stimulating tissue regeneration following radiation - induced apoptosis , and other physiological changes , as well as introducing dna mutations , it is plausible that the overall effect of irradiation on age - specific mortality would affect both the number of steps and the transition rate . here , the effects of radiation and cr were not included in the model itself , since a sufficient quantity of data are not currently available to formalize the relationship .
however , having demonstrated an effect of cr on both the parameters of this simple model , we can begin to consider the types of experimental data that would be valuable for incorporating radiation exposure and cr directly into the model .
the most pertinent questions pertaining to cr would be :
what is the relationship between the level of cr and the induction of protective effects , and is there a threshold? what is the effect of the interval between radiation exposure and the onset of cr? what is the effect of the duration of cr ?
what is the relationship between the level of cr and the induction of protective effects , and is there a threshold ? what is the effect of the interval between radiation exposure and the onset of cr ? what is the effect of the duration of cr ? in one study , adult trp53 mice ( which are highly predisposed to cancer development )
were assigned to either a calorie - restricted diet or a one - day - a - week fasting regimen , and both diets were associated with a delay in cancer - related death.17 as discussed above , if radiation - mediated cancer induction and cr - mediated cancer suppression are indeed independent of one another , existing data on cr regimens could be used to formulate a model to examine optimal strategies for offsetting both radiation - induced and spontaneous cancer risk .
such approaches have been used to model lung cancer risk after smoking cessation to understand the competing effects of smoking levels , duration , and time since quitting.18 analogously , comparing the effectiveness of long - term / lifetime cr versus short - term intense cr after irradiation might provide insight into the nature of the additional steps implied by the model discussed here , and such animal experiments are now being undertaken .
ultimately , the impractical nature of extreme cr gives rise to the question of whether complementary stressors or mimetics could be used instead .
much research has been undertaken to find cr mimetics,19 and candidates which prove effective will likely be applicable to post - radiation risk mitigation .
we are investigating the effect of addition of dietary supplements ( such as the plant phenol resveratrol , one candidate cr mimetic20 ) to the standard laboratory diet of mice . we are also undertaking experiments to determine whether radiation - induced tumors arising in calorie - restricted and -unrestricted animals show any differences in key carcinogenic pathways at the molecular level .
any differences in tumor phenotype may indicate pathways where cr interacts with the accumulation or selection of mutations . in parallel , we have established experiments utilizing environmental enrichment for laboratory mice ( play equipment , increased housing space , nesting materials ) following radiation exposure to explore whether other simple lifestyle changes may mitigate radiation - induced cancer . it is not expected that any one of these measures alone will have a dramatic impact on cancer incidence , but lifestyle changes that are safe , simple , and beneficial represent low - hanging fruit that might be included in post - disaster counselling and public health campaigns .
human populations invariably have additional cancer risk factors,21 such as smoking , alcohol consumption , sedentary lifestyles , and associated obesity , which are difficult to reproduce in experimental animals .
however , it is likely that existing public health recommendations for reducing cancer risk will be similarly effective in offsetting or mitigating low dose radiation - induced cancer risk . ultimately , at the radiation exposure levels relevant to large populations in the wake of environmental contamination , the increased risk to the individual is small compared to the background risk of cancer in the population .
simple lifestyle changes which can be proven to help offset both spontaneous and radiation - induced risk might provide significant public health benefits , both in terms of decreased cancer burden and improved mental health and well - being .
|
the risk of radiation - induced cancer adds to anxiety in low - dose exposed populations .
safe and effective lifestyle changes which can help mitigate excess cancer risk might provide exposed individuals the opportunity to pro - actively reduce their cancer risk , and improve mental health and well - being . here
, we applied a mathematical multi - stage carcinogenesis model to the mouse lifespan data using adult - onset caloric restriction following irradiation in early life .
we re - evaluated autopsy records with a veterinary pathologist to determine which tumors were the probable causes of death in order to calculate age - specific mortality .
the model revealed that in both irradiated and unirradiated mice , caloric restriction reduced the age - specific mortality of all solid tumors and hepatocellular carcinomas across most of the lifespan , with the mortality rate dependent more on age owing to an increase in the number of predicted rate - limiting steps .
conversely , irradiation did not significantly alter the number of steps , but did increase the overall transition rate between the steps .
we show that the extent of the protective effect of caloric restriction is independent of the induction of cancer from radiation exposure , and discuss future avenues of research to explore the utility of caloric restriction as an example of a potential post - irradiation mitigation strategy .
|
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
|
exacerbations of copd are associated with accelerated loss of lung function , poor quality of life , and mortality.1,2 these events can be predicted by numerous clinical factors , including prior exacerbations , airflow obstruction , symptom burden , gastroesophageal reflux , and leukocytosis.3 it is important to detect copd exacerbations early and minimize their severity .
patients with copd frequently experience significant decreases in oxygen saturation during exercise , attributed to the imbalance between oxygen delivery and exercise - induced demand.4 exercise - induced oxygen desaturation ( eid ) is reported to be associated with hospitalization and mortality in patients with copd.5 the 6-minute walking test ( 6mwt ) has been suggested as the preferred measure to identify patients with copd and eid.6 eid occurs frequently during the 6mwt in patients with copd.7 eid has been related to forced expiratory volume in 1 second ( fev1 ) , diffusion capacity of lung carbon monoxide ( dlco ) , amount of emphysema , and baseline oxygen saturation.810 pulmonary hypertension ( ph ) is an important factor contributing to acute exacerbation of copd.11 ph appears when airflow limitation is severe , and is associated with chronic hypoxemia .
pulmonary vascular remodeling in copd is the main cause of increased pulmonary artery ( pa ) pressure , and is thought to result from the combined effects of hypoxia , inflammation , and capillary loss in severe emphysema.12 the presence of ph has been shown to increase the hospitalization rate and mortality of patients with copd.13,14 computed tomography ( ct)-detected pa enlargement is independently associated with acute exacerbations of copd.15 the pa - to - aorta ( pa : a ) ratio measured by ct scan outperforms echocardiography for diagnosing resting ph in patients with severe copd.16 a pa : a > 1 indicates lower oxygen saturation at rest than a pa : a < 1.15 however , there are no reports on the association between pa : a and eid in patients with copd .
we hypothesized that pa : a correlates with the presence of eid and that 6mwt results are useful for predicting the risk of having a pa : a > 1 .
the present study aimed to examine the relationship between pa : a and eid and develop a simple screening tool by determining the appropriate cutoff score on the 6mwt to predict a pa : a > 1 in patients with copd .
this study analyzed regularly treated outpatients with copd between 2014 and 2015 at the kobe city medical center west hospital .
a total of 64 patients with copd were included after applying the exclusion criteria in this study ( figure 1 ) .
the criteria for diagnosing copd were a smoking history ( 20 pack - years ) and postbronchodilator fev1/forced vital capacity ( fvc ) < 70% .
furthermore , we used the following inclusion criteria to define copd clinically , all of which had to be fulfilled : symptoms , including cough , sputum production , wheezing , dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721 study - exclusion criteria were as follows : history of lung surgical procedures , exacerbation - related hospitalization 3 months before 6mwt , and patients on long - term oxygen therapy .
this examination included an assessment of body weight , height , and medical history ( eg , pulmonary embolism and sleep apnea syndrome ) , gold ( global initiative for chronic obstructive lung disease ) grade 04 , history of acute exacerbations of copd within the previous year , copd assessment test , level of dyspnea ( using the modified medical research council dyspnea scale ) , postbronchodilator spirometry , dlco , 6mwt ( according to international recommendations ) , emphysema area , and pa enlargement on ct .
body mass index ( bmi ) was calculated as weight in kilograms divided by height in meters squared .
gold 0 was defined as current and former smokers with a normal postbronchodilator ratio of fev1:fvc exceeding 0.7 and an fev1 of at least 80% , symptoms , including cough , sputum production , wheezing , and dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721 the 6mwt was performed according to the 2002 american thoracic society guidelines.22 participants were asked to walk indoors on a flat , round , 25 m walking course supervised by a physician and physical therapist .
a pulse oximeter ( wristox 3150 ; nonin medical , plymouth , mn , usa ) with a finger probe measured peripheral oxygen saturation ( spo2 ) during 6mwt , and 6mwt - analysis software ( wristox 2 ; star product , tokyo , japan ) was used . in addition , a modified borg scale was used to quantify the levels of dyspnea perceived by subjects at each minute during the 6mwt .
eid was defined as a nadir spo2 < 90% , spo2 88% , and spo2 4%.2325 one reviewer , blinded to hemodynamic information , analyzed ct scans ( optima ct 660 discovery ; ge healthcare , little chalfont , uk ) .
measurements of the diameter of the main pa and the diameter of the aorta at the level of the bifurcation were used to calculate the pa : a ratio , as previously reported.1416 in cases where the aorta was not uniform in diameter , two measurements were taken 90 apart and the larger diameter used .
pa was measured on the line that joins the origin of the left pa and the center of the adjacent ascending aorta on the axial section at the level of pa bifurcation.26 ct - measured relative pa enlargement was defined as pa : a > 1 ( figure 2).1416 results are expressed as counts or median ( interquartile range ) .
cohen s -coefficient measured intraobserver and interobserver agreements for ct measurements of the pa : a ratio .
bivariate analyses were performed with the use of pearson s test for categorical data and the mann
spearman s rank - correlation coefficient was determined for relationships between the pa : a ratio , lung - function parameters , 6mwt parameters , and ct parameters .
receiver operating characteristic ( roc ) curves were used to determine the threshold values with the best sensitivity and specificity to predict pa : a > 1 , with the best being defined as the point on the roc curve with the shortest distance from the upper - left corner .
sensitivity , specificity , positive / negative predictive value , and positive / negative likelihood were calculated for lung - function parameters and 6mwt parameters of exacerbation - risk factors on the basis of a previous study.6,27,28 all statistical analyses were performed with ezr ( saitama medical center , jichi medical university , saitama , japan ) , which is a graphical user interface for the r project ( r foundation for statistical computing , vienna , austria).29 more precisely , it is a modified version of r commander designed to add statistical functions frequently used in biostatistics , and p - values < 0.05 were considered statistically significant .
this study analyzed regularly treated outpatients with copd between 2014 and 2015 at the kobe city medical center west hospital .
a total of 64 patients with copd were included after applying the exclusion criteria in this study ( figure 1 ) .
the criteria for diagnosing copd were a smoking history ( 20 pack - years ) and postbronchodilator fev1/forced vital capacity ( fvc ) < 70% .
furthermore , we used the following inclusion criteria to define copd clinically , all of which had to be fulfilled : symptoms , including cough , sputum production , wheezing , dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721 study - exclusion criteria were as follows : history of lung surgical procedures , exacerbation - related hospitalization 3 months before 6mwt , and patients on long - term oxygen therapy .
this examination included an assessment of body weight , height , and medical history ( eg , pulmonary embolism and sleep apnea syndrome ) , gold ( global initiative for chronic obstructive lung disease ) grade 04 , history of acute exacerbations of copd within the previous year , copd assessment test , level of dyspnea ( using the modified medical research council dyspnea scale ) , postbronchodilator spirometry , dlco , 6mwt ( according to international recommendations ) , emphysema area , and pa enlargement on ct .
body mass index ( bmi ) was calculated as weight in kilograms divided by height in meters squared .
gold 0 was defined as current and former smokers with a normal postbronchodilator ratio of fev1:fvc exceeding 0.7 and an fev1 of at least 80% , symptoms , including cough , sputum production , wheezing , and dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721 the 6mwt was performed according to the 2002 american thoracic society guidelines.22 participants were asked to walk indoors on a flat , round , 25 m walking course supervised by a physician and physical therapist .
subjects were encouraged using standard methodology every minute of the 6mwt . a pulse oximeter ( wristox 3150 ; nonin medical , plymouth , mn , usa ) with a finger probe measured peripheral oxygen saturation ( spo2 ) during 6mwt , and 6mwt - analysis software ( wristox 2 ; star product , tokyo , japan ) was used .
in addition , a modified borg scale was used to quantify the levels of dyspnea perceived by subjects at each minute during the 6mwt .
90% , spo2 88% , and spo2 4%.2325 one reviewer , blinded to hemodynamic information , analyzed ct scans ( optima ct 660 discovery ; ge healthcare , little chalfont , uk ) .
measurements of the diameter of the main pa and the diameter of the aorta at the level of the bifurcation were used to calculate the pa : a ratio , as previously reported.1416 in cases where the aorta was not uniform in diameter , two measurements were taken 90 apart and the larger diameter used .
pa was measured on the line that joins the origin of the left pa and the center of the adjacent ascending aorta on the axial section at the level of pa bifurcation.26 ct - measured relative pa enlargement was defined as pa : a > 1 ( figure 2).1416
this examination included an assessment of body weight , height , and medical history ( eg , pulmonary embolism and sleep apnea syndrome ) , gold ( global initiative for chronic obstructive lung disease ) grade 04 , history of acute exacerbations of copd within the previous year , copd assessment test , level of dyspnea ( using the modified medical research council dyspnea scale ) , postbronchodilator spirometry , dlco , 6mwt ( according to international recommendations ) , emphysema area , and pa enlargement on ct .
body mass index ( bmi ) was calculated as weight in kilograms divided by height in meters squared .
gold 0 was defined as current and former smokers with a normal postbronchodilator ratio of fev1:fvc exceeding 0.7 and an fev1 of at least 80% , symptoms , including cough , sputum production , wheezing , and dyspnea , smoking history ( 20 pack - years ) , existence of emphysema on chest ct , and a physician diagnosis of copd.1721
the 6mwt was performed according to the 2002 american thoracic society guidelines.22 participants were asked to walk indoors on a flat , round , 25 m walking course supervised by a physician and physical therapist .
a pulse oximeter ( wristox 3150 ; nonin medical , plymouth , mn , usa ) with a finger probe measured peripheral oxygen saturation ( spo2 ) during 6mwt , and 6mwt - analysis software ( wristox 2 ; star product , tokyo , japan ) was used .
in addition , a modified borg scale was used to quantify the levels of dyspnea perceived by subjects at each minute during the 6mwt .
one reviewer , blinded to hemodynamic information , analyzed ct scans ( optima ct 660 discovery ; ge healthcare , little chalfont , uk ) .
measurements of the diameter of the main pa and the diameter of the aorta at the level of the bifurcation were used to calculate the pa : a ratio , as previously reported.1416 in cases where the aorta was not uniform in diameter , two measurements were taken 90 apart and the larger diameter used .
pa was measured on the line that joins the origin of the left pa and the center of the adjacent ascending aorta on the axial section at the level of pa bifurcation.26 ct - measured relative pa enlargement was defined as pa : a > 1 ( figure 2).1416
cohen s -coefficient measured intraobserver and interobserver agreements for ct measurements of the pa : a ratio .
bivariate analyses were performed with the use of pearson s test for categorical data and the mann
spearman s rank - correlation coefficient was determined for relationships between the pa : a ratio , lung - function parameters , 6mwt parameters , and ct parameters .
receiver operating characteristic ( roc ) curves were used to determine the threshold values with the best sensitivity and specificity to predict pa : a > 1 , with the best being defined as the point on the roc curve with the shortest distance from the upper - left corner .
sensitivity , specificity , positive / negative predictive value , and positive / negative likelihood were calculated for lung - function parameters and 6mwt parameters of exacerbation - risk factors on the basis of a previous study.6,27,28 all statistical analyses were performed with ezr ( saitama medical center , jichi medical university , saitama , japan ) , which is a graphical user interface for the r project ( r foundation for statistical computing , vienna , austria).29 more precisely , it is a modified version of r commander designed to add statistical functions frequently used in biostatistics , and p - values < 0.05 were considered statistically significant .
the current analysis comprised 64 patients who were separated into groups on the basis of pa : a > 1 ( n=29 ) and 1 ( n=35 ) .
the -values for intraobserver and interobserver agreements for detecting pa : a > 1 were 0.87 ( 95% confidence interval [ ci ] 0.740.99 ) and 0.75 ( 95% ci 0.580.91 ) , respectively .
differences in the pa : a ratio between both groups were driven by the diameter of pa ( 2.9 [ 2.73.3 ] cm in pa : a 1 vs 3.7 [ 3.53.9 ] cm in pa : a > 1 , p=0.002 ) , because no differences were detected in the diameter of aortae ( 3.7 [ 3.43.9 ] cm vs 3.5 [ 3.33.7 ] cm , p=0.20 ) .
there were no significant differences between the two groups with regard to age , sex , bmi , pack - years , modified medical research council dyspnea scale , gold , copd assessment test , baseline pulse rate , or baseline modified borg scale ( p>0.05 ) .
on the other hand , there were significant differences between the two groups with regard to fev1 ( 71.6% [ 60.5%80.8% ] vs 52.6% [ 39.6%72.1% ] , p=0.013 ) , fvc ( 82.3% [ 50.3%93.6% ] vs 75.8% [ 42.7%86% ] , p=0.04 ) , fev1:fvc ratio ( 68% [ 61%73.3% ] vs 53.8% [ 48.8%69.4% ] , p=0.023 ) , dlco ( 72.5% [ 55.5%82.9% ] vs 44.6% [ 37.7%49.6% ] , p=0.005 ) , bode ( bmi , obstruction [ airflow ] , dyspnea , and exercise performance ) index ( 2 [ 13 ] vs 4 [ 25 ] , p<0.001 ) , 6mw distance ( 6mwd ; 450 m [ 400510.5 ] vs 325 m [ 238466 ] , p<0.001 ) , baseline spo2 ( 97% [ 95%97.5% ] vs 95% [ 93%96% ] , p=0.001 ) , lowest spo2 ( 92% [ 91%94% ] vs 86% [ 84%88% ] , p<0.001 ) , highest modified borg scale result ( 2 [ 05 ] vs 5 [ 25 ] , p=0.04 ) , low - attenuation area ( laa ; 6.8% [ 2.8%14.7% ] vs 25.4% [ 11.3%33.4% ] , p<0.001 ) , and history of acute exacerbations of copd within the previous year ( 1 [ 2.9% ] vs 7 [ 24.1% ] , p=0.019 ) ( table 1 ) .
the pa : a ratio demonstrated a significant linear correlation with lowest spo2 ( r=0.68 , r=0.46 ; p<0.001 ) , dlco ( r=0.61 , r=0.37 ; p<0.001 ) , 6mwd ( r=0.43 , r=0.18 ; p<0.001 ) , bode index ( r=0.41 , r=0.17 ; p<0.001 ) , baseline spo2 ( r=0.36 , r=0.13 ; p=0.003 ) , laa ( r=0.36 , r=0.13 ; p=0.004 ) , fvc ( r=0.34 , r=0.12 ; p=0.006 ) , fev1 ( r=0.29 , r=0.08 ; p=0.019 ) , and highest pulse rate ( r=0.26 , r=0.07 ; p=0.035 ) ( table 2 ) .
using roc curves , the threshold values with the best cutoff point , sensitivity , and specificity to predict pa : a > 1 were determined for spo2 during the 6mwt ( best cutoff point 89% , area under curve [ auc ] 0.94 , 95% ci 0.881 ) , dlco ( best cutoff point 51% , auc 0.87 , 95% ci 0.780.96 ) , 6mwd ( best cutoff point 388 m , auc 0.75 , 95% ci 0.620.87 ) , and bode index ( best cutoff points 4 , auc 0.74 , 95% ci 0.610.87 ) ( table 3 , figure 3 ) .
the performance data on the 6mwt and lung function for predicting pa enlargement are depicted in table 4 .
spo2 < 90% , spo2 88% , and spo2 4% during 6mwt were 94.3 ( 80.899.3 ) , 97.1 ( 85.199.9 ) , and 45.7 ( 28.863.4 ) , respectively , for specificity , 93.1 ( 77.299.2 ) , 95.8 ( 78.999.9 ) , and 59.6 ( 44.373.6 ) , respectively , for positive predictive value , and 16.2 ( 4.262.8 ) , 27.7 ( 4193.3 ) , and 1.8 ( 1.32.4 ) , respectively , for positive likelihood ratios .
first , we found a strong association between the pa : a ratio and lowest spo2 during the 6mwt .
for this reason , a consistent finding in patients with copd is the close relationship between severity of hypoxemia and pa pressure or pulmonary vascular resistance , supporting a major role for alveolar hypoxia.30 alveolar hypoxia causes constriction of the resistance pas , and sustained alveolar hypoxia induces pulmonary vascular remodeling.28 pathological studies of lung specimens from patients with copd have shown extensive pulmonary vascular remodeling , with prominent intimal thickening and medial hypertrophy . for this reason , chronic alveolar
hypoxia plays an important role in pulmonary vascular remodeling.28 in a previous study , patients with pa : a > 1 showed lower resting spo2 , higher usage rates of supplemental oxygen , and an indirect association with eid.15 in the present study , the lowest spo2 during the 6mwt to predict pa : a > 1 was considered to be a beneficial result based on the roc curves .
these results suggest that the lowest spo2 during the 6mwt is a very helpful measure and screening test for the pa : a ratio .
for example , it might be possible to easily screen for pulmonary artery expansion by means of the six - minute walking test in a home - care situation , where it would be difficult to perform ct imaging .
second , with regard to the relationship between pa : a ratio and lung function , correlations were observed among fev1 , dlco , and laa .
one of the factors that may play a role in causing ph to advance in patients with copd is the destruction of lung parenchyma , leading to loss of part of the pulmonary vascular bed,30,31 and the burden of airway remodeling influencing pa - pressure increase.28 a previous study included patients with airflow - obstruction severity greater than moderate , and our study included mild airflow obstruction and smokers with normal spirometry.15 therefore , regardless of the severity of airflow obstruction , pa enlargement may be progressing .
undiagnosed copd is a problem worldwide.18 gold 0 has been reported to be an exacerbation risk ; therefore,18 early detection and not just spirometry evaluation is important from multiple perspectives.18,32 from the viewpoint of early detection of pa enlargement , a definition of eid as spo2 < 90% may be a good start .
however , these findings may imply that pa : a > 1 is one of the multiple risk factors for acute copd exacerbations .
one reason for this is that pa : a is associated with ph16 and ph is also a risk factor for acute copd exacerbations.33 furthermore , a previous study reported an association between the pa : a ratio and acute copd exacerbation.15 these results suggest that screening for the pa : a ratio without ct using the 6mwt may indicate the risk of acute copd exacerbations at an early stage .
this study had some limitations , including small size , single - center experience , and retrospective design .
in addition , this study also included copd subjects who did not fit the gold criteria .
furthermore , because healthy controls do not have respiratory symptoms and there are no control data for the measurement items pertaining to such individuals , healthy controls were not included in the present study .
however , it has been reported that the presence of clinical symptoms and low dlco in smokers , even with normal spirometry , increases the risk of progression to airflow obstruction in copd.1720 therefore , the present study s results during the 6mwt could be useful to screen for ph at an early copd stage , even in gold 0 patients . finally , according to a previous study , left ventricular dysfunction causes pa enlargement .
however , echocardiography was not performed in all subjects , and this information could not be included because it was unavailable from the medical history , although we observed clinically relevant associations between ct - measured pa : a ratios and 6mwt results .
the current study s findings suggest that there is a strong association between pa : a ratio and lowest spo2 during the 6mwt .
lowest spo2 during the 6mwt is a very helpful measurement to predict pa : a ratios on ct , and lowest spo2 of < 89% during the 6mwt is excellent to screen for ph in copd .
|
purposepulmonary hypertension and exercise - induced oxygen desaturation ( eid ) influence acute exacerbation of copd .
computed tomography ( ct)-detected pulmonary artery ( pa ) enlargement is independently associated with acute copd exacerbations .
associations between pa to aorta ( pa : a ) ratio and eid in patients with copd have not been reported .
we hypothesized that the pa : a ratio correlated with eid and that results of the 6-minute walk test ( 6mwt ) would be useful for predicting the risk associated with pa : a > 1.patients and methodswe retrospectively measured lung function , 6mwt , emphysema area , and pa enlargement on ct in 64 patients with copd .
the patients were classified into groups with pa : a 1 and > 1 .
receiver - operating characteristic curves were used to determine the threshold values with the best cutoff points to predict patients with pa : a > 1.resultsthe pa : a > 1 group had lower forced expiratory volume in 1 second ( fev1 ) , forced vital capacity ( fvc ) , fev1:fvc ratio , diffusion capacity of lung carbon monoxide , 6mw distance , and baseline peripheral oxygen saturation ( spo2 ) , lowest spo2 , highest modified borg scale results , percentage low - attenuation area , and history of acute copd exacerbations 1 year , and worse bode ( body mass index , airflow obstruction , dyspnea , and exercise ) index results ( p<0.05 ) . predicted pa : a > 1 was determined for spo2 during 6mwt ( best cutoff point 89% , area under the curve 0.94 , 95% confidence interval 0.881 ) .
spo2 < 90% during 6mwt showed a sensitivity of 93.1 , specificity of 94.3 , positive predictive value of 93.1 , negative predictive value of 94.3 , positive likelihood ratio of 16.2 , and negative likelihood ratio of 0.07.conclusionlowest spo2 during 6mwt may predict ct - measured pa : a , and lowest spo2 < 89% during 6mwt is excellent for detecting pulmonary hypertension in copd .
|
Introduction
Patients and methods
Study design and patient selection
Clinical characterization
Assessments
Six-minute walking test
Measuring the PA:A ratio
Statistical analysis
Results
Discussion
Limitations
Conclusion
|
patients with a preexisting episcleral encircling element who underwent implantation of a silicone agv to treat intractable glaucoma during january 2009 to september 2010 at a large , tertiary care ophthalmic hospital were identified by means of operative records .
postsurgical follow - up of less than 6 weeks and inability to measure intraocular pressure ( iop ) were the exclusion criteria .
the conjunctival incision was made at the corneal limbus or at 4 - 5 mm behind and parallel to the limbus , at the operating surgeon 's discretion , for approximately 100 in the supero - temporal quadrant .
the agv ( model fp7 , new world medical , rancho cucamonga , la , usa ) was primed by injecting 1 - 2 ml balanced salt solution .
the plate of agv was placed over the episcleral encircling element ; either encircling band or scleral buckle [ figure 1 ] .
the plate was secured to the underlying sclera with 8 - 0 nylon suture material ( m / s gn corporation ltd . ,
yamanashi , japan ) such that its anterior edge lied 8 mm behind the corneal limbus .
this was followed by placement of the silicone tube into anterior chamber or pars plana region through a 23-gauge needle track .
the anterior part of the tube was covered with previously prepared human donor scleral patch graft .
fibrin sealant ( tisseel kit , baxter ag , vienna , austria ) or 8 - 0 nylon suture material was used to secure the scleral patch graft .
the overlying conjunctiva was sutured with 8 - 0 polyglactin suture material ( ethicon inc . ,
if there was limited mobility of the conjunctiva due to prior surgeries , relaxing incisions were given in the conjunctiva and/or amniotic membrane transplantation was considered .
the eye was inspected for any leaks after the anterior chamber was inflated using balanced salt solution .
the base plate of ahmed glaucoma valve being placed over the episcleral encircling element that is indicated by arrows the donor scleral tissue preserved in absolute alcohol was used in every case .
the tissue was cleaned of all the uveal tissue attachments , washed thoroughly with balanced salt solution and cut into the desired size ( 4 - 5 4 - 5 mm ) .
the data collection included information on patient demography , diagnosis of glaucoma , prior ocular surgeries , measurement of visual acuity , iop , number of antiglaucoma medications at pre - agv and every post - agv follow - up visit , duration between placement of episcleral encircling element and agv , and complications , if any .
we measured iop either by applanation tonometer , namely , goldmann tonometer ( haag - streit , switzerland ) , a hand - held perkin 's tonometer ( haag - streit , essex , uk ) , or by tonopen xl ( reichert ophthalmic instruments , walden ave .
the cause ( s ) for low vision and postoperative reduction in visual acuity , if any , were also recorded .
surgical success was defined as a final iop between 5 and 22 mmhg without ( complete success ) or with topical antiglaucoma medication ( s ) ( qualified success ) and without any vision threatening complication .
paired t - test was used to compare measurements of iop and number of antiglaucoma medications at the preoperative and the final visits .
data analysis was done using spss software version 15.0 for windows ( spss inc . ,
patients with a preexisting episcleral encircling element who underwent implantation of a silicone agv to treat intractable glaucoma during january 2009 to september 2010 at a large , tertiary care ophthalmic hospital were identified by means of operative records .
postsurgical follow - up of less than 6 weeks and inability to measure intraocular pressure ( iop ) were the exclusion criteria .
the conjunctival incision was made at the corneal limbus or at 4 - 5 mm behind and parallel to the limbus , at the operating surgeon 's discretion , for approximately 100 in the supero - temporal quadrant .
the agv ( model fp7 , new world medical , rancho cucamonga , la , usa ) was primed by injecting 1 - 2 ml balanced salt solution .
the plate of agv was placed over the episcleral encircling element ; either encircling band or scleral buckle [ figure 1 ] .
the plate was secured to the underlying sclera with 8 - 0 nylon suture material ( m / s gn corporation ltd . ,
yamanashi , japan ) such that its anterior edge lied 8 mm behind the corneal limbus .
this was followed by placement of the silicone tube into anterior chamber or pars plana region through a 23-gauge needle track .
the anterior part of the tube was covered with previously prepared human donor scleral patch graft .
fibrin sealant ( tisseel kit , baxter ag , vienna , austria ) or 8 - 0 nylon suture material was used to secure the scleral patch graft .
the overlying conjunctiva was sutured with 8 - 0 polyglactin suture material ( ethicon inc . , aurangabad , india ) .
if there was limited mobility of the conjunctiva due to prior surgeries , relaxing incisions were given in the conjunctiva and/or amniotic membrane transplantation was considered .
the eye was inspected for any leaks after the anterior chamber was inflated using balanced salt solution .
the base plate of ahmed glaucoma valve being placed over the episcleral encircling element that is indicated by arrows
the tissue was cleaned of all the uveal tissue attachments , washed thoroughly with balanced salt solution and cut into the desired size ( 4 - 5 4 - 5 mm ) .
the data collection included information on patient demography , diagnosis of glaucoma , prior ocular surgeries , measurement of visual acuity , iop , number of antiglaucoma medications at pre - agv and every post - agv follow - up visit , duration between placement of episcleral encircling element and agv , and complications , if any .
we measured iop either by applanation tonometer , namely , goldmann tonometer ( haag - streit , switzerland ) , a hand - held perkin 's tonometer ( haag - streit , essex , uk ) , or by tonopen xl ( reichert ophthalmic instruments , walden ave .
the cause ( s ) for low vision and postoperative reduction in visual acuity , if any , were also recorded .
surgical success was defined as a final iop between 5 and 22 mmhg without ( complete success ) or with topical antiglaucoma medication ( s ) ( qualified success ) and without any vision threatening complication .
paired t - test was used to compare measurements of iop and number of antiglaucoma medications at the preoperative and the final visits .
data analysis was done using spss software version 15.0 for windows ( spss inc . ,
fourteen patients underwent implantation of agv in eyes with a preexisting episcleral encircling element during the study period .
two eyes ( 16.6% ) had undergone placement of a circumferential scleral buckle in the supero - temporal quadrant . in one of these eyes with scleral buckle
the shunt plate was additionally covered with two layers of cryo - preserved human amniotic membrane to reduce the chances of exposure of this implant in the postoperative period .
the mean ( standard deviation ) duration between placement of episcleral encircling element and implantation of agv was 30.5 ( 33.8 ) months ; median , 11.5 months .
the mean follow - up was 37.4 ( 22.9 ) weeks ; median , 33.5 weeks .
the mean preoperative iop was 31.4 ( 7.9 ) mmhg ; median , 32mmhg with an average of 2.8 ; median , 3 medications . at last postoperative follow - up , the mean iop decreased to 12.5 ( 3.5 ) mmhg
the postoperative reduction in iop and in number of antiglaucoma medications was statistically significant ( p < 0.001 , paired t - test ) .
demographic / pre - operative data corneal graft failure occurred in three eyes in late postoperative period following implantation of agv ( cases 4 , 7 , 9 ) . in one of these eyes , the agv tube was placed in the pars plana region . in the remaining two eyes ,
the agv tube was placed in the anterior segment , however , there was no tube - cornea touch .
the mean duration between placement of agv and corneal graft failure was 7.3 ( 5.3 ) months ; median , 8.5 months .
tube erosion occurred in another two eyes in late postoperative period ( cases 10 , 11 ) .
case 12 reported reduced vision at sixth postoperative week and was found to have a rhegmatogenous retinal detachment .
in this series , we describe implantation of the silicone plate agv in eyes with a preexisting episcleral encircling element
. this novel use of ahmed valve can be helpful in the management of intractable glaucoma after the encircling procedure .
described the use of a silicone tube to shunt aqueous from the anterior segment to the fibrous capsule surrounding a previously placed episcleral encircling element . using the existent fibrous capsule around the encircling element as an aqueous reservoir obviated the need for temporary restriction of the aqueous flow to allow encapsulation .
early postoperative complications attributable to hypotony were few and self - limiting . however , eight instances of fibrous obstruction of the distal tube opening occurred within 4 months of the initial procedure .
suh et al . also reported high overall success rate with the same technique , but they studied only seven eyes .
modified the technique by using a valved ( plate less ) krupin - denver tube . no case of early postoperative hypotony occurred , although two ( 29% ) of seven eyes experienced distal tube occlusion necessitating surgical revision .
scott et al . described an alternative approach to manage refractory glaucoma in eyes with a preexisting encircling band .
they inserted 250- ( 7 eyes [ 44% ] ) or 350-mm ( 9 eyes [ 56% ] ) baerveldt drainage devices behind or over a preexisting encircling band and over the adjacent recti muscles .
the fibrous capsule overlying the encircling band was excised in the quadrant of surgical implantation .
nine patients ( 56% ) achieved complete success and seven patients ( 44% ) achieved qualified success at 1 year postoperatively .
implant migration or exposure and diplopia did not develop in any patient over the median follow - up of 22.9 months .
however , it might be unsafe to insert a large sized implant in the presence of an encircling band in all four quadrants due to the effects of crowding
. it may be that this study with limited number of patients does not reflect the true outcome . the incidence of postoperative diplopia has been shown to be significantly higher with the baerveldt than any other implant .
this is the reason smith et al . trimmed the wings of baerveldt implants to debulk them before insertion in eyes with a preexisting episcleral encircling element .
the valve abutted against the anterior portion of the scleral band and was sutured into position .
the valve function of the implant was retained to reduce postoperative hypotony . however , the reduction in the surface area of the trimmed drainage implant is of concern as the extent of iop reduction with glaucoma drainage implants has been demonstrated to be directly proportional to the surface area of the fibrous capsule .
additionally , it may be difficult to mobilize adequate conjunctiva to cover the implant in eyes with a preexisting episcleral encircling element , especially a scleral buckle .
an implant with minimum - sized end - plate that is sufficient to achieve iop control is preferable in such eyes . increasing the surface area of
a randomized controlled clinical trial showed comparable failure rates at 1-year for both agv and 350 mm baerveldt implants .
therefore , agv should be preferred over baerveldt implant in eyes with a preexisting episcleral encircling element .
moreover , the base plate of the newer silicone ahmed valve ( model fp7 ) is thinner ( 0.9 mm ) and has the same surface area ( 184 mm ) compared with the earlier polypropylene model s2 ( thickness 1.9 mm ) .
the silicone base plate is also flexible and has a tapered profile for easy insertion .
these features allowed us to insert a silicone agv over the episcleral element in our patients .
one can also make use of a long , curved surgical scissor for better access to the supero - temporal quadrant , and guide the base plate of the agv into position over the partly open blades of the scissor .
two patients in our series had preexisting scleral buckle in the supero - temporal quadrant ( cases 8 and 12 ) .
however , the procedure was difficult in one eye ( case 12 ) due to multiple prior surgeries .
we gave a couple of relaxing incisions in the surrounding conjunctiva for adequate wound closure .
the shunt plate was additionally covered with two layers of cryo - preserved human amniotic membrane , as described by amini et al .
to reduce the chances of exposure of the implant in the postoperative period . despite these efforts , the anterior edge of the scleral patch graft covering the implant tube did expose in the early postoperative period
one of them had prior corneal graft failure ( case 9 ) . in two of these three eyes ,
the agv tube was placed in the anterior segment , however , there was no demonstrable tube - cornea touch .
found corneal graft failure in 24 ( 43.6% ) out of 55 eyes that had undergone both penetrating keratoplasty ( pk ) and either baerveldt or ahmed glaucoma implant .
also found poor long - term survival of corneal grafts in their study of 77 eyes that underwent pk following agv implantation .
the grafted corneas failed in 46 [ 59.1% , ( 95% confidence interval : 47.5 - 71.2% ) ] eyes at 3 years .
prior pk and stromal vessels , but not tube - cornea touch , did increase the risk of graft failure .
erosion of drainage tube through the overlying conjunctiva was seen in two eyes despite meticulous conjunctival closure and the use of a scleral patch graft .
both eyes underwent early repair as tube erosion carries with it increased risk of endophthalmitis .
he was a high myope , suffered from posttraumatic retinal detachment and developed secondary glaucoma following retinal detachment repair .
retinal detachment as a complication of glaucoma drainage device has been reported in as many as 16% cases with varied risk factors . nevertheless ,
considering the presence of multiple risk factors for retinal detachment , the complication may not necessarily be related to the implantation of agv in our case .
conventional filtration surgery such as trabeculectomy was either not possible or carried an unacceptably high risk of failure in these eyes .
known frequent complications such as early postoperative hypotony , shallow anterior chamber , tube obstruction , and conjunctival wound leak were not detected in this series .
even so , the overall postsurgical complications do appear frequent in this series ; however , many patients maintained useful vision that they could potentially have lost since all had uncontrolled glaucoma prior to surgery .
the retrospective design and the heterogeneity of the cases are major limitations of this study .
the sample size is small and therefore , we did not analyze complete and qualified success separately .
however , the clinical situations in which the procedures were done were extremely severe and until now none of the proposed treatments have gained general acceptance .
this study , for the first time , shows that the silicone , flexi - plate ahmed valve can be effectively implanted in eyes with a preexisting episcleral encircling element keeping in mind the possibility of significant postoperative complications .
|
background : to describe the use of ahmed glaucoma valve ( agv ) in the management of intractable glaucoma in eyes with a preexisting episcleral encircling element.materials and methods : this is a retrospective , consecutive , noncomparative study .
the study included 12 eyes of 12 patients with a preexisting episcleral encircling element that underwent implantation of silicone agv to treat intractable glaucoma during january 2009 to september 2010.results:the mean patient age was 25.6 ( standard deviation 17.1 ) years .
five ( 41.6% ) patients were monocular .
the indications for agv were varied .
the mean duration between placement of episcleral encircling element and implantation of agv was 30.5 ( 33.8 ) months .
the mean follow - up was 37.4 ( 22.9 ) weeks .
preoperatively , the mean intraocular pressure ( iop ) was 31.4 ( 7.9 ) mmhg and the mean antiglaucoma medications were 2.8 . at the final postoperative follow - up , the mean iop was 12.5 ( 3.5 ) mmhg and the mean number of antiglaucoma medications was 0.8 ( p < 0.001 ) .
the complications observed over the follow - up period did include corneal graft failure in three eyes , tube erosion in two eyes and rhegmatogenous retinal detachment in one eye.conclusion:agv is an effective option in the management of intractable glaucoma in eyes with a preexisting episcleral encircling element keeping in mind the possibility of significant postoperative complications .
|
Materials and Methods
Study design
Surgical procedure
Preparation of donor scleral graft
Data collection and analysis
Results
Discussion
|
hydroxyl radical attack on dna most frequently leads to base damages that result in generating a range of derivatives [ 13 ] .
aerobic organisms , within the course of evolution , developed a range of adaptive mechanisms inducing synthesis of anti - oxidative enzymes and/or enzymes repairing oxidative damages of dna [ 57 ] .
oxidative damage to nucleic acid has been associated with a number of pathologies including cancer and neurodegenerative and cardiovascular diseases [ 810 ] . to date , more than 20 different types of oxidative modifications of bases have been identified .
of all the modified bases , the processes which repair 8-oxogua are perhaps best understood , and may be regarded as a template for the processes which repair other lesions . to combat the deleterious biological effect of the presence of 8-oxogua , cells have developed specific mechanisms to remove this lesion from cellular dna . in mammalian cells ,
the first level of this protection is human mut t homologue ( hmth1 ) which hydrolyses 8-oxodgtp ( a potential substrate for dna polymerase ) , thereby eliminating it from the nucleotide pool .
the second level of defence is specific glycosylases that initiate base excision repair ( ber ) .
finally , human mut y homologue ( hmyh ) removes adenine that is mis - paired with 8-oxogua .
most recently , we proposed that nucleotide excision repair ( ner ) , which involves the removal of a lesion - containing oligonucleotide , may compliment the go system , based upon evidence that oxidative dna damage may be repaired by this route .
however , there is very little evidence that 8-oxodg is a direct product of dna repair itself ( ie , released as the deoxynucleoside , rather than the base , from dna ) .
it is generally accepted that products of cellular repair of oxidatively damaged dna , such as modified bases and nucleosides : 8-oxo-7,8-dihydroguanine ( 8-oxogua ) and 8-oxo-7,8-dihydro-2-deoxyguanosine ( 8-oxodg ) are excreted in urine .
an assay was performed to analyze levels of 8-oxogua and 8-xoxdg in urine samples with regard to diet . in case of the examined group
, a conclusion was drawn that diet does not determine excretion of these biomolecules in urine . in another study , different amounts ( up to 25 mg ) of 15n labeled oxidatively modified dna were absorbed orally by volunteers . throughout 2 weeks , blood and urine samples were collected .
no 15n 8-oxogua or 8-oxodg were detected either in urine and or in dna of monoclonal cells in venous blood obtained from the same subjects taking part in the study , demonstrating that diet has no influence on the level of these damages . in both known works by faure et al . and erhola et al . , no rise of levels in 8-oxodg excretion in urine was observed despite unequivocal evidence for mass reduction of treated tumors . in certain reports , a clear rise of 8-oxodg in urine excretion was observed after radio - chemotherapy or radiotherapy itself [ 2123 ] .
however , measuring excretion of repair product in urine exclusively may be misleading since it provides no information on the oxidative state of the organism ( damage rate vs. repair rate ) in cellular dna and reports only the mean value of damage repair in the past .
the aim of the work was to investigate whether the levels of markers of oxidative damages of dna : excreted in urine 8-oxo-7,8-dyhydro-2-deoxyguanosine ( 8-oxodg ) and 8-oxo-7,8-dyhydroguanine ( 8-oxogua ) as well as the level of 8-oxodg in dna of venous blood leukocytes differ in a population of healthy subjects when compared with cancer patients .
hydroxyl radical attack on dna most frequently leads to base damages that result in generating a range of derivatives [ 13 ] .
aerobic organisms , within the course of evolution , developed a range of adaptive mechanisms inducing synthesis of anti - oxidative enzymes and/or enzymes repairing oxidative damages of dna [ 57 ] .
oxidative damage to nucleic acid has been associated with a number of pathologies including cancer and neurodegenerative and cardiovascular diseases [ 810 ] . to date , more than 20 different types of oxidative modifications of bases have been identified .
of all the modified bases , the processes which repair 8-oxogua are perhaps best understood , and may be regarded as a template for the processes which repair other lesions . to combat the deleterious biological effect of the presence of 8-oxogua , cells have developed specific mechanisms to remove this lesion from cellular dna . in mammalian cells ,
the first level of this protection is human mut t homologue ( hmth1 ) which hydrolyses 8-oxodgtp ( a potential substrate for dna polymerase ) , thereby eliminating it from the nucleotide pool .
the second level of defence is specific glycosylases that initiate base excision repair ( ber ) .
finally , human mut y homologue ( hmyh ) removes adenine that is mis - paired with 8-oxogua .
most recently , we proposed that nucleotide excision repair ( ner ) , which involves the removal of a lesion - containing oligonucleotide , may compliment the go system , based upon evidence that oxidative dna damage may be repaired by this route .
however , there is very little evidence that 8-oxodg is a direct product of dna repair itself ( ie , released as the deoxynucleoside , rather than the base , from dna ) .
it is generally accepted that products of cellular repair of oxidatively damaged dna , such as modified bases and nucleosides : 8-oxo-7,8-dihydroguanine ( 8-oxogua ) and 8-oxo-7,8-dihydro-2-deoxyguanosine ( 8-oxodg ) are excreted in urine .
an assay was performed to analyze levels of 8-oxogua and 8-xoxdg in urine samples with regard to diet . in case of the examined group
, a conclusion was drawn that diet does not determine excretion of these biomolecules in urine . in another study , different amounts ( up to 25 mg ) of 15n labeled oxidatively modified dna were absorbed orally by volunteers . throughout 2 weeks , blood and urine samples were collected .
no 15n 8-oxogua or 8-oxodg were detected either in urine and or in dna of monoclonal cells in venous blood obtained from the same subjects taking part in the study , demonstrating that diet has no influence on the level of these damages .
, no rise of levels in 8-oxodg excretion in urine was observed despite unequivocal evidence for mass reduction of treated tumors . in certain reports , a clear rise of 8-oxodg in urine excretion was observed after radio - chemotherapy or radiotherapy itself [ 2123 ] .
however , measuring excretion of repair product in urine exclusively may be misleading since it provides no information on the oxidative state of the organism ( damage rate vs. repair rate ) in cellular dna and reports only the mean value of damage repair in the past .
the aim of the work was to investigate whether the levels of markers of oxidative damages of dna : excreted in urine 8-oxo-7,8-dyhydro-2-deoxyguanosine ( 8-oxodg ) and 8-oxo-7,8-dyhydroguanine ( 8-oxogua ) as well as the level of 8-oxodg in dna of venous blood leukocytes differ in a population of healthy subjects when compared with cancer patients .
analysis of daily excretion of 8-oxogua and 8-oxodg with urine was done in a study group consisting of 222 patients with malignant cancer ( iii and iv degree of clinical stage ) .
leukocytes from peripheral blood samples for analysis of 8-oxodg level were obtained from 179 patients from among the study group .
control peripheral blood samples for analysis of 8-oxodg in leukocytes were obtained from 134 healthy volunteers . from this group ,
85 urine samples were taken for the measurement of daily excretion of 8-oxogua and 8-oxodg in urine .
the patients had various malignant tumors : , head and neck cancer ( n=45 ) , breast cancer ( n=32 ) , colon cancer ( n=25 ) , lung cancer ( n=37 ) , uterine cancer ( n=15 ) , ovarian cancer ( n=39 ) , testicular cancer ( n=7 ) , prostate cancer ( n=11 ) , gastrointestinal cancer ( n=11 ) .
the patients were asked to abstain from vitamin supplementation for at least 1 month before the chemotherapy started and during the course of the treatment , and only these patients qualified .
the control group was chosen in such a way that the following criteria matched the patient group : eating habits , age , body weight , sex , and smoking status .
the study was approved by the medical ethics committee of the collegium medicum nicolaus copernicus university bydgoszcz , poland , ( in accordance with good clinical practice , warsaw 1998 ) , and all the patients gave informed consent .
venous blood samples ( 18 ml ) from the patient and volunteer groups were collected .
the blood was carefully applied on top of histopaque 1119 solution ( sigma - aldrich inc . ; st .
louis , mo ) and leukocytes were isolated by centrifugation according to the procedure specified by the manufacturer .
determination of 8-oxodg by the mean of hplc / ec technique was as described previously .
urine sample preparation , hplc purification and gc / ms analysis were conducted as described earlier .
mann - whitney testing for independent groups with abnormal distribution was performed . for normal distribution ,
analysis of daily excretion of 8-oxogua and 8-oxodg with urine was done in a study group consisting of 222 patients with malignant cancer ( iii and iv degree of clinical stage ) .
leukocytes from peripheral blood samples for analysis of 8-oxodg level were obtained from 179 patients from among the study group .
control peripheral blood samples for analysis of 8-oxodg in leukocytes were obtained from 134 healthy volunteers . from this group ,
85 urine samples were taken for the measurement of daily excretion of 8-oxogua and 8-oxodg in urine .
the patients had various malignant tumors : , head and neck cancer ( n=45 ) , breast cancer ( n=32 ) , colon cancer ( n=25 ) , lung cancer ( n=37 ) , uterine cancer ( n=15 ) , ovarian cancer ( n=39 ) , testicular cancer ( n=7 ) , prostate cancer ( n=11 ) , gastrointestinal cancer ( n=11 ) .
the patients were asked to abstain from vitamin supplementation for at least 1 month before the chemotherapy started and during the course of the treatment , and only these patients qualified .
the control group was chosen in such a way that the following criteria matched the patient group : eating habits , age , body weight , sex , and smoking status .
the study was approved by the medical ethics committee of the collegium medicum nicolaus copernicus university bydgoszcz , poland , ( in accordance with good clinical practice , warsaw 1998 ) , and all the patients gave informed consent .
venous blood samples ( 18 ml ) from the patient and volunteer groups were collected .
the blood was carefully applied on top of histopaque 1119 solution ( sigma - aldrich inc . ; st .
louis , mo ) and leukocytes were isolated by centrifugation according to the procedure specified by the manufacturer .
determination of 8-oxodg by the mean of hplc / ec technique was as described previously .
urine sample preparation , hplc purification and gc / ms analysis were conducted as described earlier .
mann - whitney testing for independent groups with abnormal distribution was performed . for normal distribution ,
significantly elevated levels of 8-oxogua and 8-oxodg excreted in urine daily as well as 8-oxodg in dna of leucocytes in venous blood were observed in cancer patients as compared with healthy subjects , with considerable statistic significance ( table 1 ) . for the whole patient population ( n=222 ) , the median values of 8-oxogua and 8-oxodg in urine samples were 12.44 ( interquartile range : 8.1420.33 ) [ nmol/24 hr ] and 6.05 ( 3.1215.38 ) [ nmol/24 hr ] , respectively .
the median values of 8-oxogua and 8-oxodg in urine samples of the control group ( n=85 ) were 7.7 ( 4.6510.15 ) [ nmol/24 hr ] and 2.2 ( 1.72.8 ) [ nmol/24 hr ] , respectively .
the level of 8-oxodg in dna isolated from leukocytes in the patient group ( n=179 ) and of the control group ( n=134 ) was 4.93 ( 3.469.27 ) per 10 dg and 4.46 ( 3.825.31 ) per 10 dg , respectively .
certain amounts of oxidatively modified bases are present in every cell , reflecting the balance between ros attacking dna in the course of many metabolic processes and damage repair of these molecules by specific enzymes repairing dna .
it is not known at present what is the endogenous level of these potentially mutagenic damages . according to the reports of authors applying different analytical techniques , the values range from 0.2 to several modifications/10 pairs of bases for healthy cells .
they are removed in the process of repair and excreted in urine in unchanged state .
a balance between producing ros which induce oxidative dna damages and removing these damages was observed in a cell ( background level ) . according to some authors , the levels of these modifications
it is possible to estimate the extent of repair on the level of the whole organism while analyzing the amount of oxidative dna damages in urine .
high values of oxidative damages of dna excreted in urine indicate an intensified level of oxidative stress , but they may also reflect high efficiency of repair systems of these damages ( oxidative stress may be high , yet repair mechanisms remove its effects ) . however , combining the background level specific for every patient and analyzing 8-oxogua and 8-oxodg in excreted urine may clearly reflect information about dna repair mechanisms .
the results presented in the present study comparing amounts of 8-oxogua and 8-oxodg excreted in urine daily as well as amounts of 8-oxodg in venous blood dna leucocytes in patients with diagnosed cancer when compared with healthy subjects demonstrate higher levels of these modifications in cancer patients ( figures 13 ) .
significantly elevated levels of analyzed markers of oxidative damage dna may reflect the oxidative stress situation which accompanies cancer .
several studies have analyzed 8-oxogua and 8-oxodg in urine in different types of cancer patients and control groups , reporting elevated level of 8-oxogua in urine of cancer patients and in a control group of smoking subjects , and the level of 8-oxodg in dna isolated from venous blood leucocytes was higher in the patient group .
these findings suggest that in cancer patients repair mechanisms of oxidative damage to dna are less efficient ( the concentration of modified nucleoside / base in urine reflects dna damages of the whole organism , and the level of 8-oxodg in cellular dna reflects balance between processes generating damages and the ability of their repair ) .
compared a group of cancer patients and healthy volunteers with regard to the amount of 8-oxogua and 8-oxodg excreted in urine , demonstrating elevated levels of oxidatively modified base in patients when compared with healthy subjects by 50% .
our work indicates the levels of these derivatives elevated to such an extent can not be related to its increase in cancer cells exclusively .
the results suggest that oxidative stress in patients with cancer , demonstrated by elevated levels of these modifications in urine , may be typical not only of affected tissue but also of other tissues and even the whole organism . from practical point of view , a test that would enable determination of background levels of basic markers of oxidative stress ( 8-oxogua and 8-oxodg in urine and 8-oxodg in dna in leukocytes ) , might be applied as an additional and helpful marker for early detection of the development of cancer .
|
summarybackgroundthe broad spectrum of oxidative damage dna biomarkers : urinary excretion of 8-oxodg ( 8-oxo-7,8-dihydro-2-deoxyguanosine ) , 8-oxogua ( 8-oxo-7,8-dihydroguanine ) as well as the level of oxidative damage dna in leukocytes , was analyzed in cancer patients and healthy subjects.material/methods222 cancer patients and 134 healthy volunteers were included in the analysis , using methodologies which involve hplc ( high - performance liquid chromatography ) prepurification followed by gas chromatography with isotope dilution mass spectrometry detection and hplc / ec.resultsfor the whole patient population ( n=222 ) the median values of 8-oxogua and 8-oxodg in urine samples were 12.44 ( interquartile range : 8.1420.33 ) [ nmol/24 hr ] and 6.05 ( 3.1215.38 ) [ nmol/24 hr ] , respectively .
the median values of 8-oxogua and 8-oxodg in urine samples of the control group ( n=85 ) were 7.7 ( 4.6510.15 ) [ nmol/24 hr ] and 2.2 ( 1.72.8 ) [ nmol/24 hr ] , respectively .
the level of 8-oxodg in dna isolated from leukocytes of the patient population ( n=179 ) and of the control group ( n=134 ) was 4.93 ( 3.469.27 ) per 106 dg and 4.46 ( 3.825.31 ) per 106 dg , respectively.conclusionsthe results suggest that oxidative stress in cancer patients , demonstrated by augmented amounts of these modifications in urine , could be typical not only for affected tissue but also for other tissues and even the whole organism .
an assay that enables the determination of levels of basic markers of oxidative stress might be applied in clinical practice as an additional , helpful marker to diagnose cancer .
|
Background
Oxidatively modified bases
Diet as a potential source of 8-oxoGua and 8-oxodG in urine
Cell death as a potential source of 8-oxoGua and 8-oxodG in urine
Purpose
Material and Methods
Patients
Isolation of leukocytes from venous blood
DNA isolation and 8-oxodG determination in DNA isolates
Urine analysis
Statistical analysis
Results
Discussion
Conclusions
|
it is predicted that by 2025 , the prevalence of hypertension ( htn ) will increase by 60% to a total of 1.56 billion worldwide suggesting that htn remains a major public health problem .
htn in sub - saharan africa ( ssa ) has also been on the rise with reports indicating higher values in urban settings compared to rural settings [ 24 ] .
the prevalence of htn in ssa ranges between 14.5% in rural eritrea , 32.9% in semiurban ghana , and 40.1% in urban south africa .
likewise , adequate blood pressure ( bp ) control has been on the decline , ranging between 1.7% in rural ghana , 4% in urban slum dwellers in nigeria , and 21.5% in urban kenya . in cameroon
, the prevalence of htn spans from 5.7% in rural settings through 21.9% in semiurban to 47.5% in urban milieu , with a national average survey of 31.0% . despite the relatively better bp control in urban over rural settings , levels of adequate bp control as low as 2% to 27.5% ( in men ) and 38.7% ( in women )
even though much has been done so far to reduce the incidence of hypertension in urban areas , this has not been the same in rural areas , with public health policies aimed at controlling hypertension mainly directed towards the large cities .
most of the studies done so far in these urban areas can not therefore give a true estimate of the extent to which the rural population is affected by this major cardiovascular disease risk factor .
an analysis of hypertension and its risk factors in rural cameroon will therefore help guide health policy decisions and provide baseline data for future studies aimed at addressing this problem .
we therefore had the following as objectives : determining the prevalence of hypertension in the adult population in the moliwe health area in rural cameroon ; identifying the risk factors associated with htn in this rural setting ; determining the extent of bp control in known hypertensive patients in this locality .
a community - based cross - sectional descriptive and analytical study was conducted over a 6-month period ( june 20 to december 28 , 2013 ) in the moliwe health area ( mha ) .
this principally rural health area is found in the limbe health district of the south west region of cameroon .
a multistage sampling method was used to select participants from the 5 villages / settlements of the health area : bonadikombo , wotutu , ewongo , moliwe , and tomatal .
the sample selected was self - weighted through the probability proportional to size method used . the data of 2005 cameroon census was used as a guide to the sampling frame with households considered as the primary sampling units . using a random start
, a systematic sample of households was then selected from a list of the approximate cumulative number of households of all the villages .
this gave an estimate of the number of households to be selected from each village .
as the secondary sampling units , two participants were then recruited from each selected household by simple random sampling from a list of eligible households occupants produced by the respective household heads .
three hundred and twenty - nine participants were thus recruited from bonadikombo , 113 from wotutu , 112 from moliwe , 96 from ewongo , and 83 from tomatal .
a combined estimate of the prevalence of htn in rural rwanda , tanzania , and malawi of 22% was used .
we recruited 733 participants by systematic and simple random sampling methods . at each selected household ,
a questionnaire was administered to each recruited participant to collect information on age , sex , level of education , marital status , status of htn , treatment of htn , smoking , quantity of alcohol intake , physical activity , diabetes status , and family history of htn .
blood pressure measurements were also done at the home of the participants by a casually dressed clinician not in white coat .
two measurements ( in mmhg ) were taken on the right arm using an automated electronic bp machine ( omron m3 hem-7200-e omron matsusaka co. ltd . , kyoto , japan ) through the standard procedure ( jnc 7 recommendations ) . heights in meters ( m ) and weights in kilograms ( kg )
were measured using standard procedures and were used to determine the body mass index ( bmi ) as follows : bmi = weight ( kg)/[height ( m ) height ( m ) ] . who steps surveillance manual
the data was entered into epi info statistical software , version 7.0 ( cdc / who , atlanta , usa ) .
frequencies and means were obtained for appropriate variables ; chi - square and fischer 's exact tests were used as appropriate to test for associations between binary and categorical variables .
a logistic regression model was built to assess the effect of factors found to be significantly associated with hypertension while controlling for potential confounders .
an ethical clearance was obtained from the institutional review board of the faculty of health sciences , university of buea .
administrative approvals were obtained from the regional delegate of public health as well as from the local chiefs , quarter heads , and camp presidents concerned .
all patients found to have elevated blood pressure values were advised and referred to the nearest health facility for proper management and follow - up .
of the 733 participants , 479 ( 39.5% ) were between 20 and 29 years .
the male to female ratio was 1 : 1.2 , 35.9% had primary education , and 50.5% were married .
less than half of the participants ( 47.3% ) had normal bmi and 15% had sedentary life style . a small proportion , ( 2.9% )
were known diabetics and 32.7% reported a family history of hypertension ( table 1 ) .
of the 733 participants recruited , 228 were hypertensive , giving a prevalence of 31.1% ( 95% ci : 27.834.6 ) .
sixty - six of the 228 hypertensive participants ( 28.9% , 95% ci : 23.235.3 ) knew their status prior to our study .
of these 66 known hypertensive participants already aware of their status , 14 of them ( 21.2% , 95% ci : 12.133.3 ) had their blood pressures well controlled even though as high as 47 of them ( 71% , 95% ci : 58.781.7 ) were on antihypertensive treatment at that moment ( figure 1 ) .
age 40 and above , obesity , smoking , alcohol consumption , diabetes , low educational level , and marriage were found to be associated with hypertension ( table 2 ) . after adjusting for all significant factors using logistic regression , only age 40 and above , obesity , low educational level , and marriage
there was a progressive increase in the prevalence of htn with age when age below 40 was used as the reference : from 3 folds in the 4059 years , through 8.3 folds in the 6079 years , to 11.6 folds in 80 years age groups .
people who were obese had 2.8 times the odds of being hypertensive compared to the nonobese .
those who never went to school had 6.7 times the odds of having htn compared to those who had at least high school education ( table 3 ) . since the new recommendations for htn diagnosis and management by jnc 7 in 2003 , no study had been done in rural cameroon . the last published rural study on htn was as far back as 1998 .
the 31.1% prevalence of htn observed in the mha is similar to the 31.0% reported in cameroon by kingue et al . and the 32.9% reported by amoah in semiurban ghana but higher than that reported elsewhere [ 3 , 7 , 8 , 12 , 16 , 17 ] .
however , our value was lower than 44.7% reported by williams et al . in rural ghana and 40% reported by chow et al . in rural and urban high , middle , and low income countries .
our study revealed low hypertension awareness similar to the 32.3% observed in rural ghana by addo et al . and the 32.6% observed by dzudie et al . in urban cameroon .
however , this value is higher than the 11% reported in rural cameroon by mbanya et al .
this variation between rural and urban settings could be explained by poor knowledge of the disease , lesser access to health facilities , and poverty , in rural compared to urban communities .
age was identified to be strongly associated with htn , supporting the findings of other studies [ 9 , 12 , 2123 ] .
this is because as people get older , their blood vessels become harder and they are likely to have decreased baroreceptor sensitivity , increased responsiveness to sympathetic nervous system stimuli , altered renal and sodium metabolism , and an altered renin - aldosterone relationship thereby predisposing them to high blood pressure .
obesity , which is one of the common pathways between diabetes and htn , was also found to be strongly associated with htn in our study , as earlier documented [ 8 , 26 ] .
low educational status and being married were strongly linked with htn in our study as reported in previous studies [ 21 , 27 , 28 ] . a poor understanding of the disease and its risk factors and the stress experienced my married couples , respectively ,
could put these groups at a higher risk . on the other hand , gender , family history of htn , overweight , and physical inactivity
smoking , alcohol consumption , and diabetes mellitus which were associated with htn on bivariate analysis were not significantly associated with hypertension on multivariate analysis by logistic regression .
the proportion of known hypertensives who were currently on antihypertensive treatment in our study was similar to the 64.9% reported in ghana but much higher than the 12.5% reported in cameroon
. the high proportion on antihypertensives may be due to the increase in awareness of htn and the availability and access to antihypertensive medications in cameroon lately compared to 16 years ago . despite this considerable proportion of hypertensive patients being on treatment ,
reported as low as 3% in rural nigeria and yuvaraj et al . reported 12.5% in rural india
however , higher proportions have been reported ( 33.1% and 63.2% ) among hypertensives in urban and semiurban settings in cameroon , respectively , 56.8% in mexico , and 75.9% in urban india .
in this context noncompliance to treatment is multifactorial in aetiology with reasons such as inadequate knowledge of htn , intolerance of side effects of medications , and low purchasing power .
this study was cross - sectional which means participants declared hypertensive may not necessarily be hypertensive clinically .
also , our study was done in a small rural area whose findings may not necessarily apply to all rural cameroonian or sub - saharan african communities . moreover , some known risk factors such as dyslipidaemia , salt intake , and psychosocial and socioeconomic status were not assessed in our study .
nevertheless , the large sample size , varied sampling method , and execution of standard measurement procedures make the findings of this study robust and accurate .
also , the white coat effect which could potentially produce an overestimation of the actual prevalence of hypertension in this setting was minimized since the casually dressed clinician without a white coat did the measurements from the participants ' homes .
our study revealed that about one out of three adults in the moliwe health area could be hypertensive with only a quarter of them being aware of their status and a fifth of those on treatment having their blood pressure well controlled .
this reveals how very much a cardiovascular risk the rural population may be exposed to .
there is , therefore , need for massive improvement in awareness through education and repeated patient follow - up in these rural settings alongside the urban ones .
also , the importance of further research in other rural communities to assess trends and risk factors of hypertension and the extent of blood pressure control can not be overemphasized .
|
background . despite the increasing trends suggesting that hypertension is a growing public health problem in developing countries , studies on its prevalence , associated risk factors , and extent of blood pressure control have been inequitably done in urban and rural communities in these countries .
we therefore aimed to determine the prevalence of hypertension and extent of blood pressure control in rural cameroon .
methods .
this was a community - based cross - sectional study conducted in rural cameroon ( the moliwe health area ) .
participants aged 21 years and above were recruited by a probability proportional to size multistage sampling method , using systematic sampling for household selection and random sampling for participant selection .
blood pressure , weight , and height were measured by standard methods .
hypertension was defined as bp 140/90 mmhg .
results .
the prevalence of hypertension among the 733 participants recruited was 31.1% ( 95% ci : 27.834.6 ) and 71% ( 95% ci : 58.781.7 ) of these hypertensive patients were newly diagnosed . only 21.2% ( 95% ci : 12.133.3 ) of known hypertensives had a well controlled bp .
age , obesity , low educational status , and being married were associated with htn after adjusting for confounders .
conclusions . the high prevalence of hypertension and inadequate bp control among known hypertensives in rural cameroon warrants greater sensitization and regular screening to reduce hypertension - related morbidity and mortality .
|
1. Background
2. Materials and Methods
3. Results and Discussion
4. Conclusions
|
granuloma annulare ( ga ) was first described in 1895 by colcott - fox who reported an 11-year - old girl with a ring eruption of the fingers .
the condition is a benign , usually self - limited granulomatous disease of the dermis and subcutaneous tissue .
clinically , the condition is characterized by asymptomatic , flesh - colored or erythematous - brown papules , frequently arranged in a ring or annular pattern on the distal extremities
. it may be skin - colored erythematous or violaceous measuring 1 - 5 cm in diameter .
several clinical variants of ga have been reported , namely localized , generalized or disseminated , targetoid , giant , subcutaneous and perforating ga .
we report an 18-month - old male child with multiple , progressive ga over the lower extremities .
an 18-month - old male child presented with multiple annular lesions over lower extremities and buttocks .
initially it started as a small papule over the left thigh , substantially attaining the present size .
there was no history of fever , pain , drug intake or recent vaccination . on examination ,
on cutaneous examination , closely set , skin - colored , firm , smooth papules 2 - 3 mm in size arranged in a ring - like fashion to form annular plaques measuring approximately 3 3 cm ( eight lesions ) were present over dorsal aspect of foot [ figures 1 and 2 ] , both legs and buttocks .
granuloma annulare over the left foot granuloma annulare over the right ankle routine hematological , biochemical and urine examinations were normal .
venereal disease research laboratory test , human immunodeficiency virus 1 and 2 , hepatitis surface antigen test were negative and a biopsy showed features consistent with ga .
ga is a benign , relatively common cutaneous disease that classically presents as closely set , skin - colored , firm , smooth papules 2 - 3 mm in size arranged in a ring - like fashion to form annular lesions .
most cases occur before the age of 30 years and it is approximately twice more common in females .
it often favors sacral sites such as the dorsa of the hands , elbows and feet but involvement of the palms appears to be rare .
occasional familial cases are described with the occurrence in twins , siblings , and members of successive generations .
attempts to identify an associated hla sub - type have yielded disparate results in different population groups .
linear ga , a follicular pustular form and popular umbilicated lesions in children have also been described .
the condition manifests as numerous ( at least 10 , often hundreds to thousands ) small , asymptomatic , erythematous , violaceous , brown- or skin - colored papules .
rarely , ga may be complicated by nerve involvement as a result of granulomatous inflammation surrounding cutaneous nerves and perineural infiltrates of histiocytes in the dermis .
the differential diagnoses of localized ga include tineacorporis , pityriasis rosea , pityriasis rotunda , erythema annulare centrifugum , nummular eczema , discoid lupus erythematosus , psoriasis , necrobiosis lipoidica , morphea , hypertrophic lichen planus and erythema chronicum migrans .
treatment options include topical or intralesional corticosteroids , imiquimod cream , topical calcineurin inhibitors ( tacrolimus , pimecrolimus ) , cryotherapy , and pulsed dye laser .
|
granuloma annulare is a benign , self - limiting , inflammatory and granulomatous disease of unknown etiology occurring in both adults and children .
an 18-month - old male child had multiple progressive annular plaques over the lower extremities .
clinical and histopathological features were consistent with granuloma annulare .
localized granuloma annulare is the most common form in children .
we report a young child with multiple , progressive granuloma annulare over the lower extremities .
|
Introduction
Case Report
Discussion
|
we analyzed all 140 mrsa isolates we obtained during a previous study ( 2 ) and 50 other isolates selected from our collection of nosocomial mrsa isolates obtained in 2003 .
characteristics of these 190 isolates are shown in table 1 . * ni , nosocomial infection acquired at national taiwan university hospital ( ntuh ) ; nc , nosocomial colonization at ntuh ; ieoh , mrsa infection detected at ntuh within 48 h after transfer from another hospital .
bl , blood ; ur , urine ; sp , sputum ; pu , pus ; wo , wound ; ns , nostril ; ct , catheter tip ; st , stool .
pulsed - field gel electrophoresis patterns were interpreted according to procedures previously reported ( 7,8 ) .
thirty - four isolates belonged to pulsotype a , 49 to pulsotype b , 69 to pulsotype c , 6 to pulsotype d , 2 to pulsotype e , 3 to pulsotype f , 11 to pulsotype k , 5 to pulsotype l , 8 to pulsotype m , and 3 ( all isolated in 2003 ) to 3 minor pulsotypes .
all isolates were tested by sccmec element typing and multilocus sequence typing ( mlst ) ( 9 ) and were analyzed for the panton - valentine leukocidin ( pvl ) gene ( 10 ) and drug susceptibility to erythromycin , clindamycin , gentamicin , amikacin , ciprofloxacin , levofloxacin , tetracycline , trimethoprim - sulfamethoxazole , rifampin , and vancomycin by using the disk diffusion method ( 11 ) .
isolates with pulsotype a are sequence type 254 ( st254 ) ; those with pulsotype b are st241 ; those with pulsotypes c , k , and l are st239 ; and those with pulsotypes d , e , f , and m are st59 , st 254 , st30 , and st5 , respectively .
all mrsa isolates with pulsotypes a , d , e , and f have the type iv sccmec element .
however , only isolates with pulsotypes d and f , as well as 2 isolates from 2003 with 2 minor pulsotypes , have the pvl gene .
isolates with pulsotypes b and c have the type iii sccmec element , and isolates with pulsotype m have the type ii sccmec element . *
p , pulsotype ; scc , staphylococcal cassette chromosome ; mlst , multilocus sequence typing ; pvl , panton - valentine leukocidin ; n , no ; y , yes . ox , oxacillin ; em , erythromycin ; cl , clinidamycin ; gm , gentamicin ; am , amikacin ; cp , ciprofloxacin ; lv , levofloxacin ; tc , tetracycline ; ts , trimethoprim - sulfamethoxazole ; rf , rifampin ; va , vancomycin .
results of mlst and typing of sccmec elements of the 3 isolates with 3 minor pulsotypes obtained in 2003 are shown in table 2 .
the correlation between sccmec element typing and mlst in this study corresponds to findings of previous reports ( 6,1214 ) .
enright et al . identified st254-iv mrsa isolates in germany and the united kingdom ( 12 ) , and chongtrakool et al . identified st239-iii and st241-iii mrsa isolates in several asian countries ( 14 ) .
we demonstrate that the predominant mrsa clone at ntuh in early 1990s had the type iv sccmec element . however , the predominant mrsa clones at ntuh from 1994 to 2003 had the type iii sccmec element .
these findings differ from those of wisplinghoff et al . , who reported that that the sccmec element in predominant mrsa clones at their institute changed from type iii in 1984 to 1988 to type i in 1989 to 1998 ( 6 ) .
mrsa isolates of pulsotypes b and c are more resistant than isolates of pulsotype a to certain antimicrobial drugs , especially fluoroquinolones and trimethoprim - sulfamethoxazole ; mrsa isolates with pulsotype c are more resistant to clindamycin but less resistant to rifampin than those with pulsotype b ( table 2 ) . from 1993 through 2000 , annual use of fluoroquinolones increased 3 at ntuh ; however , use of trimethoprim - sulfamethoxazole , clindamycin , and rifampin did not change ( 15 ) .
therefore , the shift of predominant mrsa clones , which also led to the shift in types of sccmec elements at ntuh , might be caused by selective pressure from antimicrobial drugs , especially fluoroquinolones .
the mrsa clone ( pulsotype a ) that predominated in 1992 and 1993 at ntuh has the type iv sccmec element .
although the first study of mrsa with the type iv sccmec element reported that this element was found in community - acquired mrsa ( ca - mrsa ) ( 5 ) , some studies have reported mrsa isolates with this element in a hospital environment ( 12 ) . however , to our knowledge , these reports did not demonstrate that mrsa isolates with the type iv sccmec element became predominant among all mrsa isolates in an institution , especially before the mid-1990s .
furthermore , 4 st59 mrsa isolates obtained in 1994 and 1996 and 3 st30 mrsa isolates obtained in 1992 and 1993 have the type iv sccmec element and pvl gene .
recently , st59 mrsa isolates were found to cause ca - mrsa infection in taiwan ( 13 ) . among these st59
ca - mrsa isolates , some had the type iv sccmec element , and others had the type v sccmec element ( 13 ) . although the type iv sccmec element could be transferred to ca - mrsa clones with other genetic backgrounds , our finding supports the possibility that st59 mrsa isolates with the sccmec element type iv in taiwan may originate from hospital strains but transfer into ca - mrsa strains .
recently reported the results of sccmec typing of 615 mrsa isolates obtained in 1998 and 1999 from 11 asian countries ( 14 ) .
the st239-iii , st241-iii , st254-ii , and st5-ii mrsa isolates were prevalent in many asian countries .
however , the st254-iv , st30-iv , and st59-iv mrsa isolates from our study were not found in other asian countries .
in addition , st254-iv mrsa isolates have been found in germany and the united kingdom ( 12 ) . whether st254-iv mrsa isolates in our study
were transmitted from those in germany or the united kingdom by international travel requires further study .
the first mrsa isolate with the type iv sccmec element in our hospital appeared as early as 1992 . the sccmec element carried by predominant mrsa clones changed from type iv to type iii sccmec element during the period 19922003 at ntuh .
because the major mrsa clones isolated in 19942003 are more resistant to antimicrobial drugs , especially fluoroquinolones and trimethoprim - sulfamethoxazole , than those obtained in 1992 and 1993 , this shift may be caused by selective pressure from indiscriminate use of antimicrobial drugs .
|
to determine the predominant staphylococcal cassette chromosome ( scc ) mec element in methicillin - resistant staphylococcus aureus , we typed 190 isolates from a hospital in taiwan .
we found a shift from type iv to type iii sccmec element during 19922003 , perhaps caused by selective pressure from indiscriminate use of antimicrobial drugs .
|
The Study
Conclusions
|
fine - needle aspiration ( fna ) is the standard method used to determine treatment plans for thyroid nodules .
based on the bethesda system , the most generally accepted system for reporting thyroid cytology , the benign category implies a less than 3% risk of malignancy .
follow - up ultrasound ( us ) is recommended when the nodule has benign cytologic result .
repeat fna is recommended when a nodule shows significant growth or morphologic transformation with suspicious
however , the practical risk of malignancy in nodules with benign cytology varies in each institute , ranging from 2% to 18% , and it has even been reported to have gone up to 62% . therefore , some investigators recommend routine repeat fna for thyroid nodules with benign cytology [ 7 , 8 ] . considering cost - effectiveness and diagnostic value , repeat fna has been considered when the nodule shows any suspicious feature on the initial us [ 9 , 10 ] . however , known us features associated with malignancy show an extremely variable probability of malignancy .
microcalcifications , marked hypoechogenicity , and irregular or spiculated margin show a high risk of malignancy , while solid composition and hypoechogenicity show a relatively low positive predictive value ( ppv ) [ 1113 ] .
based on these results , each suspicious us feature may not be considered as an equal risk factor for malignancy .
radiologists specialized in breast imaging have been confronted with the same problem in the core needle biopsy of a breast lesion .
correlation of pathologic results with sonographic findings has been used in some institutions to verify that the lesion was adequately sampled .
this approach was suggested owing to the wide range of false negative rates of this category .
based on different malignancy rates of suspicious us features in thyroid nodules and considering approaching steps in management of breast lesions , we conjecture that an imaging - cytology correlation can be a better diagnostic approach for patient management than initial us features in a thyroid nodule with benign cytology .
therefore , we investigated the role of imaging - cytology correlation to reduce the false negative rates of cytology at thyroid nodules as compared with the use of initial us features .
the institutional review board of the severance hospital approved of this retrospective study and required neither patient approval nor informed consent for our review of patients ' images and records .
however , written informed consents were obtained from all patients for us - guided fnas ( us - fnas ) prior to each procedure as a daily practice . our institutional registry for the thyroid nodule was settled since 2006 including all patients with thyroid nodules who underwent us examinations and us - fnas at our institution . from march 2006 to december 2006 , 3119 consecutive thyroid nodules in 2866 patients underwent us - fnas . among them , we included 667 nodules in 649 patients ( men , 83 ; women , 566 ) , which fulfilled the following criteria : ( a ) they had no history of prior fna on the same nodule ; ( b ) they were reported as benign ( category ii ) in the initial fna .
nondiagnostic , atypia or follicular lesions of undetermined significance , follicular neoplasm or suspicious for a follicular neoplasm ,
suspicious for malignancy , and malignant were excluded ; ( c ) they were equal to or larger than 1 cm ; ( d ) they underwent further evaluation such as follow - up us , follow - up fna , or thyroid surgery .
in nodules which had not underwent operation , determinative cytologic reports ( category ii or category vi ) on follow - up us were used as standard reference . if a nodule decreased in size on follow - up us , the nodule was also included as a benign nodule ; ( e ) there were available radiologic reports that included an additional radiologist 's opinion about the concordance or discordance between imaging and cytologic results in postbiopsy correlation ( figure 1 ) .
mean lesion size of the thyroid nodules was 20.7 mm ( range , 1070 mm ) .
median follow - up of 601 nodules in 584 patients which were included based on followup fna or us without surgical pathology was 1509 days from the date of initial fna to the last followup ( iqr , 1522 days ; range , 1722744 days ) .
all us examinations were performed using a 7 to 15 mhz linear array transducer ( hdi 5000 ; philips medical systems , bothell , wash ) or a 5 to 12 mhz linear probe ( iu22 , philips medical systems ) by 1 of 5 board - certified radiologists with 1 to 12 years of experience in thyroid imaging .
all us - fnas were performed by the same radiologist who performed the us examinations .
us features of all thyroid nodules that underwent us - fnas were prospectively recorded by the previously described methods .
us features suspicious for malignancy were determined using previously published criteria from our institution : marked hypoechogenicity , microlobulated or irregular margin , microcalcifications , and taller than wider shape .
when overall echogenicity of a nodule was darker than that of the surrounding strap muscle , it was defined as
marked hypoechogenicity to differentiate it from hypoechogenicity based on the parenchymal echogenicity of the thyroid gland .
only calcifications equal to or less than 1 mm in diameter were indicated . if microcalcifications were detected with macrocalcifications , the lesion was considered to have microcalcifications as a worrisome finding .
if hyperechoic foci accompanied comet - tail artifacts on conventional us , they were considered as colloids . an anteroposterior to transverse dimension ratio greater than 1 was defined as taller than wider shape .
us - fnas were performed on either thyroid nodules with suspicious assessment or the largest nodule among nodules with probably benign assessment on us .
if there were multiple nodules with suspicious us findings in one patient or if the patient or physician requested a biopsy of a benign - looking nodule coexisting with a nodule showing suspicious us features , fnas were performed on multiple nodules in one patient .
a free - hand biopsy technique was used with either a 23-gauge needle attached to a 20 ml disposable plastic syringe and an aspirator or a 23-gauge needle attached to a 2 ml disposable plastic syringe , depending on the performing radiologist 's preference .
each lesion was aspirated at least twice , and the aspirated materials were expelled onto a slide and immediately placed in 95% alcohol for papanicolaou staining .
an inadequate specimen was defined as less than 6 groups of cells containing more than 10 cells .
adequate specimens were categorized as benign , indeterminate , suspicious for malignancy , or malignant samples .
the radiologist who performed fna routinely reviewed the initial us images within a week of the fna after the cytologic results were reported .
for benign cytologic results , radiologists who performed the us - fnas decided and reported whether the cytology was concordant or discordant with the imaging findings .
as researchers at our institution always try to assess lesions based on their most worrisome finding , the saved images should represent these worrisome us features .
the final conclusion was not derived from the number of suspicious us features but from the subjective decision made by the radiologist who performed the us - fna . in our institution , concordant lesions included some nodules which had suspicious us features on the initial us but were acceptable for the benign cytology in postbiopsy image review as well as the nodules without features suspicious for malignancy on the initial us .
concordant benign thyroid nodules were recommended for follow - up by us after one year .
in contrast , discordant lesions included nodules which were initially suspected for malignancy on us and were still thought to be suspicious for cancer even after obtaining benign cytology .
repeat fnas were usually recommended for discordant benign thyroid nodules after 612 months . among the 667 nodules that met all the inclusion criteria , 586 nodules ( 87.9% , 586 of 667 )
were reviewed by radiologists who had more than three years of experience in thyroid imaging and fna whereas the remaining nodules were managed by less experienced radiologist .
we compared the clinical characteristics of patients between benign and malignant nodules by using the test for categorical variables and independent t - test for continuous variables .
we also compared the risk of malignancy as well as the clinical characteristics between concordant and discordant nodules by using or fisher 's exact test for categorical variables and independent t - test for continuous variables .
the baseline characteristics were also compared between patients with included and excluded nodules among thyroid nodules equal to or larger than 1 cm with the same methods .
the risk of malignancy was calculated for several subgroups classified according to initial us features and imaging - cytology concordance . using the generalized estimating equation , we compared the risk of malignancy in thyroid nodules with initially benign cytologic results with those of the remaining subgroups and also compared the risk of malignancy of thyroid nodules among subgroups .
logistic regression analysis was performed to assess the odds ratio for the risk of malignancy .
statistical analysis was performed using commercial statistical software ( sas version 9.1 , sas inc . , cary , nc , usa ) .
among 667 nodules with initially benign cytologic results , 656 nodules were benign ( 98.4% ) and 11 nodules were malignant ( 1.6% ) based on cytopathology ( table 1 ) .
the mean age of patients with malignant nodules was not significantly different from that of patients with benign nodules ( p = 0.277 ) .
the mean size of malignant nodules ( 17.6 12.5 mm ) was not significantly different from that of benign nodules ( 20.7 10.1 mm , p = 0.315 ) .
there were 70 nodules with initial suspicious us features and 597 nodules without initial suspicious us features .
the risk of malignancy was higher in nodules with initial suspicious us features ( 11.4% , 8 of 70 ) than in nodules without initial suspicious us features ( 0.5% , 3 of 597 ; p < 0.001 , table 1 )
. when reviewing us images after initial fna results were reported , 40 out of 70 nodules which had suspicious features on initial us evaluation were finally concluded as concordant with benign cytology ( figures 2 and 3 ) .
therefore , in 667 nodules with benign cytology , 637 nodules were concordant with cytology , whereas 30 nodules were discordant with benign cytology .
the reasons that 40 nodules with revised radiologic diagnosis after imaging - cytologic correlation were initially classified as suspicious nodules were microcalcifications ( n = 16 ) , microlobulated or irregular margin ( n = 9 ) , taller than wider shape ( n = 3 ) , or marked hypoechogenicity ( n = 1 ) in order of frequency , respectively , and more than one characteristic of the above features in 11 nodules . between the concordant and discordant group , gender of the patients was not significantly different ( p = 0.159 ) .
the patients with discordant nodules were significantly older than other patients with concordant nodules ( 53.5 10.5 years versus 48.9 12.0 years ; p = 0.038 ) .
the mean size of discordant nodules was significantly smaller than that of concordant nodules ( 16.0 6.6 mm versus 20.9 10.2 mm ; p < 0.001 ) .
3% ; 7 of 30 ) than in the concordant group ( 0.6% , 4 of 637 ; p
about 44.5% ( 534 of 1201 ) of 1 cm or larger nodules with benign cytology in initial fna were excluded because they had neither standard reference , such as follow - up us , follow - up fna , or thyroid surgery , nor available radiologist 's additional reports regarding imaging - cytologic correlation .
the mean age of patients with included nodules was statistically different from the other patients ( 49.1 12.0 versus 50.7 13.1 years ; p = 0.033 ) .
patient gender ( p = 0.392 ) and mean nodule size ( p = 0.601 ) were not significantly different between included and excluded nodules .
there were 60 nodules with suspicious findings in the initial us evaluation of excluded nodules ( 11.2% , 60 of 534 ) , and the proportion was not significantly different from that of included nodules ( 10.5% , 70 of 667 ; table 2 ) . when comparing the risk of malignancy between benign cytology alone and each subgroup by a combination of benign cytology with initial us findings or postbiopsy concordance , all combinations had significantly different risk values from cytology alone ( table 3 , figure 4 ) .
also , when comparing the risk of malignancy between discordant lesions and lesions with suspicious features on initial us , the former ( 23.3% , 7 of 30 ) was significantly higher than the latter ( 11.4% , 8 of 70 ) . however , there was no significant difference in the risk of malignancy between concordant lesions ( 0.6% , 4 of 637 ) and lesions without suspicious features on initial us ( 0.5% , 3 of 597 ; p = 0.438 ) ( figure 4 ) .
although fna is a widely used tool for the diagnosis of thyroid nodules , the most significant problem it has is false negative results which bring out misses and delays in treatment of the cancer .
errors in cytologic reports have arisen from the overinterpretation of nondiagnostic specimens as diagnostic ones [ 23 , 24 ] .
therefore , many reports discussed the differentiation of a nondiagnostic specimen from a diagnostic one in the cytologic interpretation of thyroid fna [ 1 , 3 ] .
diagnostic errors of thyroid fna can also be caused by the mistakes of cytopathologists and the inherent nature of thyroid nodules due to overlapping cytologic criteria among hyperplastic adenomatoid nodule in goiter , follicular adenoma , well - differentiated follicular carcinoma , and follicular variant of papillary carcinoma .
moreover , reported false negative rates are variable among institutions and operators due to variable sampling skills [ 5 , 6 ] .
several guidelines recommend follow - up us in thyroid nodules with benign cytology unless the nodule shows significant growth or morphologic change in follow - up us [ 1 , 3 , 4 ] .
however , it has been argued that follow - up might be not enough in some nodules because of the inevitable false negative diagnosis and the possible risk of delayed treatment [ 6 , 22 ] . to reduce false negative results of thyroid fnas ,
there have been two suggested approaches ; first , routine repeat fna in thyroid nodules with benign cytology [ 26 , 27 ] and , second , selective repeat fna [ 8 , 10 , 28 , 29 ] . in the aspect of cost - effectiveness , it is more rational to consider performing follow - up fna selectively for nodules with a high - risk of malignancy rather than performing a total inspection of cytologically benign nodules in initial fna . based on several reports , the rate of malignancy in benign thyroid nodules with suspicious us features was 3.747.1% which was significantly higher than that of benign thyroid nodules without suspicious us features ( table 4 ) [ 9 , 1518 ] .
although the us criteria applied to each study had subtle differences , initial us features may be reliable factors in determining whether to repeat fna or not .
going one step further from simply matching cytologic results against imaging findings evaluated before biopsy , the postbiopsy correlation of us features with cytologic results could be an alternative in determining whether the nodule should be reaspirated to confirm its cytology or not .
imaging - pathologic correlation after biopsy has been found to be useful in validating biopsy results of breast lesions , and discordance has been suggested as an indication for excision because of its higher upgrade rate than that of concordant lesions [ 3032 ] .
however , there has been no organized study that applies imaging - cytology correlation to patient management and considers how to accept results of postbiopsy correlation in regard to reducing false negative diagnosis in thyroid nodules . in this study , 1.6% of nodules with benign cytology in initial fna
were finally proven to be malignant . as expected , the malignancy rate of thyroid nodules ( 11.4% ) with suspicious features on initial us was significantly higher than that of nodules ( 0.5% ) without suspicious features on initial us , and the malignancy rate of nodules ( 23.3% ) with discordant imaging findings was also significantly higher compared to concordant nodules ( 0.6% ) in postbiopsy imaging - cytologic correlations .
furthermore , the rate of malignancy was higher in the nodules showing imaging - cytology discordance compared to nodules showing suspicious feature on the initial us .
however , there was no significant difference in the risk of malignancy between concordant nodules in postbiopsy correlation and nodules without suspicious features on initial us .
this result lets us conclude that imaging - cytology correlation is a more effective approach than using initial us features alone when deciding follow - up management in patients with cytologically benign thyroid nodules without a statistical increase in missing malignancy . in this study
, 40 of 70 nodules with suspicious features on initial us were determined as concordant with benign cytology after postbiopsy imaging - cytology correlation .
although many descriptions of each suspicious us feature are present , interobserver and intraobserver variability still exist for the us assessment of thyroid nodules . among us
characteristics , margin and calcification showed relatively less consistency between observers and nodules in most patients whose radiologic assessments were changed after obtaining benign cytology were initially assumed as suspicious nodule due to calcification ( 16 of 40 ) , margin ( 9 of 40 ) , or multiple features ( 11 of 40 ) including them in our study .
also , there have been difficulties in deciding whether a thyroid nodule shows echogenic spots on us .
echogenic spots can be due to microcalcifications related to cancer or crystals related to colloids .
therefore , postbiopsy imaging - cytology correlation can be a good diagnostic approach in deciding whether to repeat fna or not at a thyroid nodule with benign cytology .
first , some nodules were excluded in analysis despite having benign cytologic results due to loss of follow - up and absence of additional reports .
however , the initial us assessment was not significantly different between included nodules and excluded nodules which were 1 cm or larger with benign cytology in the initial fna .
second , interobserver and intraobserver variability among radiologists are possible in the interpretation of us images and among cytologists , especially when reviewing follicular lesions .
third , there might be a bias arising from the postbiopsy review process itself which was based on saved images instead of on a review in real - time us .
although we always tried to save any images showing worrisome us findings and the postbiopsy review was preferably done within a week of biopsy by the performer , an observer bias might not have been completely removed from the final results .
fourth , suspicious us features such as calcification , margin , vascularity , and echogenicity have been differently applied to thyroid nodules by various guidelines and different institutions .
fifth , most ( 87.9% ) of the nodules in this study were reviewed by highly experienced radiologists in thyroid imaging .
repeat fna can be effectively limited to patients with cytologically benign thyroid nodules showing discordance in imaging - cytology correlation , which reduces unnecessary repeat aspirations as well as decreasing false negative results .
|
objective . to determine the role of imaging - cytology correlation in reducing false negative results of fine - needle aspiration ( fna ) at thyroid nodules
.
methods . this retrospective study included 667 nodules 1 cm or larger in 649 patients diagnosed as benign at initial cytologic evaluation and that underwent
follow - up ultrasound ( us ) or fna following a radiologist 's opinion on concordance between imaging and cytologic results .
we compared the risk of malignancy of nodules classified into subgroups according to the initial us features and imaging - cytology correlation .
results . among included nodules ,
11 nodules were proven to be malignant ( 1.6% ) in follow - up fna or surgery .
the malignancy rate was higher in nodules with suspicious us features ( 11.4% ) than in nodules without suspicious us features ( 0.5% , p < 0.001 ) . when a thyroid nodule had discordant us findings on image review after having benign fna results , malignancy rate increased to 23.3% , significantly higher than that of nodules with suspicious us features ( p < 0.001 )
.
however , no significant difference was found in the risk of malignancy between the nodules without suspicious us features ( 0.5% ) and imaging - cytology concordant nodules ( 0.6% , p = 0.438 ) .
conclusions .
repeat fna can be effectively limited to patients with cytologically benign thyroid nodules showing discordance in imaging - cytology correlation after initial biopsy , which reduces unnecessary repeat aspirations .
|
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions
|
congenital heart defects / diseases contribute substantially to morbidity and mortality in childhood , especially in the third - world countries where facilities for management are often deficient.1 about 35,000 infants ( 1 out of every 125 ) are born with heart defects every year in the united states.2 the incidence in the tropics is not expected to differ from the 8 - 10 per 1000 live - births documented in the developed world.3 the defect may be so slight that the baby may be asymptomatic for many years after birth , or so severe that it is life threatening .
heart defects are the leading cause of birth defect - related deaths.4 however ; the use of echocardiography has improved description of congenital heart diseases and their early diagnosis.5 this , has led to dramatic increases in survival of children with serious heart defects .
cor triatriatum is a rare congenital cardiac anomaly , in which a fibromuscular membrane divides the atrium in two . it was first reported in 1868.6 cor triatriatum , a heart with 3 atria ( triatrial heart ) , is a congenital anomaly in which the left atrium ( cor triatriatum sinistrum ) or right atrium ( cor triatriatum dextrum ) is divided into 2 parts by a fold of tissue , a membrane , or a fibromuscular band.7 however , variable types of subtotal cor triatriatum are also noted , with only the right or left pulmonary veins draining into the upper chamber.8 cor triatriatum represents 0.1% of all congenital cardiac malformations and may be associated with other cardiac defects in as many as 50% of cases .
examples of associated cardiac defects include atrial septal defect , persistent left superior vena cava with an unroofed coronary sinus , partial anomalous pulmonary venous connection , ventricular septal defect and more complex cardiac lesions such as tetralogy of fallot , atrioventricular canal and double outlet right ventricle .
associated bicuspid pulmonary valve , aortic valve atresia and heterotaxy have also been described.9 congenital pulmonary vein stenosis is a very rare association with cor triatriatum.10 the morbidity and mortality of cor triatriatum sinistrum is high in those who are symptomatic in infancy .
this is due to the severely restrictive opening in the accessory membrane and the association with major cyanotic or acyanotic congenital heart lesions .
significant sequel is unusual with cor triatriatum dextrum as it is not commonly associated with life - threatening symptoms or major congenital cardiac defects .
a female baby seen on the second day of life was delivered by a 25-year - old primiparous youth corper .
the mother attended antenatal clinic in federal medical centre owerri from the 16 week of gestation .
however , during the 8 week of pregnancy , the mother had threatened abortion for which some drugs were given ( injections and tablets names unknown ) at a hospital in lagos
. she also had some liquid herbal preparation at 8 months of gestation ; other drugs she took were essentially haematinics .
she gave birth to a 3.6-kg baby girl who cried immediately after , and breastfeeding commenced .
the child was transferred to the special care baby unit from the postnatal ward on account of history of fever on day 2 of life .
examination revealed a full - term newborn , febrile ( 38.1 ) with a respiratory rate of 100 cycles per minute , acyanotic , heart rate of 110 beats / min heart
however , the child completed a course of antibiotics ( ampicillin sulbactam and gentacin ) and , with clinical improvement , was discharged home . at follow - up at the 2 week of life ,
the child weighed 3.5 kg , heart rate was 130 beat / min and a grade 3 pansystolic murmur was heard , following which a chest radiograph and electrocardiograph ( ecg ) was requested . at 1 month of life , a review of ecg showed : heart rate : 128 beats / min , t-18 , which suggested supraventricular arrhythmia , abnormal right superior axis deviation .
murmur was still grade 3/6 , pansystolic maximal at the left - lower sternal edge .
echocardiograph was subsequently requested and the result showed that a 2d echocardiogram was done [ figure 1 ] with the following results : echocardiogram showing the distal chamber ( x ) and the left atrium ; the right atrium ( ra ) , left ventricle ( lv ) , and right ventricle ( rv ) separate left atrium with dividing membranous running in the direction of long axis of left atrium close to the lateral wall .
mitral valve draining the larger , more medial chamber , normal atrio - ventriculo - arterial connection .
high velocity turbulent flow in the main artery noted ( pa vmax = 335 cm / s pg = 45 mm ) .
secundum atrial septal defect [ figure 1 ] the child is presently being followed - up regularly . during her last visit , at 7 months of age
, she weighed 9.6 kg , length was 73 cm , occipito - frontal circumference was 47 cm and mid - upper - arm circumference was 15 cm ( all within normal for age ) .
cardiovascular examination revealed a child with heart rate of 120 beats / min , apex beat located at the 4 left intercostals space , with a grade 3/6 systolic murmur heard loudest at the upper left sternal edge .
the respiratory rate was 42 cycles / min and no organs palpably enlarged on digestive system examination .
this was the first case of a five - chambered heart seen and managed in the federal medical centre owerri .
the case demonstrates the need for comprehensive evaluation of patients and provision of cardiac investigative facilities ( x - ray , ecg and echocardiograph machines ) in health facilities . in our patient , it was an incidental finding at a routine follow - up clinic .
subsequent investigative evaluations led to the confirmation of the diagnosis of a five - chambered heart in a child who has remained asymptomatic .
the dearth of facilities in our hospital is obvious here , where the patient had to be referred to the university of nigeria teaching hospital enugu for echocardiograph .
clinical manifestations are often delayed due to the presence of a large opening ; and include an asymptomatic murmur .
finding is mostly incidental on routine cardiac imaging ; it therefore highlights the need for exhaustive evaluation and management because such conditions , although rare , can still be found here . in conclusion
, we reported a case of an asymptomatic congenital - heart defect a five - chambered heart - cor triatriatum , an extremely rare condition .
|
five - chambered heart is extremely rare in children .
we report a case of asymptomatic five chamber heart detected in infancy .
the patient is 2-day - old and managed in a special care baby unit ( scbu ) for neonatal sepsis . during routine follow - up at the age of 1 month
, she was found to have an asymptomatic murmur .
echocardiograph reported five - chambered heart , concluding that it is cor triatriatum , supravalvular pulmonary stenosis and secundum atrial septal defect .
the child is still been followed - up and is still asymptomatic at 7 months .
five - chambered heart , although rare , can occur even if asymptomatic .
|
INTRODUCTION
CASE REPORT
DISCUSSION
|
, we will provide an update on clinical features , pathogenesis , and treatment options .
clinically nc is characterized by closely arranged , dilated follicular openings with keratinous plugs resembling classical comedones .
it is a rare type of an epidermal nevus usually with predilection for the face and neck area .
comedo nevus. nc can occur congenitally or develop later in life , most commonly at the age of approximately 10 years .
nc is part of the nevus comedonicus syndrome ( ncs ) , a term coined by engber in 1978 [ 1 , 3 ] .
the prevalence of nc has been estimated from 1 in 45,000 to 1 in 100,000 , with no gender or racial preference [ 1 , 3 ] . in 50% of patients ,
the condition develops shortly after birth and in the majority of patients lesions appear before the age of 10 [ 46 ] .
single cases of delayed development of nc and ncs at later age have been reported [ 46 ] .
nc is usually asymptomatic and most commonly it affects the face and neck area and , by exception , other anatomical regions , including genital area , palms , and soles .
clinically , nc presents with grouped , dilated , plugged follicular ostia in a honeycomb pattern .
their appearance resembles black dots but the material can not be easily removed mechanically in contrast to acne comedones [ 1 , 7 ] .
nc lesions might present with various patterns of distribution : unilateral , bilateral , linear , interrupted , segmental , or blaschkoid ( figs . 1 , 2 and 3 ) .fig .
1nevus comedonicus : plaque - like lesion with wide open follicles on the neck ( 44-year - old caucasian male)fig .
2stages of treatment of a 29-year - old asian male with linear nevus comedonicus of the leg with multiple follicular openings .
3nevus comedonicus of the neck with pseudoepithelial epidermal hyperplasia in a 39-year - old caucasian nevus comedonicus : plaque - like lesion with wide open follicles on the neck ( 44-year - old caucasian male ) stages of treatment of a 29-year - old asian male with linear nevus comedonicus of the leg with multiple follicular openings . a presentation of linear nevus comedonicus with multiple follicular openings .
d with compression garment to avoid postsurgical lymphedema nevus comedonicus of the neck with pseudoepithelial epidermal hyperplasia in a 39-year - old caucasian two types of nc have been identified ; the first is non - pyogenic nc with acne - like characteristics and a second type characterized by formation of cysts , papules , pustules , and abscesses in various stages of development [ 8 , 9 ] .
the former type is also known as munro s acne nevus and separated by some authors from nc .
differential diagnoses are shown in table 1.table 1histopathological differential diagnoses of nevus comedonicusnevus comedonicustrichofolliculomadilated pore of winerpilar sheath acanthomafolliculocystic and collagen comedo hamartomahair cortexlocalization head+++ face++++ neck++ trunk(+)+++ limbs+folliclesnumerous open folliclesopen
mother folliclesnumerous open folliclessolitary lesionscysts+++hair shafts+ , velluslamellated keratinothersmassive epithelial proliferations without folliclesabundant collagen formation histopathological differential diagnoses of nevus comedonicus the major histopathologic symptoms are large grouped , dilated follicular ostia devoid of hair shafts but filled with keratin layers ( fig .
one may observe singular rudimentary glands , which are not , however , obligatory as they may be absent .
small cysts , cystic invaginations , and occasionally large cysts may be seen in histopathologic investigation ; the variable cystic structures are lined by keratinizing squamous epithelium [ 1 , 7 ] .
hyperkeratosis ( epidermolytic hyperkeratosis ) and acanthosis of the epidermis may be present but not para- or dyskeratosis [ 8 , 1013].fig .
4histopathology of nevus comedonicus . a overview ( hematoxylin and eosin stain [ he ] 2 ) .
b details with large follicles containing lamellated keratin but absent hair shafts ( he 4 ) histopathology of nevus comedonicus .
a overview ( hematoxylin and eosin stain [ he ] 2 ) .
b details with large follicles containing lamellated keratin but absent hair shafts ( he 4 ) an increased number of langerhans cells had been observed by electron microscopy . more abundant tonofilaments in the upper part of the stratum spinosum and numerous keratohyalin granules were also noticed [ 11 , 12 ] .
the arrector pili muscles were incompletely differentiated and demonstrated intracellular glycogen particles [ 11 , 12 ] .
filaggrin , which is usually located in the granular epidermal layer , occurs in the whole epidermis of closed comedones suggesting its role in their development [ 14 , 15 ] .
interaction between fibroblast growth factor ( fgf ) and fgf receptor-2 ( fgfr2 ) is a crucial pathway for mesenchymal epithelial interplay in pilosebaceous unit development .
fgfr2b is exclusively found in epithelial cells including epidermal keratinocytes and sebocytes [ 15 , 16 ] .
overstimulation of fgfr2 signaling with increased expression of interleukin-1 may be involved in nc pathogenesis .
somatic mutations like ser252trp substation have been identified in individual patients with segmental acne [ 1618 ] .
-secretase found in human hair follicle epithelium is another candidate for nc since the absence of this enzyme results in complete conversion of hair follicles to epidermal cysts .
ncs ( orpha64754 ) belongs to the group of epidermal nevus syndromes including schimmelpenning syndrome , phacomatosis pigmentokeratotica , angora hair nevus syndrome , and becker nevus syndrome among others where a genetic basis has yet not been identified [ 3 , 4 , 20 ] .
ncs is a neurocutaneous disorder which consists of nc with skeletal , ocular , and central nervous system abnormalities .
the most common symptoms include cataracts , scoliosis , fused vertebrae , spina bifida , and delayed mental development [ 5 , 6 ] . in addition , seizures , paresis , dysgenesis of corpus callosum , electroencephalographic abnormalities , limb deformities such as absence of the fifth finger , polysyndactyly or clinodactyly , oligodontia , and other skin disorders such as ichthyosis , leukoderma , lichen striatus , linear morphea , sturge - weber syndrome , hemangiomas , and inverse acne have been reported in ncs [ 1 , 3 , 4 , 2022 ] .
ncs is associated with epithelial tumors such as trichoepithelioma , pilar sheath tumor or dilated pore of winer , keratoacanthoma , syringocystadenoma papilliferum , hidradenoma papilliferum , and very rarely basal cell carcinoma or squamous cell carcinoma [ 2326 ] .
the prevalence of nc has been estimated from 1 in 45,000 to 1 in 100,000 , with no gender or racial preference [ 1 , 3 ] . in 50% of patients ,
the condition develops shortly after birth and in the majority of patients lesions appear before the age of 10 [ 46 ] .
single cases of delayed development of nc and ncs at later age have been reported [ 46 ] .
nc is usually asymptomatic and most commonly it affects the face and neck area and , by exception , other anatomical regions , including genital area , palms , and soles . clinically , nc presents with grouped , dilated , plugged follicular ostia in a honeycomb pattern .
their appearance resembles black dots but the material can not be easily removed mechanically in contrast to acne comedones [ 1 , 7 ] .
nc lesions might present with various patterns of distribution : unilateral , bilateral , linear , interrupted , segmental , or blaschkoid ( figs . 1 , 2 and 3 ) .fig .
1nevus comedonicus : plaque - like lesion with wide open follicles on the neck ( 44-year - old caucasian male)fig .
2stages of treatment of a 29-year - old asian male with linear nevus comedonicus of the leg with multiple follicular openings . a presentation of linear nevus comedonicus with multiple follicular openings .
3nevus comedonicus of the neck with pseudoepithelial epidermal hyperplasia in a 39-year - old caucasian nevus comedonicus : plaque - like lesion with wide open follicles on the neck ( 44-year - old caucasian male ) stages of treatment of a 29-year - old asian male with linear nevus comedonicus of the leg with multiple follicular openings .
d with compression garment to avoid postsurgical lymphedema nevus comedonicus of the neck with pseudoepithelial epidermal hyperplasia in a 39-year - old caucasian two types of nc have been identified ; the first is non - pyogenic nc with acne - like characteristics and a second type characterized by formation of cysts , papules , pustules , and abscesses in various stages of development [ 8 , 9 ] .
the former type is also known as munro s acne nevus and separated by some authors from nc .
differential diagnoses are shown in table 1.table 1histopathological differential diagnoses of nevus comedonicusnevus comedonicustrichofolliculomadilated pore of winerpilar sheath acanthomafolliculocystic and collagen comedo hamartomahair cortexlocalization head+++ face++++ neck++ trunk(+)+++ limbs+folliclesnumerous open folliclesopen
mother folliclesnumerous open folliclessolitary lesionscysts+++hair shafts+ , velluslamellated keratinothersmassive epithelial proliferations without folliclesabundant collagen formation histopathological differential diagnoses of nevus comedonicus
the major histopathologic symptoms are large grouped , dilated follicular ostia devoid of hair shafts but filled with keratin layers ( fig . 4 ) .
at some locations in the bases of the follicular invaginations one may observe singular rudimentary glands , which are not , however , obligatory as they may be absent .
small cysts , cystic invaginations , and occasionally large cysts may be seen in histopathologic investigation ; the variable cystic structures are lined by keratinizing squamous epithelium [ 1 , 7 ] .
hyperkeratosis ( epidermolytic hyperkeratosis ) and acanthosis of the epidermis may be present but not para- or dyskeratosis [ 8 , 1013].fig .
4histopathology of nevus comedonicus . a overview ( hematoxylin and eosin stain [ he ] 2 ) .
b details with large follicles containing lamellated keratin but absent hair shafts ( he 4 ) histopathology of nevus comedonicus .
a overview ( hematoxylin and eosin stain [ he ] 2 ) .
b details with large follicles containing lamellated keratin but absent hair shafts ( he 4 ) an increased number of langerhans cells had been observed by electron microscopy . more abundant tonofilaments in the upper part of the stratum spinosum and numerous keratohyalin granules were also noticed [ 11 , 12 ] .
the arrector pili muscles were incompletely differentiated and demonstrated intracellular glycogen particles [ 11 , 12 ] .
filaggrin , which is usually located in the granular epidermal layer , occurs in the whole epidermis of closed comedones suggesting its role in their development [ 14 , 15 ] .
interaction between fibroblast growth factor ( fgf ) and fgf receptor-2 ( fgfr2 ) is a crucial pathway for mesenchymal epithelial interplay in pilosebaceous unit development .
fgfr2b is exclusively found in epithelial cells including epidermal keratinocytes and sebocytes [ 15 , 16 ] .
overstimulation of fgfr2 signaling with increased expression of interleukin-1 may be involved in nc pathogenesis .
somatic mutations like ser252trp substation have been identified in individual patients with segmental acne [ 1618 ] .
-secretase found in human hair follicle epithelium is another candidate for nc since the absence of this enzyme results in complete conversion of hair follicles to epidermal cysts .
ncs ( orpha64754 ) belongs to the group of epidermal nevus syndromes including schimmelpenning syndrome , phacomatosis pigmentokeratotica , angora hair nevus syndrome , and becker nevus syndrome among others where a genetic basis has yet not been identified [ 3 , 4 , 20 ] .
ncs is a neurocutaneous disorder which consists of nc with skeletal , ocular , and central nervous system abnormalities .
the most common symptoms include cataracts , scoliosis , fused vertebrae , spina bifida , and delayed mental development [ 5 , 6 ] . in addition , seizures , paresis , dysgenesis of corpus callosum , electroencephalographic abnormalities , limb deformities such as absence of the fifth finger , polysyndactyly or clinodactyly , oligodontia , and other skin disorders such as ichthyosis , leukoderma , lichen striatus , linear morphea , sturge - weber syndrome , hemangiomas , and inverse acne have been reported in ncs [ 1 , 3 , 4 , 2022 ] .
ncs is associated with epithelial tumors such as trichoepithelioma , pilar sheath tumor or dilated pore of winer , keratoacanthoma , syringocystadenoma papilliferum , hidradenoma papilliferum , and very rarely basal cell carcinoma or squamous cell carcinoma [ 2326 ] .
being benign , nc does not require aggressive treatment , except for aesthetic reasons or in complicated cases .
conservative options include emollients and moisturizer , topical corticosteroids ( for inflammatory lesions ) , keratolytics such as salicylic acid , or 12% ammonium lactate solution .
some reports showed better improvement of nc by topical tazarotene as monotherapy or in combination with topical mometasone furoate or calcipotriene [ 2931 ] .
oral retinoids , such as isotretinoin , were found to be ineffective in most cases but may be an option in widespread systematized inflammatory variants [ 32 , 33 ] . there have , as yet , been no published studies of other retinoids , such as topical adapalene and systemic or topical bexarotene . of potential interest are fgfr inhibitors with antivascular activity .
interleukin-1 inhibitors can be classified into receptor inhibitors , such as anakinra , and monoclonal antibodies against the interleukin itself . currently , there are no data available for nc .
another interesting drug target is -secretase ; stimulators of this enzyme such as general control nonderepressible 2 ( gcn2)a subunit of eukaryotic translation factor 2 kinase might be a future therapeutic option .
localized nc can be removed by surgical excision with good aesthetic results in contrast to superficial shaving , comedo extraction , and dermabrasion techniques ( fig .
if linear nc is surgically removed on the limbs , complex decongestive therapy including manual lymph drainage and compression garments supports the healing process ( fig .
2d ) . larger lesions may need transplantation for defect closure , or another option is the use of repeat - filling or self - filling osmotic tissue expanders to obtain a resurplus of skin for defect closure [ 38 , 39 ] .
laser treatments with 2,940-nm erbium yag , 10,600-nm ultrapulsed co2 , or 1,450-nm diode lasers have shown improvement in single patients [ 4042 ] .
erbium yag laser treatment is often followed by delayed relapses . in contrast to ablative lasers , the 1,450 nm diode shrinks sebaceous glands and reduces seborrhea .
the combination of 1,450-nm diode laser with 1,550-nm erbium - doped fiber laser has potential for treating nc .
with this review we provided an update on clinical features , pathogenesis and treatment option of nc . nc is a rare epidermal nevus .
new data are available on signaling pathways in acne - related disorders that may have an impact on nc as well .
in contrast to the acne - related comedones , the epithelium is often hyperkeratotic and sometimes acanthotic and comedo extraction is not as easily achieved as with acne comedones .
dermatologists should be aware of potential association of nc with skin tumors and extracutaneous findings such as ncs .
new developments in laser technology may become an alternative in near future . with a more detailed understanding in molecular pathogenesis , targeted therapy
|
the intention of this review on nevus comedonicus ( nc ) is to update on clinical features , pathogenesis , and therapy .
nc is a rare epidermal nevus type .
it is part of the nevus comedonicus syndrome , a neurocutaneous disorder with ocular , skeletal , and central nervous symptoms .
recently , acne - related signaling pathways and somatic mutations of tyrosine kinase receptors have been identified and may play a role in nc pathogenesis .
on preexistent nc secondary skin tumors can develop , which are often benign .
treatment options of nc include topical therapy , laser , and surgery .
|
Introduction
Epidemiology
Clinical Characteristics
Histopathology
Association with Genetic Syndromes and Other Diseases
Treatment
Conclusion
|
the " gold standard " for assessing fibrosis , liver biopsy , is recommended prior to the initiation of antiviral therapy ; in addition , it is vital for monitoring fibrosis progression . unfortunately , this procedure is invasive , prone to complications , including hemorrhage and death , and has a high risk of sampling error .
biochemical markers for liver fibrosis ( fibrotest ) and necroinflammatory features ( actitest ) are an alternative to liver biopsy , in patients with chronic hepatitis c . since the first publication , which included a validation period , those tests have been validated in different populations by the same reference laboratory and by an independent group .
the tests combine five components ( 2-macroglobulin , haptoglobin , apolipoprotein a1 , -glutamyl transpeptidase ( ggt ) , and total bilirubin ) for fibrotest and same plus alanine aminotransferase ( alt ) for actitest .
the aim of this study was to assess the inter - laboratory variability of fibrotest and actitest , including their six serum liver components , in patients with chronic liver disease , and to identify factors associated with that variability .
our concern was to assess whether the analytical methods adapted on the different analyzers were associated with significant variability in fibrotest and/or actitest values .
moreover , we aimed to compare the variability of fibrotest and actitest in relation to the method of expressing enzymatic activity ; in particular , in terms of absolute values or as multiples of the upper limit of normal . since we and others have demonstrated that current definitions of normal values may be inappropriate [ 8 - 10 ]
in routine practice , the definition of the upper limit of normal ( uln ) of alt and ggt varies between laboratories , but is rarely detailed . because numerous medical guidelines make reference to alt and ggt expressed as multiples of the uln ( uln units ) , variations in the definition of normal may have important practical consequences .
the main characteristics of the included patients are outlined in table 1 . according to each patient and laboratory , details of the fibrotest and actitest assays
there was no significant difference between centers for fibrotest using ggt expressed in international units [ mean ( sd ) = 0.57 ( 0.26 ) , range = 0.480.65 , f - ratio = 0.27 , p = 0.27 ] .
for fibrotest using ggt expressed in uln units [ mean ( sd ) = 0.55 ( 0.27 ) , range = 0.450.68 , f - ratio = 1.26 , p = 0.27 ] , there was a significant difference between three centers ( center 5 had higher means values than center 2 and 4 ; p = 0.02 for both comparisons ) .
fibrotest and actitest variability according to laboratories ( centers ) and units of enzymatic expression : international units ( iu ) and upper limit of normal ( unl ) . characteristics of included patients hcv hepatitis c virus ; hiv human immunodeficiency virus ; sd standard deviation .
there was no significant difference between centers for actitest using alt and ggt expressed in international units [ mean ( sd ) = 0.32 ( 0.26 ) , range = 0.380.53 , f - ratio = 1.21 , p = 0.30 ] and for actitest using alt and ggt expressed in uln units [ mean ( sd ) = 0.44 ( 0.27 ) , range = 0.270.43 , f - ratio = 0.81 , p = 0.59 ) .
the details of the liver proteins and total bilirubin assays according to each patient and laboratory are outlined in figure 2 .
there were no significant differences according to testing center for any of these assays ( between centers or versus the reference center ) : ( 2-macroglobulin [ mean ( sd ) = 2.89 ( 1.16 )
g / l , range = 2.693.33 , f - ratio = 0.72 , p = 0.67 ] , haptoglobin [ mean ( sd ) = 0.98 ( 0.58 )
g / l , range = 0.921.03 , f - ratio = 0.07 , p = 0.99 ) , apolipoprotein a1 [ mean ( sd ) = 1.30 ( 0.51 ) g / l , range = 1.161.42 , f - ratio = 1.21 , p = 0.30 ] and bilirubin [ mean ( sd ) = 28.8 ( 66 ) micromol / l , range = 15.851.1 , f - ratio = 0.51 , p = 0.85 ] .
one analyzer ( advia ) gave lower mean apoliprotein a1 levels [ 1.06 ( 0.43 )
g / l ) than the other analyzers [ 1.33 ( 0.52 ) g / l ; p = 0.02 ] .
the details of the alt and ggt assays , according to each patient and laboratory and expressed in international or uln units , are given in figure 3 .
there was no significant difference between centers for alt expressed in international units [ mean ( sd ) = 70 ( 47 ) iu / ml , range = 5786 , f - ratio = 1.30 , p = 0.25 ] .
however , when the assays used pyridoxal phosphate as in the reference center , the mean alt was higher [ 78 ( 50 ) iu / ml ] than assays not using pyridoxal phosphate [ 60 ( 42 ) iu / ml ; p = 0.003 ] .
for alt expressed in uln units [ mean ( sd ) = 48 ( 37 ) , range = 3771 , f - ratio = 1.65 , p = 0.12 ] , there was a significant difference between center 1 and all centers ( p = 0.009 vs center 2 , p = 0.008 vs center 3 , p = 0.04 vs center 4 , p = 0.04 vs center 5 , p = 0.02 vs center 6 , p = 0.03 vs center 7 , p = 0.01 vs center 10 and p = 0.001 vs center 11 ) .
there were no significant differences between centers for ggt expressed in international units [ mean ( sd ) = 130 ( 158 ) iu / ml , range = 5786 , f - ratio = 1.30 , p = 0.25 ] or in uln units [ mean ( sd ) = 109 ( 121 ) iu / ml , range = 78154 , f - ratio = 1.46 , p = 0.17 ] .
however , and despite the use of the same szasz method , one automate ( dade behring rxl ) gave higher ggt mean values [ 165 ( 200 ) iu / ml ] than the others [ 120 ( 143 ) iu / ml ; p = 0.06 ] .
alanine aminotransferase ( alt ) and -glutamyl transpeptidase ( ggt ) variability according to laboratory and units of enzymatic expression : international units ( iu ) and upper limit of normal ( unl ) .
passing - bablok linear regression analyses of all samples between laboratories and the reference center are summarized in table 2 .
the intercept and slope between the reference center and other laboratories were excellent for the three proteins with only one decrease for apolipoprotein a1 in a single center using the advia analyzer .
for ggt , centers using the rxl analyzer had a higher slope ( greater than 1 ) . passing - bablok analysis between laboratories and reference center ( lab 1 ) for each component concordance rates ( kappa statistics ) among laboratories
are given in table 3 ; all were statistically significant . when ggt and alt were expressed in international units , fibrotest and actitest kappa statistics were all greater than 0.50 with only 0.8% cases ( 3 out of the 384 comparisons ) with a discordance of more than one fibrosis stage .
in contrast , when ggt and alt were expressed in uln units , fibrotest and actitest kappa statistics were lower than 0.50 in 11 comparisons ( out of the 16 comparisons versus the reference laboratory ) with 5% of cases ( 21 out of the 384 comparisons ) with a discordance of more than one fibrosis stage or greater than one activity grade .
concordance rates ( kappa statistics ) of laboratories with reference center ( lab 1 ) , according to the expression of ggt and alt activities
this study showed that the variability of fibrotest and actitest values , among nine different laboratories , was acceptable and without clinical consequences for the prediction of the stage of liver fibrosis or grade of activity .
this finding is important since it confirms that those tests can be routinely computed from the results of the six individual components obtained by non - centralized measurements .
online assessment is available using the website . to guarantee the quality of this assessment , it was necessary to identify the factors associated with the variability of the six components .
this study confirms that the expression of alt and ggt in multiples of the upper limit of reference values should be avoided . despite efforts to standardize enzymatic assay methods , homogeneity of alt results
has not been achieved as attested by external quality controls , and identical limits of reference values can not be defined .
many clinicians believe that expression of the results as multiples of the upper limits of reference ranges can reduce inter - laboratory variability .
our study confirms previously observed results , that this method of expression is , in fact , worse than that using international units both for alt and ggt . in our reference center ,
the reference limit recorded was similar to the described mean value from a recent study and lower than in the other laboratories .
if actitest was expressed in a standardized way , using the upper limit of each laboratory for ggt and alt , this induced lower concordance rate than actitest using international units . to increase inter - laboratory coherence in the results of enzymatic activities , standardized assays against a reference method should be employed , with calibration of the assay using a commutable enzymatic material .
the values of this calibrator must be assigned by a reference method . for proteins and bilirubin assays
this was anticipated for 2-macroglobulin since the same analyzer was used in all laboratories . although the use of three different analyzers , haptoglobin has the best homogeneity .
in fact , the assays of these two proteins are standardized against the crm 470 reference material .
this reference product is now used in different measurement procedures to attain results numerically the same , whatever the clinical conditions . for apolipoprotein a1 ,
this is due to the use of a different reference material to standardize the assay .
overall , the data from the laboratories were linearly related with the reference center with a slope close to 1 and a non - significant analytical imprecision ; there were few pairs of assays outside the confidence limits and the samples were adequately distributed over the investigated range .
as previously observed , when ordinary linear regression ( in combination with correlation analysis in the passing bablock method ) gave poor estimates , in particular for ggt and alt assays , we found several analytical reasons for the poor performance .
enzymatic measurement with the szasz method ( standardized against the original for ggt ) , and with the standardized method according to the international federation of clinical chemistry ( using pyridoxal phosphate for alt ) , would probably reduce the variability .
because of their predictive values and their reproducibility in different populations , biochemical markers could be used as surrogate markers for liver biopsy both for the initial decision of liver biopsy and for the follow - up of chronic hepatitis c patients . to date ,
liver biopsy has been considered mandatory for the management of patients infected by hepatitis c virus ( hcv ) .
reviews of morbidity and mortality of intercostal liver biopsy observed a mean occurrence of pain in 30 % of patients , 3 out of 1,000 endured severe adverse events , and 3 out of 10,000 died .
even liver biopsy is dependant on the inter- and intra - observer ( pathologist ) differences .
there are also potential problems with liver biopsy sampling variation . in a study with three consecutive samples through a single entry site ,
only 50 % of patients with cirrhosis were scored as cirrhosis on the three samples .
it is therefore possible that biochemical markers such as those described may provide a more accurate ( quantitative and reproducible ) picture of fibrogenic events occurring within the liver . furthermore , and because treatment is now so effective in patients with genotype 2 or 3 infection , the utility of biopsy in this setting could be challenged .
when ggt and alt are expressed in international units , fibrotest and actitest can be computed from different laboratories with acceptable variability . to increase inter - laboratory coherency , standardized methods and enzymatic calibration
expression of alt and ggt in multiples of the upper limit of reference values should be avoided .
the serum of 24 informed patients ( 21 with chronic hepatitis c and 3 with decompensated alcoholic cirrhosis ) were prospectively collected in the department of hepato - gastroenterology of the piti - salptrire hospital , in paris , france .
sera were separated in the above reference laboratory , conserved at + 4c and distributed to ten different laboratories , in france , within 24 hours . for two laboratories ,
serum was missing for at least one patient ; therefore , these laboratories have been excluded from the core analysis .
sensitivity analyses including these two excluded laboratories did not change the results or conclusions ( data not shown ) .
characteristics of the analyzer , reagents and analytical methods employed used in the nine included laboratories are detailed in table 4 .
eleven different analyzers were used . for the measurement of alt activity , five laboratories used a standardized method according to the ifcc , with pyridoxal phosphate , and four without pyridoxal phosphate . for the measurement of ggt activity ,
the nine laboratories used the szasz method ; including in four a recommended method of standardization .
laboratory analyzers and biochemical methods bn2 , rxl , vitros 250 : dade behring , marburg , germany .
immage , cx5 , cx7 : beckman coulter , brea , california , usa . advia 1650 : bayer diagnostics , tarrytown , new jersey , usa .
analytical measurements of 2-macroglobulin and haptoglobin were standardized against the certified international reference material 470 ( crm 470 ) .
apolipoprotein a1 assays adapted on the different analyzers were standardized against the reference material of world health organization - international federation of clinical chemistry sp1 - 01 ( who - ifcc sp1 - 01 ) , except on the advia - bayer - analyzer ( advia ) .
total bilirubin was assayed by diazoreactions methods . statistical analysis used multiple measure variance analyses and passing - bablok linear regression analyses for the comparison of inter - laboratory results , and kappa statistics for the predicted histological features .
multiple comparisons used bonferroni ( versus control ) and tukey - kramer multiple - comparison tests .
number cruncher statistical systems software was used . the linear relationship between laboratories and reference center
were assessed with confidence limits for the slope and the intercept and the number of pairs out of bounds ; they were used to determine whether there was only a chance difference between the slope and 1 and between the intercept and 0 .
ph , fib , dm , ga , db , pcl , gd , dd , tk , ms , dt and et performed the assays .
|
backgroundbiochemical markers for liver fibrosis ( fibrotest ) and necroinflammatory features ( actitest ) are an alternative to liver biopsy in patients with chronic hepatitis c. our aim was to assess the inter - laboratory variability of these tests , and their 6 components ( -glutamyl transpeptidase , alanine aminotransferase , 2-macroglobulin , haptoglobin , apolipoprotein a1 , and total bilirubin ) and to identify factors associated with this variability.resultsserum of 24 patients with chronic hepatitis c or severe alcoholic liver disease were prospectively recorded and analyzed in one reference center and in 8 additional laboratories .
when -glutamyl transpeptidase and alanine aminotransferase were expressed in international units , there was no significant difference between laboratories in the results of fibrotest or actitest ; kappa statistics were greater than 0.50 with only 0.8% of cases ( 3/384 ) with a discordance of more than one stage .
the main factor significantly associated with variability was the expression of -glutamyl transpeptidase and alanine aminotransferase , as multiples of upper limit of reference values .
the use of standardized method with pyridoxal phosphate reduced the variability of alanine aminotransferase expression , and standardized original szasz method reduced the variability of -glutamyl transpeptidase expression.conclusionsthe variability of fibrotest and actitest was acceptable without clinical consequences for the prediction of the stage of liver fibrosis and grade of activity .
standardized methods and assay calibration should be used and expression of alanine aminotransferase and -glutamyl transpeptidase in multiples of the upper limit of reference values should not be employed .
|
Background
Results
Discussion
Conclusions
Methods
Authors' contribution
Acknowledgements
|
with the exception of mammals , the function of the members of the atad2 protein family remains mostly undefined in multicellular eukaryotes .
the first reported activity of atad2 ( atpase family aaa+ domain - containing protein 2 ) is its ability to act as a co - activator of estrogen and androgen receptors ( zou et al .
other studies later defined atad2 as an e2f and mll co - factor stimulating e2f - dependent cell proliferation ( revenko et al . , 2010 ) and also as a myc partner ( ciro et al . , 2009 ) . these transcriptional activities of atad2 have been , at least partly , attributed to its two conserved domains ( revenko et al .
indeed , a conserved feature of atad2 and of its homologues across species is the presence of a two partite aaa+ atpase ( atpases associated with diverse cellular activities ) domain that mediates protein multimerization and of a bromodomain that is responsible for its binding to his - tones ( boussouar et al . , 2013 ) .
this high conservation of the domain organization of the protein in the vast majority of eukaryotes , including unicellular organisms such as yeasts , is also indicative of its conserved functional features .
functional studies of atad2 family members carried out in mammals were mostly in cancer settings and have recently been reviewed ( boussouar et al . , 2013 ) .
however , in addition to these studies , there is a relatively rich literature on yta7 ( yeast tat - binding analog 7 ) , the unique atad2 homologue expressed in the budding yeast saccharomyces cerevisiae .
this review considers all the molecular data available on yta7 in the light of our knowledge on atad2 .
the data reported highlight the implication of yta7 not only in gene expression , as expected , but also in genome organization , as a possible histone chaperone acting at boundary sites and regulating transcription .
1a ( left ) , illustrates the strong conservation of this protein family within the eukaryotic kingdom , including yeast .
these proteins all share a two - partite aaa+ atpase domain , which is found n - terminal to a bromodomain .
these include chordata and the fission yeast schizosaccharomyces pombe that possess two atad2-like proteins known as atad2a and b and abo1 and 2 , respectively .
it is of note that the vast majority of the published functional studies concerns the atad2a ( also termed atad2 ) member in both human and mouse .
the high sequence similarity between these paralogs argues in favor of a possible functional redundancy across all species .
other eukaryotes such as plants ( arabidopsis thaliana , zea mays ) , the worm caenorhabditis elegans , and the budding yeast s. cerevisiae possess only one gene encoding an atad2-like protein .
intriguingly , in drosophila melanogaster and tetrahymena thermophila no gene encoding a homologue of atad2 has been identified . in these two organisms ,
the closest relative proteins belong to the valosin - containing protein / transitional endoplasmic reticulum atpase ( vcp / tera family ) , which is also a conserved protein family that , to a certain extent , shares a similar domain organization with atad2 family members , although the amino - acid sequence aside the aaa+ atpase domain is poorly conserved .
xenopus laevis is quite peculiar , since it possesses a gene expressing a shorter version of atad2-like protein .
this protein contains only the first aaa+ atpase domain and the n - terminal part of the second one .
the existence of organisms that have lost part or the totality of atad2-like proteins might indicate that a concomitant functional adaptation has occurred to compensate for the lack of atad2 functions in these organisms .
this observation also points to the existence of possible redundant functional pathways in various eukaryotic cells that express atad2 family members .
atad2-like proteins have their highest sequence similarity within the two aaa+ atpase domains and the bromodomain ( fig .
the aaa+ atpase domain is found in all kingdoms of living organisms , in proteins possessing many different cellular functions .
atad2-like proteins contain two aaa+ atpase domains both located in their n - terminal part .
atp binding and atpase activities were demonstrated for atad2 ( zou et al . , 2007 ) .
additionally , mutations in the first aaa+ atpase domain that affect atp binding and hydrolysis impact on the property of mouse atad2 and human atad2 to oligomerize , to bind to acetylated histone h4 ( caron et al . , 2010 ) and to co - activate transcription ( zou et al . , 2007 ) , indicating that this domain is critical for atad2 functions .
since the amino - acid sequence of the aaa+ atpase domain is highly similar among atad2-like proteins , it is likely that the activity and function of this domain are also conserved in other eukaryotes . in agreement with such a possibility
, mutations in the first aaa+ atpase domain of the s. cerevisiae yta7 protein affect many of its functions ( kurat et al .
, 2011 ; lombardi et al . , 2011 ) , as described in the next sections .
atad2-like proteins contain also a putative bromodomain , a module known to bind acetylated lysine in histones and other proteins ( filippakopoulos et al . ,
the binding to acetylated histones was demonstrated for mouse atad2 and human atad2 , which show a preferential binding to acetylated histones h3 and h4 ( caron et al . , 2010 ; revenko et al . ,
bromodomains have a three - dimensional structure that consists of four helix bundles ( z , a , b and c ) with two interhelical za and bc loops , containing several amino - acids necessary to form an hydrophobic pocket for the interaction with acetylated lysines ( dhalluin et al . , 1999 ) .
the overall conservation of the bromodomain amino - acid sequence in atad2-like proteins suggests that its global architecture and its capacity to bind to histones may be conserved .
nonetheless , alignment of the yta7 bromodomain with other yeast bromodomains revealed that residues critical for binding acetylated histones are missing , pointing out that the specific binding to acetylated histones is probably not true for all atad2-like proteins ( jambunathan et al . , 2005 ) .
in vitro histone pull - down experiments have indeed shown that yta7 binds histones but in an acetylation - independent manner ( gradolatto et al .
interestingly , similar pull - down experiments , using truncated forms of yta7 , revealed a second region that binds histones .
this region is located in the n - terminal part of yta7 , upstream of the aaa+ atpase domains ( fig .
1b ) , and contains a stretch of acidic residues that may be responsible for electrostatic interactions with charged and unmodified lysine and arginine residues in histones ( gradolatto et al . , 2009 ) .
remarkably , a patch of acidic residues in the n - terminal part of the protein seems to be a common feature of all atad2-like proteins , suggesting that the function of this negatively charged region in binding histones may be conserved .
mutations changing the nature of this region would be informative both in relation to the importance of this domain in contributing to atad2-like protein functions , as well as to know whether this region influences the capacity and/or specificity of the bromodomain of yta7 to bind histones . finally , members of the atad2 family also have in common a fourth region of around 60 amino - acids located at the extreme c - terminal part of the protein ( fig .
this domain , which is highly conserved among atad2-like proteins , does not correspond to any annotated domain and is not found in any other type of proteins .
the analysis of this newly identified and conserved domain should establish its function within atad2 proteins .
interestingly , upstream of this c - terminal domain there is another region conserved in atad2b proteins , but not in the atad2a / atad2 paralogs , indicating that this domain may play an important role in attributing a specific function to the atad2b proteins . here
again , the analysis of this newly identified and conserved region , possibly using the powerful yeast genetics , could provide some clues to the function of atad2-like proteins .
a genome - wide chromatin localization approach using chromatin immunoprecipitation ( chip)-coupled to chip ( chip - chip ) analysis demonstrated that yta7 binds to all histone genes ( gradolatto et al . , 2008 ) .
however , the effects of a deletion of the yta7 gene on the level of histone transcripts are not clear yet .
( 2008 ) reported that yta7 is a histone gene repressor based on the fact that yta7 cells present a precocious increase in mrnas of all histone genes after -factor block ( g1-phase arrest ) .
2009 ) proposed that yta7 could be an activator of histone genes transcription , since a deletion of the yta7 gene correlates with a decrease in hta1 transcripts that is concomitant with a decreased recruitment of rna polymerase ii to both the promoter region and the orf of hta1 ( kurat et al . , 2011 ) .
more recent studies have shown that the importance of yta7 in regulating histone transcription may in fact differ depending on the analyzed histone gene .
indeed , in yta7 cells hta1 transcripts are significantly reduced , while in these same cells hht1 , hhf1 or hta2 transcript levels remain unchanged , despite the fact that yta7 binds to all these histone loci ( lombardi et al . , 2011 ;
transcription of the histone genes is tightly regulated throughout the cell cycle in order to provide the histone supply required for the replication of chromatin during the s - phase while avoiding inappropriate and toxic accumulation of neosynthesized histones during the other phases of the cell cycle ( gunjan and verreault , 2003 ) .
the localization of yta7 to the histone genes indicates a possible direct role for this protein in the phase - specific regulation of histone transcription .
interestingly , yta7 binding to the histone gene hta1 is precisely regulated during the cell cycle ( kurat et al . , 2011 )
after the loading of rna polymerase ii to histone genes in g1/s , yta7 is phosphorylated at multiple sites located in its n - terminal part by at least two different kinases cdk1 ( cyclin - dependent kinase 1 ) and ck2 ( casein kinase 2 ) .
phosphorylation of yta7 causes its release from hta1 and correlates with an efficient transcription of hta1 and other histone genes ( kurat et al . , 2011 ) .
the molecular mechanism underlying the yta7-dependent transcriptional gene activation is not completely understood , but it has been proposed that the phosphorylation - mediated removal of yta7 from a histone gene during the s - phase could be an important step to allow efficient transcriptional elongation along the histone gene . in agreement with this hypothesis , in a mutant of yta7 that can not be phosphorylated ,
the recruitment of rna polymerase ii is markedly decreased within the orf of hta1 gene but not at the promoter region ( kurat et al . , 2011 ) .
transcriptome analyses of yta7 cells revealed that the expression of inducible genes is also deregulated and that yta7 probably acts as a histone chaperone ( lombardi et al .
chip experiments have shown that yta7 localizes to the promoters and 5orf of early meiotic genes and galactose genes while they are induced .
moreover , the presence of yta7 limits the nucleosome density in these genic regions , possibly by promoting histone h3/h4 eviction ( fig .
in agreement with the existence of an yta7-mediated histone eviction activity , the deficiency in inducing gene expression in yta7 cells is rescued by a decrease in the dosage of histones h3 and h4 caused by the deletion of a pair of histone genes ( hht1hhf1 ) .
interestingly , the aaa+ atpase domain is required for this potential chaperone activity of yta7 , indicating that yta7 atp hydrolysis may be important for the eviction or degradation of histones ( lombardi et al . , 2011 ) .
in addition , yta7 was co - purified with several subunits of the rna polymerase ii ( rpb2 , rpb5 and rpb10 ) , as well as with several histone chaperones , the fact complex ( spt16 and pob3 ) and rtt106 protein ( kurat et al . , 2011 ; tackett et al . , 2005 ) .
nonetheless , it should also be pointed out that so far there is no in vitro evidence that yta7 is a bonafide histone chaperone . moreover , since in vivo experiments do not exclude the possibility that the change in histone density observed in yta7 cells is due to the activity of one of the histone chaperones interacting with yta7 , these findings await further experiments to conclude that yta7 and , by extension , other atad2-like proteins are indeed histone chaperones .
yta7 localization to histone genes promoters was also linked to a boundary activity acting at the promoters of all histone genes .
indeed , at these boundaries , yta7 prevents the spreading of the histone chaperone rtt106 from the promoter to their respective coding regions ( fillingham et al . ,
2009 ; kurat et al . , 2011 ; zunder and rine , 2012 ) ( fig .
the recruitment of rtt106 to histone gene promoters depends on asf1 and the hir complex and is not regulated through the cell cycle .
hir / asf1 binding to histone genes and the repression of their transcription rely on a specific dna sequence , the negative regulatory element , present in every histone pairs except for hta2-htb2 .
rtt106 was found to bind and act as a repressor of the histone genes targeted by hir and asf1 ( hta1-htb1 , hht1-hhf1 , hht2-hhf2 ) ( fillingham et al . , 2009 ) .
rtt106 is regulating both positively and negatively the expression of histone genes during the cell cycle . outside s - phase , when histone genes are repressed , rtt106 recruits the atp - dependent remodeling complex rsc . on the contrary , during histone gene activation in s - phase rtt106
recruits the swi / snf complex ( ferreira et al . , 2011 ) . in yta7 cells
rtt106 spreads within the coding sequence of histone genes together with the rsc complex , except for the hta2-htb2 histone pair that is not bound by hir / asf1 and rtt106 ( fillingham et al . , 2009 ) .
the spreading of rtt106 through the coding regions of histone genes is associated with a clear decrease of hta1 transcripts , suggesting that rtt106 and rsc spreading may be responsible for the transcriptional repression of hta1 gene . however , since this spreading occurs on all his - tone genes even though it is not always associated to a decrease in histone mrna levels , for instance at hht1 or hhf1 genes , the potential repressive action of rtt106 requires further investigations to be validated ( zunder and rine , 2012 ) .
in addition , it is also possible that the regulation of histone genes expression presents a certain degree of locus - specificity that would allow rtt106 spreading to repress transcription of some , but not all histone genes .
earlier evidences implicated yta7 , together with other proteins , at barriers that demarcate euchromatin from heterochromatin regions ( jambunathan et al . , 2005 ; tackett et al . ,
s. cerevisiae contains heterochromatin - like regions located at the mating type , telomeres and rrna - encoding dna loci .
the implication of yta7 in such barrier function was first found in a genetic screen aimed at identifying new genes that , when mutated , allow inappropriate spreading of silent chromatin from the mating type locus ( hmr ) over the adjacent trna gene into the neighboring genomic region which included the ade2 reporter gene ( jambunathan et al . , 2005 ) .
the boundary activity of yta7 was investigated also by using silencing assays testing the sensitivity to 5 fluoroorotic acid ( 5foa ) drug of yeast cells in which the ura3 reporter gene was ectopically incorporated either within the silenced hmr or the transcriptionally competent adjacent region ( tackett et al . , 2005 ) .
furthermore , both the bromodomain and the aaa+ atpase domains are required for yta7 barrier function ( gradolatto et al . , 2009 ; lombardi et al . , 2011 ) .
in agreement with a direct implication of yta7 in such a barrier function , it was found to co - purify with a complex localizing at the boundaries between euchromatin and heterochromatin - like regions in s. cerevisiae ( tackett et al . , 2005 ) .
nonetheless , the exact role of yta7 at these chromatin barriers remains to be determined .
for example , it would be interesting to understand if the probable histone chaperone activity of yta7 plays any important role at these barriers .
however , although data on yta7 suggest a role for this factor in the organization of the genome and chromatin dynamics , such indications are scarce for atad2 .
indeed , only one experiment that measured histone h2a mobility in a lung cancer cell line showed that a decrease in atad2 content modifies h2a turnover ( caron et al . , 2010 ) . even in this case
considering the importance of yta7 in regulating nucleosome density , it is tempting to propose that atad2 can also act as a histone chaperone , evicting histones from chromatin to avoid potentially deleterious effects associated to an increase in his - tone density . therefore atad2 and yta7 could primarily be chromatin remodelers and the above - mentioned effects on gene expression regulation could be a mere consequence of their actions on chromatin organization . alternatively , these proteins could be dual factors playing a role both in transcriptional regulation as scaffolds or as co - activators , and in chromatin remodeling as histone chaperones . in agreement with such a possibility , the acidic n - terminal region of atad2 is a histone - interacting module in the case of yta7 ( as described previously ) , but has also been shown to interact with the androgen receptor ( zou et al . , 2009 ) and
e2f transcription factor in the case of human atad2 ( revenko et al . , 2010 ) , indicating that this domain may play a central role in the potential dual function of atad2-like proteins .
it is also tempting to imagine that this domain may be involved in a cellular protective mechanism which would sense an increase in nucleosome density and in response would favor the chromatin remodeler functions of yta7 instead of its transcriptional role .
the phosphorylation of atad2/yta7 in its n - terminal region could allow switching towards a chaperone function of the protein by enabling its detachment from chromatin .
the histone binding of yta7 is mediated both by its bromodomain and the n - terminal region ( gradolatto et al . , 2009 ) , and we can therefore speculate that a functional inactivation or a lack of the n - terminal part of atad2/yta7 could transform the factor into a pure chromatin regulator . in support of this hypothesis , a spliced form of atad2 lacking this n - terminal acidic domain
is expressed exclusively in mouse spermatogenic cells ( caron et al . , 2010 ) , where one of the most extensive chromatin remodeling takes place ( goudarzi et al . , 2014 ) . however , neither in human cells nor in yeast is such n - terminally shortened protein found expressed , suggesting that , if the above hypothesis is right , in the cases of human or yeast cells , a regulatory mechanism should inactivate the transcriptional role of the n - terminal domain to transform the protein into a pure chromatin regulator . this regulation could involve changes , such as yta7 phosphorylation at its n - terminal part , which would lead to the release of the protein from histone genes ( kurat et al . , 2011 ) .
the data discussed above therefore support the idea that atad2 and yta7 follow similar regulatory rules , since the underlying activity remains conserved ( even if their mechanisms of action could be regulated differently due to specific requirements and evolutionary adaptations ) .
atad2 is one of the most conserved proteins in eukaryotes although paradoxically its function remains obscure . the remarkable conservation of its functional domains which , as shown here , goes beyond the aaa+ atpase and the bromodomain , pleads in favor of conserved activities . an almost systematic up - regulation of atad2 in all cancers ( boussouar et al . , 2013
therefore , an intriguing question is why , despite these important features , the atad2-like proteins have not been identified as critical components in the hundreds of functional screens that have been undertaken in the recent years .
one explanation is that the function of atad2/yta7 is so diverse and generalist , involved in many cellular activities , that none of them is dramatically affected by the loss of atad2/yta7 , but all work more efficiently in the presence of these proteins .
atad2 family members could therefore be important auxiliary factors in many chromatin related activities , including transcription . finally , as illustrated in this review ,
the studies done on the yeast s. cerevisiae yta7 protein are complementary to the studies conducted on atad2 in mammals , which are mostly connected to cancer . in this
regard , characterizing the function of atad2 proteins in the fission yeast schizosaccharomyces pombe , another yeast model distantly related to budding yeast and often used for studying chromatin biology , could also be informative .
this yeast possesses several pathways and his - tones modifications , like rna interference ( rnai ) and the h3k9me ( histone h3 methylated on lysine 9 ) heterochromatin mark , that are present in larger eukaryotes but absent in s. cerevisiae .
( lin-48 expression abnormal protein 1 ) , the atad2 homologue in the worm caenorhabditis elegans , was found in a screen to control the expression of repeated transgenes , suggesting that repeated sequences may also be genomic targets for atad2 proteins ( tseng et al . , 2007 ) .
hence , complementary studies on atad2 homologues conducted in the yeast s. cerevisiae or other eukaryotic model organisms shall continue to bring more valuable information about the general and conserved functions of atad2-like proteins , part of which may be deregulated in cancer .
|
atad2 , a remarkably conserved , yet poorly characterized factor is found upregulated and associated with poor prognosis in a variety of independent cancers in human .
studies conducted on the yeast saccharomyces cerevisiae atad2 homologue , yta7 , are now indicating that the members of this family may primarily be regulators of chromatin dynamics and that their action on gene expression could only be one facet of their general activity . in this review
, we present an overview of the literature on yta7 and discuss the possibility of translating these findings into other organisms to further define the involvement of atad2 and other members of its family in regulating chromatin structure and function both in normal and pathological situations .
|
INTRODUCTION
ATAD2-LIKE PROTEINS ARE CONSERVED FROM YEAST TO HUMAN
YTA7, THE UNIQUE BUDDING YEAST ATAD2-LIKE PROTEIN, IS A PROBABLE HISTONE CHAPERONE REGULATING TRANSCRIPTION OF HISTONES AND OTHER GENES
YTA7 PROTEIN AND CHROMATIN BOUNDARY ELEMENTS
FROM YTA7 TO ATAD2A: FUNCTIONAL ORTHOLOGS?
CONCLUSION
|
tuberculosis remains the most important infectious disease globally , and mycobacterium tuberculosis is thought to be present in one - third of the world 's population with 810 million new cases of active tuberculosis occurring annually worldwide . in 2013 , an estimated 9.0 million people developed tuberculosis , and 1.5 million died from the disease .
almost all cases of tuberculosis are caused by m. tuberculosis , with m. bovis contributing less than 1.4% of all pulmonary cases outside of africa and ~2.8% of cases in africa with a crude incidence of 7 cases per 100,000 population .
because of the difficulties of chemotherapy , the incidence of multidrug - resistant strains of m. tuberculosis has increased in many areas during recent decades .
this deterioration in the global situation highlights the need for new therapeutic agents against mycobacterial diseases .
recent research has shown that defensins have bactericidal activity against m. tuberculosis and indicate their potential to play a more significant role in tuberculosis control than what was previously considered . protective immunity against mycobacterial infections
. however , an efficient innate immune response may also be important in natural resistance against mycobacterial infection in addition to maintaining longer - term control of bacillary growth during latent infection .
alveolar macrophages and lung epithelial cells are the main cells to first encounter m. tuberculosis during primary infection .
studies of human airway epithelia in vivo or in vitro show that they can generate antimicrobial activity through the production of antimicrobial peptides [ 5 , 6 ] .
in addition , defensins account for a high proportion of the antibacterial activity associated with neutrophil granules [ 7 , 8 ] .
defensins act as a bridge linking innate and acquired immunity largely through their chemotactic properties .
they belong to a family of small ( 35 kda ) cationic cytotoxic and oxygen - independent peptides that are active against a wide spectrum of microorganisms including bacteria , viruses , and fungi .
this review presents a brief summary of their role in immunity with specific reference to human and animal tuberculosis and explores their potential as a novel approach to therapy or prophylaxis .
there are three major classes of defensins expressed by different cells within the vertebrate world ; - , - , and -defensins are differentiated by their structure and antimicrobial activity .
-defensins , which have been identified in humans , monkeys , and several rodent species , are particularly abundant in polymorphonuclear neutrophils ( pmns ) , certain macrophage populations , and paneth cells of the small intestine [ 8 , 10 , 11 ] .
human neutrophil -defensins ( hnps ) 14 constitute ~30% of proteins in the azurophilic granules of pmns . however , against gram - positive bacteria , hnp-1 , hnp-2 , and hnp-3 account for most of the total defensin content and have greater antimicrobial activity than hnp-4 .
in contrast , the potency of hnp-4 against gram - negative bacteria is greater than those of hnp-1 and hnp-2 .
-defensins 5 and 6 are found in paneth cells , the epithelial granulocytes of the small intestine [ 14 , 15 ] .
-defensins show activity in vitro against gram - negative and gram - positive bacteria and fungi that can be modulated by environmental conditions such as redox and ph .
in contrast to -defensins , -defensins are largely expressed in epithelial tissues [ 18 , 19 ] .
the first -defensin was identified in bovine tongue tissue , and -defensins subfamilies have now been reported in primates ( humans and apes ) , bovines , and rodents such as rats and mice [ 6 , 20 ] .
furthermore , pigs and other farm animals including birds express only -defensins [ 2022 ] .
as the most comprehensively studied , -defensins possess the widest taxonomic distribution , including invertebrates and plants , indicating an ancient point of origin .
human -defensin 1 ( hbd-1 ) is expressed constitutively by a number of body systems including the urogenital tract and respiratory tract [ 24 , 25 ] . hbd-2 was discovered in extracts of lesional scales from patients suffering from psoriasis and is expressed by inflamed skin , lung , oral mucosa , and ocular surfaces .
its expression by epithelial cells can be induced by tnf- and interleukin- ( il- ) 1 but also by bacteria .
hbd-3 is expressed mainly by the skin and tonsils , and hbd-4 is expressed by many tissues but is particularly highly expressed in the gastric antrum and testes .
the expression of hbd-1 is constitutive , whereas hbd-24 are inducible and are thought to play a crucial role against bacterial infection as part of the epithelial barrier .
proinflammatory cytokines , bacteria , and fungi have all been found to increase the expression of these defensins in cultured keratinocytes .
-defensins are the only defensin subfamily with a circular structure , which likely originated through mutation of a preexisting -defensin gene in old world monkeys .
they were first found in the monocytes and neutrophils of the rhesus monkey ( macaca mulatta ) and are the least numerous subfamily of mammalian defensins , with only three -defensins recognized ( rhesus theta defensin ( rtd ) 13 ) from studies within leukocytes .
circular defensin isoforms also exist with five identified in peripheral blood leukocytes and four in the bone marrow in the olive baboon ( papio anubis ) .
less is known about the distribution and diversity of -defensins because only 11 different -defensins have been isolated from three species of primates since their discovery in 1999 .
the antimicrobial activity of -defensins is thought to be their natural function , and they bind to and neutralize bacterial toxins [ 36 , 37 ] . for example , the human -defensin retrocyclin-1 and its analogues are active against c. albicans and l. monocytogenes and bind to anthrax lethal toxin . to date , more than 17 human defensins have been reported .
however , many more -defensins have been reported by in silico analysis , and a genome - wide computational search has identified more than 30 human uncharacterized -defensin genes with full biological significances currently undetermined .
the role of defensin chemotactic activity in initiating and regulating the immune response is now well known [ 3032 ] .
they possess chemotactic activities for immature memory t - cells and dendritic cells through the chemokine receptor ccr6 . hbd-2 and hbd-3 can combine with both cpg and host dna to form aggregates that resemble dna nets , which may enhance the intracellular uptake of cpg and self dna and activate plasmacytoid dendritic cells ( pdcs ) to promote dna - induced ifn- production in a toll - like receptor 9- ( tlr9- ) dependent manner .
subcutaneous injections of these complexes showed enhanced infiltration of inflammatory cells at the injection site , indicating a potential pathophysiological role for defensin / dna complexes in contributing to inflammation .
a recent study showed that murine -defensin 2 ( mbd-2 ) immunostaining in tuberculous mice was essentially localized to cells with dendritic morphology located near the mediastinal lymph nodes and showed a high level of gene expression .
this suggests that -defensins may play an important role in the initiation of a th1 response as a link between the innate and adaptive immune responses .
hnps can also promote b- and t - cell interactions by modulating the th1- and th2-type cytokines [ 43 , 44 ] .
the expression and production of defensins are activated by a number of routes including direct recognition of pathogen - associated molecular patterns ( pamps ) , such as lps , by tlr .
this initiates mapk- or nf-b - dependent cascades that culminate in a proinflammatory response involving the secretion of cytokines , chemokines , and defensins , which themselves also have the capacity to induce defensin secretion .
thus , infection of cells with live mycobacteria leads to the induction of tnf and hbd-2 .
defensins are also thought to contribute to the inflammatory processes by inducing histamine release by mast cells and increasing hyperresponsiveness of the airways to histamine .
besides their antimicrobial and potential proinflammatory activities , defensins also display anti - inflammatory roles by binding to c1q to inhibit activation of the classical pathway of complement activation and have been demonstrated to inhibit fibrinolysis .
some defensins are also able to limit the inhibitory action of glucocorticoids on the suppressor functions of t - lymphocytes , which was abolished after adrenalectomy .
the main mode of antibacterial action is the direct lysis of microorganisms through permeabilization of cell membranes either whilst the bacteria are extracellular or after phagocytosis .
it is believed that electrostatic binding between the arginine groups of cationic defensins and membranes rich in anionic phospholipids induces the formation of voltage - regulated channels , which causes the leakage of intracellular metabolites .
some defensins can also bind avidly to membrane glycoproteins , which may be in part responsible for their antiviral activity .
this is supported by the fact that defensins do not have antiviral activity against nonenveloped viruses .
in addition , defensins can bind to polyanionic molecules such as dna via electrostatic interaction after entering the bacterial cell .
since hnps also target genomic dna , inducing single - strand dna breaks , it has been hypothesized that adenosine 5-diphosphate ribosylation might play a regulatory role in the biological properties of arginine - rich hnps .
defensins are highly antibacterial even in micromolar concentrations for both gram - negative and gram - positive bacteria , including mycobacteria .
pathogens that colonize sites where defensins may be present in high concentrations have developed mechanisms to resist their antibacterial activity .
a number of genes responsible for defensin resistance have been identified in different pathogens . a staphylococcal gene , mprf , confers resistance to several antimicrobial peptides including defensins and related genes and has been found in the genomes of several other pathogens such as m. tuberculosis , pseudomonas aeruginosa , and enterococcus faecalis .
mprf reduces the negative charge of the membrane surface and leads to decreased binding of the cationic defensins by modifying phosphatidylglycerol with l - lysine in the membrane lipids . the phop gene in salmonella enterica serovar typhimurium
is also thought to contribute to defensin resistance because a phop mutant shows significantly greater sensitivity to defensins .
potential clinical application of defensins must thus be reviewed in the light of possible development or acquisition of resistance .
however , their nonspecific mode of action suggests that they should show promise in averting the development of resistance .
moreover , studies have demonstrated that resistance is less frequent than that observed for conventional antibiotics [ 5961 ] , and selections for resistance in susceptible strains of m. tuberculosis , pseudomonas aeruginosa , and enterococcus faecalis have failed .
in addition to their toxic effects on microorganisms , there are a number of reports indicating cytotoxicity for eukaryotic cells .
high concentrations of hnps are found in the airway secretions of patients with chronic inflammatory lung disorders .
it has been reported that hnps are cytotoxic to airway epithelial cells and can induce chemokine secretion in several cell types such as macrophages .
other studies showed that hnps are cytotoxic not only against various kinds of human and murine tumor cells but also against a wide range of normal cells , including human endothelial cells , lymphocytes , murine thymocytes , pmns , and spleen cells in a concentration - dependent ( 25100 g / ml ) and time - dependent manner [ 62 , 64 ] .
as cytotoxic molecules , hnps can cooperate with hydrogen peroxide , which is also secreted by activated neutrophils , to affect synergistic cytolytic activity in vitro .
this interaction may contribute to granulocyte - induced cytotoxicity in vivo [ 59 , 65 ] .
some studies showed that the minimum inhibitory concentration and median inhibitory concentration of hnp-1 were 2.5 g / ml and 0.8 g / ml , respectively , which are much lower than the harmful concentrations to normal cells .
this indicates that defensins have a relatively low level of cytotoxicity to normal cells at antimicrobicidal concentrations .
low cytotoxicity in vitro might be due to the presence of fetal bovine serum in the culture media , as serum proteins can protect mammalian target cells .
the inhibitory effect of fetal calf serum ( fcs ) on lysis and binding can be completely accounted for by its content of albumin .
not only could fcs prevent defensin binding , but also it removed membrane - bound defensin molecules from the targets .
several proteins that can bind to defensins have been identified , and some of them may work as defensin carriers for clearance from tissues and blood .
a high concentration of -defensins has been detected in bronchoalveolar lavage ( bal ) samples and pleural fluid from patients suffering from pulmonary tuberculosis , and significant levels of -defensins have been detected in bronchoalveolar lavage fluid from patients with m. avium - intracellulare infection .
moreover , studies of gene expression profiles by microarray of peripheral blood mononuclear cells ( pbmc ) from patients suffering from tuberculosis and m. tuberculosis - infected healthy individuals who had repeated close contact to tuberculosis patients ( such as a nurse , physician , or family member ) and were tuberculin skin test positive showed that the concentrations of effector molecules , -defensins 1 , 3 , and 4 , were upregulated in the diseased patients .
furthermore , mice infected with 1.5 10 cfu of m. tuberculosis h37rv and treated with different doses of hnp-1 injected subcutaneously showed significantly improved clearance of bacilli from the lungs , liver , and spleen .
hnp-1 at 5 g / ml killed m. avium - intracellulare in vitro at the optimal ph for bactericidal activity ( > 5 ) .
the minimal inhibitory concentration ( mic ) of hnp-1 against m. tuberculosis in vitro in one study was 2.5 g / ml , much lower than that reported in a second study ( 25 g / ml ) .
hnp was the first defensin found to be effective against nontuberculous mycobacteria , including m. avium - intracellulare .
hnp-2 and hnp-3 were as effective in killing m. avium - intracellulare in vitro as hnp-1 .
for hnp-5 , the linear peptide derived from its n - terminal fatty acylation can enhance activity against m. tuberculosis almost comparable to the native peptide .
bovine and rabbit defensins have similar or more potent antimycobacterial activity than hnps , especially against m. tuberculosis clinical isolates in vitro [ 72 , 73 ] .
in contrast , another study showed that hnp-13 are not necessarily more effective in killing m. tuberculosis even at a much higher concentration , which may be the result of differing levels of resistance in the individual strains ( m. tuberculosis erdman ) used .
defensins may play a much more significant role in immunity against mycobacteria than what was previously thought .
one can therefore speculate that defensin production , especially by epithelial cells and neutrophils , is likely to be more important early in infection before the establishment of the granuloma . based on its antimicrobial activity and function in host immunity , neutrophil - macrophage cooperation against m. tuberculosis
first , hnps clearly show antimicrobial activity against m. tuberculosis in vitro by increasing the permeability of the mycobacterial cell envelope .
second , in addition to direct antimicrobial activity , hnps secreted by neutrophils recruited into the early lesion are clearly able to act as chemotactic factors , attracting immune cells including macrophages , t - lymphocytes , mast cells , and immature memory t - cells .
third , hnps released by neutrophils recruited in the early lesion can also modulate cytokine production to influence the inflammatory response since tnf- secreted by macrophages may stimulate neutrophil mycobactericidal activity , which might be mediated simultaneously by defensins .
in vitro ,
the capacity of macrophages to control m. tuberculosis growth is improved by transfecting human monocyte - derived macrophages with the hbd-2 gene compared with non - hbd-2-transfected cells .
, hbd-1 also plays a role in immunity against m. tuberculosis by permeabilization of both the mycobacterial cell wall and the cell membrane .
m. tuberculosis infection of endothelial cells in vitro also results in hbd-1 overexpression and profound cytoskeletal rearrangement .
one study found that infection of human limbocorneal fibroblasts with m. tuberculosis , m. abscessus , and m. smegmatis results in overexpression of hbd-13 .
recent studies have emphasized the role of hbd-4 , which can be triggered through the il-1 and vitamin d receptor ( vdr ) pathways in the innate immune defense against m. tuberculosis survival in infected macrophages .
one study showed that intratracheal administration of l - isoleucine into mice infected with the antibiotic - sensitive strain h37rv or a multidrug - resistant clinical isolate can significantly upregulate -defensins 3 and 4 and decrease bacillary loads by inducing their gene expression , demonstrating that it may be possible to use defensins for treating infection by modulating their gene expression .
interestingly , more highly virulent m. bovis strains induce lower levels of murine -defensin 4 ( mbd4 ) expression than strains of lower virulence during many time points of early infection , indicating the ability to suppress induction early in infection in vivo . in experimental tuberculosis ,
expression of mbd3 and mbd4 by airway epithelial cells in the early stages of infection correlated with temporary control of mycobacterial growth .
similarly , high and stable production mbd4 during latent infection is associated with long - term control of mycobacterial proliferation .
the activity of defensins against mycobacteria coupled with their induction by infection suggests that the introduction of defensins either prophylactically or early in infection may affect the course of the disease to the benefit of the host . there
thus exists the potential for the use of defensins as new prophylactic / therapeutic agents against mycobacterial infections .
the effective in vitro activity of a number of defensins against mycobacteria combined with their beneficial chemotactic effects suggests that therapeutic or prophylactic administration of defensins or their induction in the body might lead to improvement in the course of infection and host health . to date
, there are relatively few reports on the effects of defensin administration against m. tuberculosis , but most of these show beneficial effects .
mice transfected with the -defensin 2 gene showed higher survival and lower bacterial burden after challenge .
hnp-1 injected postinfection showed significant time- and dose - dependent clearance of bacilli from lungs , livers , and spleens in mice experimentally infected with m. tuberculosis h37rv .
the hnp-1 administered to mice in that study was significantly less ( 1 and 5 g per mouse ) than the concentration required ( 50 g / ml ) for antimycobacterial activity in vitro .
the higher in vivo potency of hnps is likely due to their immune enhancing effects , such as chemotaxis of t - cells and monocytes .
human defensins are reported to show synergistic activity with antituberculosis drugs , which suggests that they may be a promising adjunct to antituberculosis chemotherapy .
a number of studies have explored the combined effect of defensins and antituberculosis drugs against intracellular mycobacteria . in vitro studies
suggest that hbd-2 is involved in reducing m. tuberculosis growth , and the combination of hnp-1 with antituberculosis drugs ( i.e. , isoniazid and rifampicin ) resulted in a significant reduction ( p < 0.001 ) in mycobacterial load .
hbd-1 has a lower activity against m. tuberculosis , and its combination with isoniazid significantly reduced m. tuberculosis growth in comparison with the peptides or isoniazid alone by permeabilization of both the mycobacterial cell wall and the cell membrane .
moreover , a protective role for -defensin against mycobacterial infection has been reported in human eosinophils .
-defensin released by eosinophils upon stimulation with lipomannan from m. bovis bcg , when used with eosinophil cationic protein , showed a synergistic inhibitory effect on mycobacterial growth inhibition .
since the peculiar mycobacterial cell envelope is considered to contribute to the resistance to conventional antimycobacterial drugs , the combination of hnps and antituberculosis drugs against m. tuberculosis h37rv not only results in increased permeability of both the mycobacterial cell wall and the cell membrane but also increases the access to intracellular targets for antituberculosis drugs .
therefore , antimicrobial peptides are potential adjuncts to chemotherapy together with conventional drugs against tuberculosis .
antimicrobial peptides can be more potent in vivo because of their immune enhancing effects by acting as a chemotactic factor and regulatory factor , interacting with immune cells like t - cells and monocytes and modulating the production of cytokines and inflammation .
studies have shown that m. bovis bcg - induced hbd-2 mrna expression in human epithelial cells can influence protection against m. tuberculosis challenge , and m. bovis bcg cell wall components ( 1830 kda ) can stimulate human pulmonary epithelial cells to express defensins .
it also shows that prime - boost bcg vaccination with -defensin 2 dna vaccines can enhance the activity against m. tuberculosis .
it is known that protection conferred by bcg against tuberculosis is variable and can maintain long - term immunity , and defensins could be important as a component part of this protection against human tuberculosis .
although -defensins have antimicrobial activity against diverse pathogens [ 35 , 93 ] , especially viruses , there is , as yet , no evidence that -defensins are involved in defense against mycobacterial infection .
rivas - santiago et al . found that vitamin d and l - isoleucine can induce the production of defensins by modulating their gene expression [ 82 , 95 ] .
therefore , future studies should focus on the mechanism by which defensin gene expression is modulated . on the other hand , although a large number of studies have discovered the antimycobacterial activity of defensins in vitro , there are fewer studies in vivo , and further study should include the activity of defensins against tuberculosis in vivo . although defensins have been examined for their clinical treatment of infections with no success , defensins have a huge clinical potential , and more research into their application is needed .
defensins are a family of antimicrobial peptides that are abundant amid an array of oxygen - independent antimicrobial proteins and peptides in neutrophil granules and secreted by epithelial cells .
the advantages and disadvantages of the various forms of defensin therapy / prophylaxis against mycobacterial infection of man and animals outlined above indicate that this could be an effective new approach to treatment and prevention of these chronic infections , which are becoming increasingly intractable to chemotherapy .
administration of defensins may have direct effects on the pathogens , stimulate innate and adaptive immunity , or be used synergistically with currently used or new chemotherapeutic agents .
the experimental work with mycobacterial infections combined with their wide spectrum of activity suggests that bacterial infections other than those caused by mycobacteria may also be amenable to this approach .
|
human tuberculosis remains a huge global public health problem with an estimated 1/3rd of the population being infected .
defensins are antibacterial cationic peptides produced by a number of cell types , most notably neutrophil granulocytes and epithelial cells .
all three defensin types ( - , - , and -defensins ) have antibacterial activities , mainly through bacterial membrane permeabilization . defensins are effective against gram - negative and gram - positive bacteria including mycobacteria and are active both intra- and extracellularly .
mycobacterial resistance has never been demonstrated although the mprf gene encoding resistance in staphylococcus aureus is present in the mycobacterium tuberculosis genome .
in addition to their antibacterial effect , defensins are chemoattractants for macrophages and neutrophils .
there are many cases for their use for therapy or prophylaxis in tuberculosis as well . in conclusion
, we propose that there is considerable scope and potential for exploring their use as therapeutic / prophylactic agents and more comprehensive survey of defensins from different species and their bioactivity is timely .
|
1. Introduction
2. Defensins in Man and Animals
3. Mechanism of Action; the Role of Defensins in Immunity
4. Antimycobacterial Activity
5. Antimycobacterial Therapy
6. Future Perspective
7. Conclusion
|
numerical investigations of cosmological spacetimes can be categorized into two broad classes of calculations , distinguished by their computational ( or even philosophical ) goals : 1 ) geometrical and mathematical principles of cosmological models , and 2 ) physical and astrophysical cosmology . in the former ,
the emphasis is on the geometric framework in which astrophysical processes occur , namely the cosmological expansion , shear , and singularities of the many models allowed by the theory of general relativity . in the latter ,
the emphasis is on the cosmological and astrophysical processes in the real or observable universe , and the quest to determine the model which best describes our universe .
the former is pure in the sense that it concerns the fundamental nonlinear behavior of the einstein equations and the gravitational field .
the latter is more complex , since it addresses the composition , organization and dynamics of the universe from the small scales ( fundamental particles and elements ) to the large ( galaxies and clusters of galaxies ) .
however , the distinction is not always so clear , and geometric effects in the spacetime curvature can have significant consequences for the evolution and observation of matter distributions .
any comprehensive model of cosmology must therefore include nonlinear interactions between different matter sources and spacetime curvature .
a realistic model of the universe must also cover large dynamical spatial and temporal scales , extreme temperature and density distributions , and highly dynamic atomic and molecular matter compositions .
in addition , due to all the varied physical processes of cosmological significance , one must draw from many disciplines of physics to model curvature anisotropies , gravitational waves , electromagnetic fields , nucleosynthesis , particle physics , hydrodynamic fluids , etc .
these phenomena are described in terms of coupled nonlinear partial differential equations and must be solved numerically for general inhomogeneous spacetimes .
the situation appears extremely complex , even with current technological and computational advances . as a result
, the codes and numerical methods that have been developed to date are designed to investigate very specific problems with either idealized symmetries or simplifying assumptions regarding the metric behavior , the matter distribution / composition or the interactions among the matter types and spacetime curvature .
it is the purpose of this article to review published numerical cosmological calculations addressing issues from the very early universe to the present ; from the purely geometrical dynamics of the initial singularity to the large scale structure of the universe .
there are three major sections : 2 where a brief overview is presented of various defining events ocurring throughout the history of our universe and in the context of the standard model ; 3 where brief summaries of early universe and fully relativistic cosmological calculations are presented ; and 4 which focuses on structure formation in the post - recombination epoch and on testing cosmological models against observations .
with current observational constraints , the physical state of our universe , as understood in the context of the standard , or friedmann - robertson - lematre - walker ( flrw ) model , can be crudely extrapolated back to the planck epoch 10 seconds after the big bang , beyond which the classical theory of general relativity is invalid due to quantum corrections . at the earliest times ,
the universe was a plasma of relativistic particles consisting of quarks , leptons , gauge bosons , and higgs bosons represented by scalar fields with interaction and symmetry regulating potentials .
it is believed that several spontaneous symmetry breaking ( ssb ) phase transitions transpired in the early universe as it expanded and cooled , including : the grand unification transition ( gut ) at 10 seconds after the big bang ( here , the strong nuclear force split off from the weak and electromagnetic forces .
this also marks an era of inflationary expansion and the origin of matter - antimatter asymmetry through baryon , charge conjugation , and charge + parity violating interactions and nonequilibrium effects . ) ; the electroweak ( ew ) ssb transition at 10 secs ( when the weak nuclear force split from the electromagnetic force ) ; and the chiral ( qcd ) symmetry breaking transition at 10 secs during which quarks condensed into hadrons .
the most stable hadrons ( baryons , or protons and neutrons comprised of three quarks ) survived the subsequent period of baryon - antibaryon annihilations , which continued until the universe cooled to the point at which new baryon - antibaryon pairs could no longer be produced .
a period of primordial nucleosynthesis followed from 10 to 10 secs during which light element abundances were synthesized to form 24% helium with trace amounts of deuterium , tritium , helium-3 , and lithium .
by 10 secs , the matter density became equal to the radiation density , identifying the start of the current matter - dominated era and the beginning of structure formation .
later , at 10 secs ( 3 10 years ) , the free ions and electrons combine to form atoms , decoupling the matter from the radiation field as the universe continued to expand and cool .
this decoupling or post - recombination epoch marks the surface of last scattering and the boundary of the observable ( via photons ) universe . assuming a hierarchical ( cdm - like ) structure formation scenario , the subsequent development of our universe is characterized by the growth of structures with increasing size .
for example , the first stars are likely to form at t 10 years from molecular gas clouds when the jeans mass of the background baryonic fluid is approximately 10
m , as indicated in figure 1 .
this epoch of pop iii star generation is followed by the formation of galaxies at t 10 years and then galaxy clusters .
the solid and dotted lines potentially track the jeans mass of the average baryonic gas component from the recombination epoch at z 10
to the current time .
a residual ionization fraction of nh+/nh 10
following recombination allows for compton interactions with photons to z 200 , during which the jeans mass remains constant at 10
m. the jeans mass then decreases as the universe expands adiabatically until the first collapsed structures form sufficient amounts of hydrogen molecules to trigger a cooling instability and produce pop iii stars at z 20 .
star formation activity can then reheat the universe and raise the mean jeans mass to above 10
m. this reheating could affect the subsequent development of structures such as galaxies and the observed lyman - alpha clouds .
the solid and dotted lines potentially track the jeans mass of the average baryonic gas component from the recombination epoch at z 10
to the current time .
a residual ionization fraction of nh+/nh 10
following recombination allows for compton interactions with photons to z 200 , during which the jeans mass remains constant at 10
m. the jeans mass then decreases as the universe expands adiabatically until the first collapsed structures form sufficient amounts of hydrogen molecules to trigger a cooling instability and produce pop iii stars at z 20 .
star formation activity can then reheat the universe and raise the mean jeans mass to above 10
m. this reheating could affect the subsequent development of structures such as galaxies and the observed lyman - alpha clouds .
the isotropic and homogeneous flrw cosmological model has been so successful in describing the observable universe that it is commonly referred to as the standard model .
furthermore , and to its credit , the model is relatively simple ; it allows for calculations and predictions to be made of the very early universe , including primordial nucleosynthesis at 10 seconds after the big bang , and even particle interactions approaching the planck scale at 10 seconds . at present , observational support for the standard model includes :
the expansion of the universe as verified by the redshifts in galaxy spectra and quantified by measurements of the hubble constant h0 = 100h km sec mpc with hubble parameter 0.5 < h < 1;the deceleration parameter observed in distant galaxy spectra ( although uncertainties about galactic evolution , intrinsic luminosities , and standard candles prevent even a crude estimate);the large scale isotropy and homogeneity of the universe based on temperature anisotropy measurements of the microwave background radiation and peculiar velocity fields of galaxies ( although the light distribution from bright galaxies seems more tenuous);the age of the universe which yields roughly consistent estimates between the look - back time to the big bang in the flrw model and observed data such as the oldest stars , radioactive elements , and cooling of white dwarf stars;the cosmic microwave background radiation suggests that the universe began from a hot big bang and the data is consistent with a black body at temperature 2.7 k;the abundance of light elements such as h , he , he and li , as predicted from the flrw model , are consistent with observations and provides a bound on the baryon density and baryon - to - photon ratio;the present mass density , as determined from measurements of luminous matter and galactic rotation curves , can be accounted for by the flrw model with a single density parameter to specify the metric topology;the distribution of galaxies and larger scale structures can be reproduced by numerical simulations in the context of inhomogeneous perturbations of the flrw models .
the expansion of the universe as verified by the redshifts in galaxy spectra and quantified by measurements of the hubble constant h0 = 100h km sec mpc with hubble parameter 0.5 < h < 1 ; the deceleration parameter observed in distant galaxy spectra ( although uncertainties about galactic evolution , intrinsic luminosities , and standard candles prevent even a crude estimate ) ; the large scale isotropy and homogeneity of the universe based on temperature anisotropy measurements of the microwave background radiation and peculiar velocity fields of galaxies ( although the light distribution from bright galaxies seems more tenuous ) ; the age of the universe which yields roughly consistent estimates between the look - back time to the big bang in the flrw model and observed data such as the oldest stars , radioactive elements , and cooling of white dwarf stars ; the cosmic microwave background radiation suggests that the universe began from a hot big bang and the data is consistent with a black body at temperature 2.7 k ; the abundance of light elements such as h , he , he and li , as predicted from the flrw model , are consistent with observations and provides a bound on the baryon density and baryon - to - photon ratio ; the present mass density , as determined from measurements of luminous matter and galactic rotation curves , can be accounted for by the flrw model with a single density parameter to specify the metric topology ; the distribution of galaxies and larger scale structures can be reproduced by numerical simulations in the context of inhomogeneous perturbations of the flrw models . because of these successes , most work in the field of physical cosmology ( see 4 ) has utilized the standard model as the background spacetime in which the large scale structure evolves , with the ambition to further constrain the cosmological parameters and structure formation scenarios through numerical simulations .
the reader is referred to for a more in - depth review of the standard model .
this section is organized to track the chronological events in the history of the early or relativistic universe ; we focus mainly on four defining moments in our current understanding of the classical picture : 1 ) the big bang singularity and the dynamics of the very early universe ; 2 ) inflation and its generic nature ; 3 ) qcd phase transitions ; and 4 ) primordial nucleosynthesis and the freeze - out of the light elements .
the late or post - recombination epoch is reserved to a separate section 4 .
belinsky , lifshitz and khalatnikov ( blk ) [ 11 , 12 ] and misner discovered that the einstein equations in the vacuum homogeneous bianchi type ix ( or mixmaster ) cosmology exhibit complex behavior and are sensitive to initial conditions as the big bang singularity is approached .
in particular , the solutions near the singularity are described qualitatively by a discrete map [ 10 , 11 ] representing different sequences of kasner spacetimes 1\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$d{s^2 } = - d{t^2 } + { t^{2{p_1}}}d{x^2 } + { t^2}^{{p_2}}d{y^2 } + { t^2}^{{p_3}}d{z^2},$$
\end{document}ds2=dt2+t2p1dx2+t2p2dy2+t2p3dz2 , with time changing exponents pi , but otherwise constrained by \documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}${p_1 } + { p_2 } + { p_3 } = p_1 ^ 2 + p_2 ^ 2 + p_3 ^ 2 = 1$\end{document}p1+p2+p3=p12+p22+p32=1 . because this discrete mapping of kasner epochs is chaotic
mixmaster behavior can be studied in the context of hamiltonian dynamics , with the hamiltonian 2\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$2{\mathcal h } = - p_\omega ^2 + p _ + ^2 + p _ - ^2 + { e^{4\omega}}(v - 1),$$
\end{document}2=p2+p+2+p2+e4(v1 ) , and a semi - bounded potential arising from the spatial scalar curvature ( whose level curves are plotted in figure 2 ) 3\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$v = 1 + { 1 \over 3}{e^{- 8{\beta _ + } } } + { 2 \over 3}{e^{4{\beta _ + } } } \left [ { \cosh ( 4\sqrt 3 { \beta _ - } ) - 1 } \right ] - { 4 \over 3}{e^{- 2{\beta _ + } } } \cosh ( 2\sqrt 3 { \beta _ -}),$$
\end{document}v=1 + 13e8++23e4+[cosh(43)1]43e2+cosh(23 ) , where e and are the scale factor and anisotropies , and p and p are the corresponding conjugate variables .
figure 2contour plot of the bianchi type ix potential v , where
are the anisotropy canonical coordinates .
seven level surfaces are shown at equally spaced decades ranging from 10
to 10 . for large isocontours
( v > 1 ) , the potential is open and exhibits a strong triangular symmetry with three narrow channels extending to spatial infinity . for v
contour plot of the bianchi type ix potential v , where
are the anisotropy canonical coordinates .
seven level surfaces are shown at equally spaced decades ranging from 10
to 10 . for large isocontours
( v > 1 ) , the potential is open and exhibits a strong triangular symmetry with three narrow channels extending to spatial infinity . for v
a solution of this hamiltonian system is an infinite sequence of kasner epochs with parameters that change when the phase space trajectories bounce off the potential walls , which become exponentially steep as the system evolves towards the singularity .
several numerical calculations of the dynamical equations arising from ( 2 ) and ( 3 ) have indicated that the liapunov exponents of the system vanish , in apparent contradiction with the discrete maps [ 17 , 31 ] .
however , it has since been shown that the usual definition of the liapunov exponents is ambiguous in this case as it is not invariant under time reparametrizations , and that with a different time variable one obtains positive exponents [ 13 , 27 ] .
although blk conjectured that local mixmaster oscillations might be the generic behavior for singularities in more general classes of spacetimes , this conjecture has yet to be established .
however , weaver et al . have , through numerical investigations , established evidence that mixmaster dynamics can occur in ( at least a restricted class of ) inhomogeneous spacetimes which generalize the bianchi type vi0 with a magnetic field and two - torus symmetry .
as noted in 3.1.1 , an interesting and ( as yet ) unresolved issue is whether or not the generic cosmological singularity is locally of a mixmaster or blk type , with a complex oscillatory behavior as the singularity is approached .
most of the other bianchi models , including the kasner solutions ( 1 ) , are characterized by either open or no potentials in the hamiltonian framework , and exhibit essentially monotonic or asymptotically velocity term dominated ( avtd ) behavior , the opposite dynamics to the complex blk oscillations .
considering inhomogeneous spacetimes , isenberg and moncrief proved that the singularity in the polarized gowdy model is avtd type .
this has also been conjectured to be the case for more general gowdy models , and numerical simulations of one - dimensional plane symmetric gowdy spacetimes support the notion .
furthermore , a symplectic numerical method has been applied to investigate the avtd conjecture in even more general spacetimes , namely t r spacetimes with u(1 ) symmetry .
although there are concerns about the solutions due to difficulties in resolving the steep spatial gradients near the singularity , the numerical calculations find no evidence of blk oscillations .
berger attributes this to several possibilities : 1 ) the blk conjecture is false ; 2 ) the simulations have not been run long enough ; 3 ) mixmaster behavior is present but hidden in the variables ; or 4 ) the initial data is insufficiently generic . in any case ,
the inflation paradigm is frequently invoked to explain the flatness ( 1 ) of the universe , attributing it to an era of exponential expansion at about 10 seconds after the big bang .
the mechanism of inflation is generally taken to be an effective cosmological constant from the dominant stress - energy of the inflaton scalar field that regulates gut symmetry breaking , particle creation , and the reheating of the universe . in order to study whether inflation can occur for arbitrary anisotropic and inhomogeneous data , many numerical simulations have been carried out with different symmetries , matter types and perturbations .
kurki - suonio et al . extended the planar cosmology code of centrella and wilson [ 22 , 23 ] ( see 3.5 ) to include a scalar field and simulate the onset of inflation in the early universe with an inhomogeneous higgs field and a perfect fluid with a radiation equation of state p = /3 .
their results suggest that whether inflation occurs or not can be sensitive to the shape of the potential . in particular , if the shape is flat enough and satisfies the slow - roll conditions ( essentially upper bounds on v/ and v/ near the false vacuum 0 ) , even large initial fluctuations of the higgs field do not prevent inflation .
they considered two different forms of the potential : a coleman - weinberg type 4\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$v(\phi ) = \lambda { \phi ^4}\left [ { \ln \left({{{{\phi ^2 } } \over { { \sigma ^2 } } } } \right ) - { 1 \over 2 } } \right ] + { { \lambda { \sigma ^4 } } \over 2}$$
\end{document}v()=4[ln(22)12]+42 which is very flat close to the false vacuum and does inflate ; and a rounder
type 5\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$v(\phi ) = \lambda { ( { \phi ^2 } - { \sigma ^2})^2}$$
\end{document}v()=(22)2 which , for their parameter combinations , does not .
goldwirth and piran studied the onset of inflation with inhomogeneous initial conditions for closed , spherically symmetric spacetimes containing a massive scalar field and radiation field sources ( described by a massless scalar field ) .
in all the cases they considered , the radiation field damps quickly and only an inhomogeneous massive scalar field remains to inflate the universe .
they find that small inhomogeneities tend to reduce the amount of inflation and large initial inhomogeneities can even suppress the onset of inflation .
suitable initial values over a domain of several horizon lengths in order for inflation to begin .
investigated the simplest bianchi model ( type v ) background that admits velocities or tilt in order to address the question of how the universe can choose a uniform reference frame at the exit from inflation , since the desitter metric does not have a preferred frame .
however if inflation persists , the wave behavior eventually freezes in and all velocities go to zero at least as rapidly as tanh r , where is the relativistic tilt angle ( a measure of velocity ) , and r is a typical scale associated with the radius of the universe .
their results indicate that the velocities eventually go to zero as inflation carries all spatial variations outside the horizon , and that the answer to the posed question is that memory is retained and the universe is never really desitter .
in addition to the inflaton field , one can consider other sources of inhomogeneity , such as gravitational waves .
although linear waves in desitter space will decay exponentially and disappear , it is unclear what will happen if strong waves exist .
shinkai & maeda investigated the cosmic no - hair conjecture with gravitational waves and a cosmological constant in 1d plane symmetric vacuum spacetimes , setting up gaussian pulse wave data with amplitudes \documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$0.02\lambda \le \max
( \sqrt i ) \le 80\lambda$\end{document}0.02max(i)80 and widths 0.08lh l 2.5lh , where i is the invariant constructed from the 3-riemann tensor and \documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}${l_h } = \sqrt { 3/\lambda}$\end{document}lh=3/ is the horizon scale .
they also considered colliding plane waves with amplitudes \documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$40\lambda \le \max ( \sqrt i ) \le 125\lambda$\end{document}40max(i)125 and widths 0.08lh l 0.1lh .
they find that for any large amplitude and/or small width with inhomogeneity in their parameter sets , the nonlinearity of gravity has little effect , and the spacetime always evolves into a desitter spacetime .
the previous paragraphs discussed results from highly symmetric spacetimes , but the possibility of inflation remains to be established for more general inhomogeneous and nonperturbative data . to address this issue , kurki - suonio et al .
investigated fully three - dimensional inhomogeneous spacetimes with a chaotic inflationary potential v( ) = /4 .
they considered basically two types of runs : small and large scale . in the small scale run
, the grid length was initially set equal to the hubble length so the inhomogeneities are well inside the horizon and the dynamical time scale is shorter than the expansion or hubble time . as a result ,
the perturbations oscillate and damp while the field evolves and the spacetime inflates . in the large scale run ,
they maintain their shape without damping and , because larger values of lead to faster expansion , the inhomogeneity in the expansion becomes steeper in time , since the regions of large and high inflation stay correlated .
the standard picture of cosmology assumes that a phase transition ( associated with chiral symmetry breaking after the electroweak transition ) occurred at approximately 10 seconds after the big bang to convert a plasma of free quarks and gluons into hadrons .
although this transition can be of significant cosmological importance , it is not known with certainty whether it is of first order or higher , and what the astrophysical consequences might be on the subsequent state of the universe .
for example , the transition may give rise to significant baryon number inhomogeneities which can influence the outcome of primordial nucleosynthesis as evidenced in the distribution and averaged light element abundances .
the qcd transition and baryon inhomogeneities may also play a significant and potentially observable role in the generation of primordial magnetic fields .
considered a first order phase transition and the nucleation of hadronic bubbles in a supercooled quark - gluon plasma , solving the relativistic lagrangian equations for disconnected and evaporating quark regions during the final stages of the phase transition .
they numerically investigated a single isolated quark drop with an initial radius large enough so that surface effects can be neglected .
the droplet evolves as a self - similar solution until it evaporates to a sufficiently small radius that surface effects break the similarity solution and increase the evaporation rate .
their simulations indicate that , in neglecting long - range energy and momentum transfer ( by electromagnetically interacting particles ) and assuming that the baryon number is transported with the hydrodynamical flux ) , the baryon number concentration is similar to what is predicted by chemical equilibrium calculations .
kurki - suonio and laine studied the growth of bubbles and the decay of droplets using a spherically symmetric code that accounts for a phenomenological model of the microscopic entropy generated at the phase transition surface . incorporating the small scale effects of the finite wall width and surface tension , but neglecting entropy and baryon flow through the droplet wall ,
they also find that evaporating droplets do not leave behind a global rarefaction wave to dissipate any previously generated baryon number inhomogeneity .
observations of the light elements produced during big bang nucleosynthesis following the quark / hadron phase transition ( roughly 10 10 seconds after the big bang ) are in good agreement with the standard model of our universe ( see 2.2 )
. however , it is interesting to investigate other more general models to assert the role of shear and curvature on the nucleosynthesis process .
rothman and matzner considered primordial nucleosynthesis in anisotropic cosmologies , solving the strong reaction equations leading to he .
they find that the concentration of he increases with increasing shear ; this is due to time scale effects and the competition between dissipation and enhanced reaction rates from photon heating and neutrino blue shifts .
their results have been used to place a limit on anisotropy at the epoch of nucleosynthesis .
kurki - suonio and matzner extended this work to include 30 strong 2-particle reactions involving nuclei with mass numbers a 7 , and to demonstrate the effects of anisotropy on the cosmologically significant isotopes h , he , he and li as a function of the baryon to photon ratio .
they conclude that the effect of anisotropy on h and he is not significant , and the abundances of he and li increase with anisotropy in accord with .
furthermore , it is possible that neutron diffusion , the process whereby neutrons diffuse out from regions of very high baryon density just before nucleosynthesis , can affect the neutron to proton ratio in such a way as to enhance deuterium and reduce he compared to a homogeneous model .
however , plane symmetric , general relativistic simulations with neutron diffusion show that the neutrons diffuse back into the high density regions once nucleosynthesis begins there thereby wiping out the effect . as a result , although inhomogeneities influence the element abundances , they do so at a much smaller degree then previously speculated .
the numerical simulations also demonstrate that , because of the back diffusion , a cosmological model with a critical baryon density can not be made consistent with helium and deuterium observations , even with substantial baryon inhomogeneities and the anticipated neutron diffusion effect .
gravitational waves are an inevitable product of the general einstein equations , and one can expect a wide spectrum of wave signals propagating throughout our universe due to shear anisotropies , primordial metric and matter fluctuations , collapsing matter structures , ringing black holes , and colliding neutron stars , for example .
the discussion here is restricted to the pure vacuum field dynamics and the fundamental nonlinear behavior of gravitational waves in numerically generated cosmological spacetimes .
centrella and matzner [ 20 , 21 ] studied a class of plane symmetric cosmologies representing gravitational inhomogeneities in the form of shocks or discontinuities separating two vacuum expanding kasner cosmologies ( 1 ) . by a suitable choice of parameters ,
the constraint equations can be satisfied at the initial time with an euclidean 3-surface and an algebraic matching of parameters across the different kasner regions that gives rise to a discontinuous extrinsic curvature tensor .
they performed both numerical calculations and analytical estimates using a green s function analysis to establish and verify ( despite the numerical difficulties in evolving discontinuous data ) certain aspects of the solutions , including gravitational wave interactions , the formation of tails , and the singularity behavior of colliding waves in expanding vacuum cosmologies . shortly thereafter , centrella and wilson [ 22 , 23 ] developed a more general plane symmetric code for cosmology , adding also hydrodynamic sources .
in order to simplify the resulting differential equations , they adopted a diagonal 3-metric of the form 6\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$${\gamma _ { ij } } = { a^2}(t , \ , z)\,\,\left({\begin{array}{*{20}c } 1 & 0 & 0 \\ 0 & { { h^2}(t , \ , z ) } & 0 \\ 0 & 0 & 1 \\ \end{array } } \right),$$
\end{document}ij = a2(t , z)(1000h2(t , z)0001 ) ,
which is maintained in time with a proper choice of shift vector .
the metric ( 6 ) allows an overall conformal factor a to simplify the initial value problem , and a dynamical transverse wave component in the variable h. the hydrodynamic equations are solved using artificial viscosity methods for shock capturing and barton s method for monotonic transport . the evolutions are fully constrained ( solving both the momentum and hamiltonian constraints at each time step ) and use the mean curvature slicing condition .
[ 1 , 3 ] , implementing more robust numerical methods and an improved parametric treatment of the initial value problem . in applications of these codes ,
centrella investigated nonlinear gravity waves in minkowski space and compared the full numerical solutions against a first order perturbation solution to benchmark certain numerical issues such as numerical damping and dispersion .
a second order perturbation analysis was also used to model the transition into the nonlinear regime .
considered small and large perturbations in the two degenerate kasner models : p1 = p3 = 0 or 2/3 , and p2 = 1 or 1/3 respectively , where pi are parameters in the kasner metric ( 1 ) . carrying out a second order perturbation expansion and computing the newman - penrose ( np ) scalars , riemann invariants and bel - robinson vector
, they demonstrated , for their particular class of spacetimes , that the nonlinear behavior is in the coulomb ( or background ) part represented by the leading order term in the np scalar and not in the gravitational wave component . for standing - wave perturbations ,
the dominant second order effects in their variables are an enhanced monotonic increase in the background expansion rate , and the generation of oscillatory behavior in the background spacetime with frequencies equal to the harmonics of the first order standing - wave solution . a unique approach to numerical cosmology ( and numerical relativity in general )
is the method of regge calculus in which spacetime is represented as a complex of 4-dimensional , geometrically flat simplices .
the principles of einstein s theory are applied directly to the simplicial geometry to form the curvature , action and field equations , in contrast to the finite difference approach where the continuum field equations are differenced on a discrete mesh .
a 3-dimensional code implementing regge calculus techniques was developed recently by gentle and miller and applied to the kasner cosmological model .
they also describe a procedure to solve the constraint equations for time asymmetric initial data on two spacelike hypersurfaces constructed from tetrahedra , since full 4-dimensional regions or lattices are used .
the new method is analogous to york s procedure where the conformal metric , trace of the extrinsic curvature , and momentum variables are all freely specifiable .
although additional work is needed to apply ( and develop ) regge calculus techniques to more general spacetimes , the early results of gentle and miller are promising .
in particular , their evolutions have reproduced the continuum kasner solution , achieved second order convergence , and sustained numerical stability .
the phrase physical cosmology is generally associated with the large ( galaxy and cluster ) scale structure of the post - recombination epoch where gravitational effects are modeled approximately by newtonian physics on an uniformly expanding , matter dominated flrw background . a discussion of the large scale structure is included in this review since any viable model of our universe which allows a regime where strongly general relativistic effects are important must match onto the weakly relativistic ( or newtonian ) regime .
also , since certain aspects of this regime are directly observable , one can hope to constrain or rule out various cosmological models and/or parameters , including the density ( 0 ) , hubble ( h0 = 100h km sec mpc ) , and cosmological ( ) constants .
due to the vast body of literature on numerical simulations of the post - recombination epoch , it is possible to mention only a small fraction of all the published papers .
hence , the following summary is limited to cover just a few aspects of computational physical cosmology , and in particular those that can potentially be used to discriminate between cosmological model parameters , even within the realm of the standard model .
the cosmic microwave background radiation ( cmbr ) , which is a direct relic of the early universe , currently provides the deepest probe of cosmological structures and imposes severe constraints on the various proposed matter evolution scenarios and cosmological parameters .
although the cmbr is a unique and deep probe of both the thermal history of the early universe and the primordial perturbations in the matter distribution , the associated anisotropies are not exclusively primordial in nature .
important modifications to the cmbr spectrum can arise from large scale coherent structures , even well after the photons decouple from the matter at redshift z 10 , due to the gravitational redshifting of the photons through the sachs - wolfe effect arising from potential gradients [ 49 , 5 ] 7\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$${{\delta t } \over t } = { \phi _ e } - { \phi _ r } - \int\nolimits_r^e { { { 2\overrightarrow l \cdot \nabla \phi } \over a}dt,}$$
\end{document}tt=erre2ladt , where the integral is evaluated from the emission ( e ) to reception ( r ) points along the spatial photon paths \documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$\vec l,\phi$\end{document}l, is the gravitational potential , t / t defines the temperature fluctuations , and a(t ) is the cosmological scale factor in the standard flrw metric . also , if the intergalactic medium ( igm ) reionizes sometime after the decoupling , say from an early generation of stars , the increased rate of thomson scattering off the free electrons will erase sub - horizon scale temperature anisotropies , while creating secondary doppler shift anisotropies . to make meaningful comparisons between numerical models and observed data , all of these effects ( and others ,
see for example 4.1.3 ) must be incorporated self - consistently into the numerical models and to high accuracy in order to resolve the weak signals .
many computational analyses based on linear perturbation theory have been carried out to estimate the temperature anisotropies in the sky ( for example see and the references cited in ) .
although such linearized approaches yield reasonable results , they are not well - suited to discussing the expected imaging of the developing nonlinear structures in the microwave background .
an alternative ray - tracing approach has been developed by anninos et al . to introduce and propagate individual photons through the evolving nonlinear matter structures .
they solve the geodesic equations of motion and subject the photons to thomson scattering in a probabilistic way and at a rate determined by the local density of free electrons in the model .
since the temperature fluctuations remain small , the equations of motion for the photons are treated as in the linearized limit , and the anisotropies are computed according to 8\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$${{\delta t } \over t } = { { \delta z } \over { 1 + z}},$$
\end{document}tt=z1+z , where 9\documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$1 + z = { { { { ( { k^\mu}{u_\mu})}_e } } \over { { { ( { k^\mu}{u_\mu})}_r}}},$$
\end{document}1+z=(ku)e(ku)r , and the photon wave vector k and matter rest frame four - velocity u are evaluated at the emission ( e ) and reception ( r ) points . applying their procedure to a hot dark matter ( hdm ) model of structure formation , anninos et al .
find the parameters for this model are severely constrained by cobe data such that 0h 1 , where 0 and h are the density and hubble parameters . in models where the igm does not reionize , the probability of scattering after the photon - matter decoupling epoch is low , and the sachs - wolfe effect dominates the anisotropies at angular scales larger than a few degrees .
however if reionization occurs , the scattering probability increases substantially and the matter structures , which develop large bulk motions relative to the comoving background , induce doppler shifts on the scattered cmbr photons and leave an imprint of the surface of last scattering .
the induced fluctuations on subhorizon scales in reionization scenarios can be a significant fraction of the primordial anisotropies , as observed by tuluie et al . .
they considered two possible scenarios of reionization : a model that suffers early and gradual ( eg ) reionization of the igm as caused by the photoionizing uv radiation emitted by decaying neutrinos , and the late and sudden ( ls ) scenario as might be applicable to the case of an early generation of star formation activity at high redshifts .
considering the hdm model with 0 = 1 and h = 0.55 , which produces cmbr anisotropies above current cobe limits when no reionization is included ( see 4.1.1 ) , they find that the eg scenario effectively reduces the anisotropies to the levels observed by cobe and generates smaller doppler shift anisotropies than the ls model , as demonstrated in figure 3 .
figure 3the top two images represent temperature fluctuations ( i.e. , t / t ) due to the sachs - wolfe effect and doppler shifts in a standard critically closed hdm model with no reionization and baryon fractions 0.02 ( plate 1 , 4 4 , rms=2.8 10 ) and 0.2 ( plate 2 , 8 8 , rms=3.4 10 ) . the bottom two plates image fluctuations in an early and gradual reionization scenario of decaying neutrinos with baryon fraction 0.02 ( plate 3 , 4 4 , rms=1.3 10 ; and plate 4 , 8 8 , rms = 1.4 10 ) .
the top two images represent temperature fluctuations ( i.e. , t / t ) due to the sachs - wolfe effect and doppler shifts in a standard critically closed hdm model with no reionization and baryon fractions 0.02 ( plate 1 , 4 4 , rms=2.8 10 ) and 0.2 ( plate 2 , 8 8 , rms=3.4 10 ) . the bottom two plates image fluctuations in an early and gradual reionization scenario of decaying neutrinos with baryon fraction 0.02 ( plate 3 , 4 4 , rms=1.3 10 ; and plate 4 , 8 8 , rms = 1.4 10 ) .
the ls scenario of reionization is not able to reduce the anisotropy levels below the cobe limits , and can even give rise to greater doppler shifts than expected at decoupling .
additional sources of cmbr anisotropy can arise from the interactions of photons with dynamically evolving matter structures and nonstatic gravitational potentials .
considered the impact of nonlinear matter condensations on the cmbr in 0 1 cold dark matter ( cdm ) models , focusing on the relative importance of secondary temperature anisotropies due to three different effects : 1 ) time - dependent variations in the gravitational potential of nonlinear structures as a result of collapse or expansion ; 2 ) proper motion of nonlinear structures such as clusters and superclusters across the sky ; and 3 ) the decaying gravitational potential effect from the evolution of perturbations in open models .
they applied the ray - tracing procedure of to explore the relative importance of these secondary anisotropies as a function of the density parameter 0 and the scale of matter distributions .
they find that the secondary temperature anisotropies are dominated by the decaying potential effect at large scales , but that all three sources of anisotropy can produce signatures of order t / t 10 as shown in figure 4 .
figure 4the top two images represent the proper motion and rees - sciama effects in the cmbr for a critically closed cdm model ( upper left ) , together with the corresponding column density of voids and clusters over the same region ( upper right ) . the bottom two images show the secondary anisotropies dominated here by the decaying potential effect in an open cosmological model ( bottom left ) , together with the corresponding gravitational potential over the same region ( bottom right ) .
the rms fluctuations in both cases are on the order of 5 10 , though the open model carries a somewhat larger signature .
the top two images represent the proper motion and rees - sciama effects in the cmbr for a critically closed cdm model ( upper left ) , together with the corresponding column density of voids and clusters over the same region ( upper right ) . the bottom two images show the secondary anisotropies dominated here by the decaying potential effect in an open cosmological model ( bottom left ) , together with the corresponding gravitational potential over the same region ( bottom right ) .
the rms fluctuations in both cases are on the order of 5 10 , though the open model carries a somewhat larger signature .
in addition to the effects discussed in the previous paragraphs , many other sources of secondary anisotropies ( such as gravitational lensing , the vishniac effect accounting for matter velocities and flows into local potential wells , and the sunyaev - zeldovich distortions from the compton scattering of cmb photons in the hot cluster medium ) can also be significant .
see reference for a more complete list and thorough discussion of the different sources of cmbr anisotropies .
observations of gravitational lenses provide measures of the abundance and strength of nonlinear potential fluctuations along the lines of sight to distant objects . since these calculations are sensitive to the gravitational potential , they may be more reliable than galaxy and velocity field measurements as they are not subject to the same ambiguities associated with biasing effects . also , since different cosmological models predict different mass distributions , especially at the higher redshifts , lensing calculations can potentially be used to confirm or discard competing cosmological models . as an alternative to solving the computationally demanding lens equations , cen et al
developed a more efficient scheme to identify regions with surface densities capable of generating multiple images accurately for splittings larger than three arcseconds .
they applied this technique to a standard cdm model with 0 = 1 , and found that this model predicts more large angle splittings ( > 8 ) than are known to exist in the observed universe .
their results suggest that the standard cdm model should be excluded as a viable model of our universe . a similar analysis for a flat low density cdm model with a cosmological constant \documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$({\omega _ 0 } = 0.3,\,\,\lambda/3h_0 ^ 2 = 0{.}7)$\end{document}(0=0.3,/3h02=0.7 ) suggests a lower and perhaps acceptable number of lensing events . however , an uncertainty in their studies is the nature of the lenses at and below the resolution of the numerical grid .
they model the lensing structures as simplified singular isothermal spheres ( sis ) with no distinctive cores .
large angle splittings are generally attributed to larger structures such as clusters of galaxies , and there are indications that clusters have small but finite core radii rcore 20 30h kpc .
core radii of this size can have an important effect on the probability of multiple imaging .
flores and primack considered the effects of assuming two different kinds of splitting sources : isothermal spheres with small but finite core radii \documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$\rho \propto { ( { r^2 } + r_{core}^2)^{- 1}}$\end{document}(r2+rcore2)1 , and spheres with a hernquist density profile r(r + a ) , where rcore 20 30h kpc and a 300h kpc . they find that the computed frequency of large - angle splittings , when using the nonsingular profiles , can potentially decrease by more than an order of magnitude relative to the sis case and can bring the standard cdm model into better agreement with observations .
they stress that lensing events are sensitive to both the cosmological model ( essentially the number density of lenses ) and to the inner lens structure , making it difficult to probe the models until the structure of the lenses , both observationally and numerically , is better known . in cdm cosmogonies ,
the fluctuation spectrum at small wavelengths has a logarithmic dependence at mass scales smaller than 10 solar masses , which indicates that all small scale fluctuations in this model collapse nearly simultaneously in time .
furthermore , the cooling in these fluctuations is dominated by the rotational / vibrational modes of hydrogen molecules that were able to form using the free electrons left over from recombination and the ones produced by strong shock waves as catalysts
. the first structures to collapse may be capable of producing population iii stars and have a substantial influence on the subsequent thermal evolution of the intergalactic medium , as suggested by figure 1 , due to the radiation emitted by the first generation stars as well as supernova driven winds . to know the subsequent fate of the universe and which structures will survive or be destroyed by the uv background , it is first necessary to know when the first stars formed .
ostriker and gnedin have carried out high resolution numerical simulations of the reheating and reionization of the universe due to star formation bursts triggered by molecular hydrogen cooling .
accounting for the chemistry of the primeval hydrogen / helium plasma , self - shielding of the gas , radiative cooling , and a phenomenological model of star formation , they find that two distinct star populations form : the first generation pop iii from h2 cooling prior to reheating at redshift z 14 ; and the second generation pop ii at z < 10 when the virial temperature of the gas clumps reaches 10 k and hydrogen line cooling becomes efficient .
star formation slows in the intermittent epoch due to the depletion of h2 by photo - destruction and reheating .
in addition , the objects which formed pop iii stars also initiate pop ii sequences when their virial temperatures reach 10 k through continued mass accretion .
the lyman - alpha forest represents the optically thin ( at the lyman edge ) component of quasar absorption systems ( qas ) , a collection of absorption features in quasar spectra extending back to high redshifts .
qas are effective probes of the matter distribution and the physical state of the universe at early epochs when structures such as galaxies are still forming and evolving .
although stringent observational constraints have been placed on competing cosmological models at large scales by the cobe satellite and over the smaller scales of our local universe by observations of galaxies and clusters , there remains sufficient flexibility in the cosmological parameters that no single model has been established conclusively .
the relative lack of constraining observational data at the intermediate to high redshifts ( 0 < z < 5 ) , where differences between competing cosmological models are more pronounced , suggests that qas can potentially yield valuable and discriminating observational data .
several combined n - body and hydrodynamic numerical simulations of the lyman forest have been performed recently [ 26 , 41 , 60 ] , and all have been able to fit the observations remarkably well , including the column density and doppler width distributions , the size of absorbers , and the line number evolution . despite the fact that the cosmological models and parameters are different in each case , the simulations give similar results , provided that the proper ionization bias is used ( bion ( bh)/ , where b is the baryonic density parameter , h is the hubble parameter and is the photoionization rate at the hydrogen lyman edge ) .
a theoretical paradigm has thus emerged from these calculations in which lyman - alpha absorption lines originate from the relatively small scale structure in pregalactic or intergalactic gas through the bottom - up hierarchical formation picture in cdm - like universes .
the absorption features originate in structures exhibiting a variety of morphologies commonly found in numerical simulations ( see figure 5 ) , including fluctuations in underdense regions , spheroidal minihalos , and filaments extending over scales of a few megaparsecs .
figure 5distribution of the gas density at redshift z = 3 from a numerical hydrodynamics simulation of the lyman - alpha forest with a cdm spectrum normalized to second year cobe observations , a hubble parameter of h = 0.5 , a comoving box size of 9.6 mpc , and baryonic density of b = 0.06 composed of 76% hydrogen and 24% helium .
the isosurfaces represent baryons at ten times the mean density and are color coded to the gas temperature ( dark blue = 3 10
k , light blue = 3 10
k ) .
the higher density contours trace out isolated spherical structures typically found at the intersections of the filaments . a single random slice through the cube is also shown , with the baryonic overdensity represented by a rainbow - like color map changing from black ( minimum ) to red ( maximum ) .
the he
mass fraction is shown with a wire mesh in this same slice . to emphasize fine structure in the minivoids ,
the mass fraction in the overdense regions has been rescaled by the gas overdensity wherever it exceeds unity .
distribution of the gas density at redshift z = 3 from a numerical hydrodynamics simulation of the lyman - alpha forest with a cdm spectrum normalized to second year cobe observations , a hubble parameter of h = 0.5 , a comoving box size of 9.6 mpc , and baryonic density of b = 0.06 composed of 76% hydrogen and 24% helium .
the isosurfaces represent baryons at ten times the mean density and are color coded to the gas temperature ( dark blue = 3 10
k , light blue = 3 10
k ) .
the higher density contours trace out isolated spherical structures typically found at the intersections of the filaments . a single random slice through the cube is also shown , with the baryonic overdensity represented by a rainbow - like color map changing from black ( minimum ) to red ( maximum ) .
the he
mass fraction is shown with a wire mesh in this same slice . to emphasize fine structure in the minivoids ,
the mass fraction in the overdense regions has been rescaled by the gas overdensity wherever it exceeds unity .
clusters of galaxies are the largest gravitationally bound systems known to be in quasi - equilibrium .
this allows for reliable estimates to be made of their mass as well as their dynamical and thermal attributes
. the richest clusters , arising from 3 density fluctuations , can be as massive as 10 10 solar masses , and the environment in these structures is composed of shock heated gas with temperatures of order 10 10 degrees kelvin which emits thermal bremsstrahlung and line radiation at x - ray energies .
also , because of their spatial size 1h mpc and separations of order 50h mpc , they provide a measure of nonlinearity on scales close to the perturbation normalization scale 8h mpc .
observations of the substructure , distribution , luminosity , and evolution of galaxy clusters are therefore likely to provide signatures of the underlying cosmology of our universe , and can be used as cosmological probes in the easily observable redshift range 0 z 1 .
thomas et al . have investigated the internal structure of galaxy clusters formed in high resolution n - body simulations of four different cosmological models , including standard , open , and flat but low density universes .
they find that the structure of relaxed clusters is similar in the critical and low density universes , although the critical density models contain relatively more disordered clusters due to the freeze - out of fluctuations in open universes at late times .
the profiles of relaxed clusters are very similar in the different simulations , since most clusters are in a quasi - equilibrium state inside the virial radius and follow the universal density profile of navarro et al .
. there does not appear to be a strong cosmological dependence in the profiles , as suggested by previous studies of clusters formed from pure power law initial density fluctuations .
however , because more young and dynamically evolving clusters are found in critical density universes , thomas et al . suggest that it may be possible to discriminate among the density parameters by looking for multiple cores in the substructure of the dynamic cluster population .
they note that a statistical population of 20 clusters can distinguish between open and critically closed universes .
the evolution of the number density of rich clusters of galaxies can be used to compute 0 and 8 ( the power spectrum normalization on scales of 8h mpc ) when numerical simulation results are combined with the constraint \documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}${\sigma _ 8}\omega _ 0^{0.5 } \approx 0.5$\end{document}800.50.5 , derived from observed present - day abundances of rich clusters .
bahcall et al . computed the evolution of the cluster mass function in five different cosmological model simulations and found that the number of high mass ( comalike ) clusters in flat , low 8 models ( i.e. , the standard cdm model with 8 0.5 ) decreases dramatically by a factor of approximately 10 from z = 0 to z 0.5 . for low 0 , high 8 models , the data results in a much slower decrease in the number density of clusters over the same redshift interval . comparing these results to observations of rich clusters in the real universe , which indicate only a slight evolution of cluster abundances to redshifts z 0.5 1
, they conclude that critically closed standard cdm and mixed dark matter ( mdm ) models are not consistent with the observed data . the models which best fit the data are the open models with low bias ( 0 = 0.3 0.1 and 8 = 0.85 0.5 ) , and flat low density models with a cosmological constant ( 0 = 0.34 0.13 and 0 + = 1 ) .
the evolution of the x - ray luminosity function , and the size and temperature distribution of rich clusters of galaxies are all potentially important discriminants of cosmological models .
investigated these properties in a high resolution numerical simulation of a standard cdm model normalized to cobe .
although the results are highly sensitive to grid resolution ( see for a discussion of the effects from resolution constraints on the properties of rich clusters ) , their primary conclusion , that the standard cdm model predicts too many bright x - ray emitting clusters and too much integrated x - ray intensity , is robust , since an increase in resolution will only exaggerate these problems .
cosmological sheets , or pancakes , form as overdense regions collapse preferentially along one axis .
originally studied by zeldovich in the context of neutrino - dominated cosmologies , sheets are ubiquitous features in nonlinear structure formation simulations of cdm - like models with baryonic fluid , and manifest on a spectrum of length scales and formation epochs .
gas collapses gravitationally into flattened sheet structures , forming two plane parallel shock fronts that propagate in opposite directions , heating the infalling gas .
sheets are characterized by essentially five distinct components : the preshock inflow , the postshock heated gas , the strongly cooling / recombination front separating the hot gas from the cold , the cooled postshocked gas , and the unshocked adiabatically compressed gas at the center .
several numerical calculations [ 15 , 51 , 8 ] have been performed of these systems which include the baryonic fluid with hydrodynamical shock heating , ionization , recombination , dark matter , thermal conductivity , and radiative cooling ( compton , bremstrahlung , and atomic line cooling ) , in both one and two spatial dimensions to ascert the significance of each physical process and to compute the fragmentation scale .
in addition , it is well known that gas which cools to 1 ev through hydrogen line cooling will likely cool faster than it can recombine .
this nonequilibrium cooling increases the number of electrons and ions ( compared to the equilibrium case ) which , in turn , increases the concentrations of h and h , the intermediaries that produce hydrogen molecules h2 .
if large concentrations of molecules form , excitations of the vibrational / rotational modes of the molecules can efficiently cool the gas to well below 1 ev , the minimum temperature expected from atomic hydrogen line cooling . because the gas cools isobarically , the reduction in temperature results in an even greater reduction in the jeans mass , and the bound objects which form from the fragmentation of h2 cooled cosmological sheets may be associated with massive stars or star clusters .
anninos and norman have carried out 1d and 2d high resolution numerical calculations to investigate the role of hydrogen molecules in the cooling instability and fragmentation of cosmological sheets , considering the collapse of perturbation wavelengths from 1 mpc to 10 mpc .
they find that for the more energetic ( long wavelength ) cases , the mass fraction of hydrogen molecules reaches h2/nh 3 10 , which cools the gas to 4 10 ev and results in a fragmentation scale of 9 10
m. this represents reductions of 50 and 10 in temperature and jeans mass respectively when compared , as in figure 6 , to the equivalent case in which hydrogen molecules were neglected .
figure 6two different model simulations of cosmological sheets are presented : ( a ) a six species model including only atomic line cooling , and ( b ) a nine species model including also hydrogen molecules . the evolution sequences in the images show the baryonic overdensity and gas temperature at three redshifts following the initial collapse at z = 5 . in each figure , the vertical axis is 32 kpc long ( parallel to the plane of collapse ) and the horizontal axis extends to 4 mpc on a logarithmic scale to emphasize the central structures .
differences in the two cases are observed in the cold pancake layer and the cooling flows between the shock front and the cold central layer .
when the central layer fragments , the thickness of the cold gas layer in the six ( nine ) species case grows to 3 ( 0.3 ) kpc and the surface density evolves with a dominant transverse mode corresponding to a scale of approximately 8(1 ) kpc . assuming a symmetric distribution of matter along the second transverse direction , the fragment masses are approximately 10 ( 10 ) solar masses .
two different model simulations of cosmological sheets are presented : ( a ) a six species model including only atomic line cooling , and ( b ) a nine species model including also hydrogen molecules . the evolution sequences in the images show the baryonic overdensity and gas temperature at three redshifts following the initial collapse at z = 5 . in each figure , the vertical axis is 32 kpc long ( parallel to the plane of collapse ) and the horizontal axis extends to 4 mpc on a logarithmic scale to emphasize the central structures .
differences in the two cases are observed in the cold pancake layer and the cooling flows between the shock front and the cold central layer .
when the central layer fragments , the thickness of the cold gas layer in the six ( nine ) species case grows to 3 ( 0.3 ) kpc and the surface density evolves with a dominant transverse mode corresponding to a scale of approximately 8(1 ) kpc . assuming a symmetric distribution of matter along the second transverse direction , the fragment masses are approximately 10 ( 10 ) solar masses
this review is intended to provide a flavor of the variety of numerical cosmological calculations that have been performed to investigate different phenomena throughout the history of our universe .
the topics discussed range from the strong field dynamical behavior of spacetime geometry at early times near the big bang singularity and the epoch of inflation , to the late time evolution of large scale matter fluctuations and the formation of clusters of galaxies .
although many problems have been addressed , the complexity of the varied physical processes in the extreme time , spatial , and dynamical scales of our universe guarantees that many more interesting and unresolved issues remain . indeed , even the background cosmological model which best describes our universe is essentially unknown .
the topology of our universe , the generic singularity behavior , the fundamental nonlinear field and gravitational wave interactions , the existence and role of a cosmological constant , the correct structure formation scenario , and the origin and spectrum of primordial fluctuations , for example , are uncertain .
however the field of numerical cosmology has matured , in the development of computational techniques , the modeling of microphysics , and in taking advantage of current computing technologies , to the point that it is now possible to perform reliable comparisons of numerical models with observed data .
|
in order to account for the observable universe , any comprehensive theory or model of cosmology must draw from many disciplines of physics , including gauge theories of strong and weak interactions , the hydrodynamics and microphysics of baryonic matter , electromagnetic fields , and spacetime curvature , for example .
although it is difficult to incorporate all these physical elements into a single complete model of our universe , advances in computing methods and technologies have contributed significantly towards our understanding of cosmological models , the universe , and astrophysical processes within them .
a sample of numerical calculations addressing specific issues in cosmology are reviewed in this article : from the big bang singularity dynamics to the fundamental interactions of gravitational waves ; from the quark - hadron phase transition to the large scale structure of the universe .
the emphasis , although not exclusively , is on those calculations designed to test different models of cosmology against the observed universe .
|
Introduction
Background
Relativistic Cosmology
Physical Cosmology
Conclusion
|
damage of the cell membrane system , especially the plasma membrane , is one of the primary events in heavy metal toxic action in plants .
disruption of membrane integrity is thought to be an effect of a complex interaction between heavy metals and functional groups of membranes .
it is well known that metal ions are easily bound to both the sulphhydryl groups of proteins and the hydroxyl part of phospholipids ( devi and prasad , 1999 ) .
they can also replace the calcium ions at essential sites on the cell membranes ( breckle and kahle , 1991 ) .
all those events result in an increase of a non - specific membrane permeability and the parallel decrease of specific transporting activities which disrupt the ionic homeostasis and , subsequently , the activities of many enzymes crucial for basic cell metabolism .
the atp - dependent proton pump of the plasma membrane has a central function in the regulation of ion homeostasis in the cytosol .
this enzyme is encoded by a multigene family ( portillo , 2000 ; arango et al . , 2003 ) and
the expression of at least some isogenes is differentially regulated according to tissue type and developmental stage ( oufattole et al . , 2000 ) .
many studies have also shown changes in the gene expression of the plasma membrane h - atpase in response to a variety of environmental factors , including salt stress ( binzel , 1995 ) , dehydration ( surowy and boyer , 1991 ) , light conditions ( harms et al . , 1994 ) , mechanical stress ( oufattole et al . , 2000 ) , and externally applied hormones ( fras et al . , 1996 ) .
increasing evidence indicates that , besides the genetic regulation of the proton pump , its activity might be fast modulated post - translationally at the protein level , mainly through reversible phosphorylation ( schaller and sussman , 1988 ; portillo , 2000 ) .
this important role in the regulation of the plasma membrane h - atpase has its autoinhibitory domain in the c - terminal region of the enzyme ( jahn et al . ,
1997 ; oecking et al . , 1997 ; baunsgaard et al . , 1998 ;
the c - terminal tail interacts with the catalytic region of the enzyme , limiting its activity ( portillo , 2000 ) .
many data have suggested that the activation of the plant plasma membrane h - atpase is mediated through phosphorylation of the thr-948 in the enzyme , which allows the binding of 14 - 3 - 3 protein ( svennelid et al . , 1999 ) .
this causes displacement of the c - terminal tail and activates the pm h - atpase ( palmgren , 2001 ; arango et al . , 2003 ) .
dephosphorylation of the enzyme protein , mediated by the specific phosphatases belonging to the pp2 and followed by the release of 14 - 3 - 3 protein from the complex , inactivates h - atpase . to date , data concerning heavy metal action on the plasma membrane h - atpase are very limited .
only a few observations have indicated that enzyme activity was decreased under heavy metal stresses ( kennedy and gonsalves , 1989 ; fodor et al . , 1995 ) .
however , no attempts were undertaken to elucidate the mechanisms involved in this inhibition . in this work ,
to explain the mechanism of metal action on the plasma membrane proton pump , the hydrolytic and transporting activities of h - atpase were measured simultaneously with the expression of genes encoding the enzyme .
moreover , the effect of cantharidin , a specific inhibitor of phosphatases , on the inhibitory action of metals on the h - atpase was determined .
since the activity of the plasma membrane proton pump depends on atp , the changes in its level were measured in plants treated with metals .
finally , the accumulation of cadmium , copper , and nickel in cucumber roots was asseyed to estimate to what degree the efficiency of metal absorption may affect the h - atpase activity .
krak ) , germinated in darkness at 25 c for 48 h , were transferred to a nutrient medium for 6 d. the nutrient solution contained 1 mm k2so4 , 0.2 mm ca(h2po4)2.h2o ; 1.5 mm caso4.0.5h2o , 0.3 mm mgso4.7h2o , and microelements 75 m ferric citrate , 10 m mnso4.5h2o , 5 m h3bo4 , 1 m cuso4.5 h2o , 0.01 m znso4.7h2o , 0.05 m na2moo4.2h2o .
after this the plants were exposed for 2 h to the 0.33 mm mes - naoh ( ph 5.5 ) solution without ( control ) or with 10 m cd , cu or ni and 100 m cd , cu or ni .
the plants were grown hydroponically with a 16 h photoperiod ( 180 mol m s ) at 25 c during the day and 22 c during the night .
plasma membrane ( pm ) vesicles were isolated from cucumber root microsomes by phase partitioning according to the procedure by larsson ( 1985 ) and modified by kobus ( 1995 ) . a 16 g phase system containing 6.2% ( w / w ) dextran t500 , 6.2% ( w / w ) polyethylene glycol 3350 ,
330 mm sorbitol , 5 mm kcl , 5 mm btp - mes ( ph 7.5 ) was used .
the plasma membranes obtained by this procedure were mainly composed of right - side - out vesicles and were used to determine the hydrolytic atpase activity .
part of the vesicles was turned to the inside - out oriented form by the method of johansson et al .
( 1995 ) and used for the measurements of atp - dependent h transport in plasma membranes .
the hydrolytic activity of the vanadate - sensitive atpase ( pm - h - atpase ) was determined according to the procedure of gallagher and leonard ( 1982 ) , as modified by sze ( 1985 ) .
the reaction mixture contained 50 g of protein ( plasma membrane ) , 33 mm tris - mes ( ph 7.5 ) , 3 mm atp , 2.5 mm mgso4 , 50 mm kcl , 1 mm nan3 , 0.1 mm na2moo4 , 50 mm nano3 , 200 m na3vo4 , and 0.02% triton x-100 .
pm h - atpase activity was expressed as the difference between the activity measured in the absence and in the presence of na3vo4 .
the pi released during the reaction was determined according to ames ( 1966 ) with 0.2% ( w / v ) sodium dodecyl sulphate included to prevent precipitation ( dulley , 1975 ) .
h transport activity was measured spectrophotometrically as the change in acridine orange absorbance at 495 nm ( a495 ) according to kobus and buczek ( 1995 ) .
the assay medium contained : pm vesicles ( about 50 g of protein ) , 25 mm btp - mes ( ph 7.5 ) , 330 mm sorbitol , 50 mm kcl , 0.1% bsa , 10 m acridine orange , and 0.05% brij 58 .
proton transport was initiated by the addition of 3 mm mg - atp . for every combination , passive proton movement through the membrane
protein was measured according to bradford ( 1976 ) in the presence of 0.02% triton x-100 with bovine serum albumin as the standard .
determination of adenosine triphosphate ( atp ) was made according to glaab and kaiser ( 1999 ) . the fresh root material ( 1 g ) was ground in liquid nitrogen and 5 cm of 4.5% perchloric acid was added and mixed until it thawed . the mixture was then supplemented with 0.125 cm 2 mm tris and centrifuged for 5 min at 5000 g. the ph of the supernatant was adjusted to 7.4 with 5 mol dm k2co3 , and recentrifuged .
firefly luciferin - luciferase assay using a td-20/20 luminometer ( turner designs , usa ) .
total and symplastic metal levels were determined spectrophotometrically ( perkin - elmer 3300 ) in fresh root tissues digested with concentrated hno3 in a microwave .
prior to the determination of the symplastic metal pool after 2 h exposure to cu , cd or ni , the roots were washed with 5 mm pbcl2 at 0 c for 30 min , in order to remove the extracellular bound ions ( harrison et al . , 1979 ) .
the apoplastic pool was calculated from the differences between the total and the symplastic metal level .
total rna was isolated from 50 mg of roots using tri reagent ( sigma ) . to evaluate the expression of the pm - h - atpase gene ( csha3 ) , semi - quantitative rt - pcr analysis with specific primers for each gene
was performed ( titan one tube rt - pcr system , roche , germany ) . as an internal standard
the following specific primers were designed for the pm - h - atpase gene ( cucumber root h - atpase csha3 , accession number ef375892 ) 5-aagtttgtggggttcatgtggaat-3 ( forward primer ) ; 5-gtaagaggaagtgactctccagtc-3 ( reverse primer ) , and for actin the primers were 5-ccgttctgtccctctacgctagtg-3 ( forward primer ) ; 5-ggaactgctctttgcagtctcgag-3 ( reverse primer ) .
for the cdna reaction 18 cycles were run ( uno ii thermoblock , biometra , germany ) , which corresponded to the exponential phase for the pm - h - atpase and actin genes .
each cycle was composed of a 30 s denaturation at 94 c , 30 s of annealing at 59 c , a 2 min 40 s extension at 68 c followed by 7 min of prolonged extension at 68 c .
the gel images were digitally captured with a sony xc - st50ce camera and analysed using the biocapt version 99 program . for western blot analysis
, 20 g of plasma membrane proteins were incubated in sds buffer containing 2% ( w / v ) sds , 80 mm dtt , 40% ( w / v ) glycerol , 5 mm pmsf , 10 mm tris , 1 mm edta , and 0.05% ( w / v ) bromophenol blue for 30 min at room temperature and separated on 7.5% sds - polyacrylamide gels ( laemmli , 1970 ) . after 1 h of electrophoresis at room temperature ( 25 ma ) proteins
were electrotransferred at room temperature ( 60 v , 200 ma ) for 1.5 h to nitrocellulose using a sigma - aldrich sv10-eb10 blotting apparatus .
transfer buffer contained 25 mm tris , 150 mm gly , and 10% ( v / v ) methanol .
to identify the plasma membrane h - atpase , the blots were incubated overnight at 8 c with monoclonal antibody against plasma membrane h - atpase , ( 46e5b11d ) kindly provided by w michalke ( universitt freiburg , germany ) .
after repeated washing the nitrocellulose membrane was incubated at room temperature for 1 h with 1:4000 diluted secondary antibody ( anti - mouse , conjugated to horseradish peroxidase , sigma - aldrich ) and visualized by staining with dab .
phosphorylation of plasma membrane h - atpase was detected with the antibody against phosphothreonine ( rabbit polyclonal to phosphothreonine , abcam ) used at a concentration of 2 g ml after overnight incubation at 8 c .
the membranes were rinsed and incubated for 1 h at room temperature with 5000-fold secondary antibody conjugated to horseradish peroxidase ( goat polyclonal to rabbit igg , abcam ) .
data reported in the figures are the results from at least three experiments performed on independent preparation .
data reported in the figures are the results from at least three experiments performed on independent preparation .
treatment of the cucumber seedlings with heavy metals ( cd , cu and ni ) changed the hydrolytic and transporting activities of the plasma membrane h - atpase ( figs 1 , 2 ) .
the effect of 10 m cd and cu was similar , and both metals decreased hydrolytic activity by about 30% ( fig .
1 , white bars ) , whereas 10 m ni increased the hydrolysis of atp in plasma membrane vesicles by about 30% ( fig .
higher concentrations of every metal ( 100 m ) in nutrient solution caused the distinct inhibition of h - atpase activity ( fig .
the greatest inhibition of atp hydrolysis was observed in plasma membranes isolated from cucumber roots treated with cd ( about 60% ) , while in the case of cu and ni it was 45% and 20% , respectively .
all metals at both concentrations affected the transporting activity of the plasma membrane proton pump in a similar manner ( fig .
effect of cd , cu and ni on the hydrolytic activity of h - atpase in plasma membrane vesicles .
the plasma membrane was isolated from the control roots ( control ) , and roots treated for 2 h with 10 m metals ( white bars ) or with 100 m metals ( grey bars ) .
hydrolytic activity of h - atpase was measured as described in the materials and methods .
effect of cd , cu and ni on the proton transport activities measured in the plasma membrane vesicles .
the plasma membranes ( 50 g of protein ) were isolated from control roots ( control ) , roots treated for 2 h with 10 m ( a , open symbols ) or with 100 m ( b , closed symbols ) of cd , cu or ni . after equilibration of membranes with the reaction medium ( at least for 5 min ) , vesicle acidification was initiated by the addition of mg - atp to give a final concentration of 3 mm .
the formation of ph gradient in the vesicles was monitored as the changes in acridine orange absorbance ( a495 ) .
the values presented are representative for the results obtained in three independent experiments with each experiment done in triplicate .
the results in the inner diagrams ( the steady - state of h - transport ) are means sd from those three independent experiments . the effect of metals on the plasma membrane proton pump could comprise the expression levels of a gene ( mrna ) as well as the enzyme protein level . to verify whether an alteration of enzyme activity under heavy metals was caused by changes in gene expression
, the level of specific transcript of cucumis sativus plasma membrane atpase ( genbank accession number ef375892 ) was determined using rt - pcr approaches .
the transcript level of the constitutively expressed actin gene was used as the internal standard .
3a , c ) and as a ratio of the pm - h - atpase gene signal in the selected line compared with the actin gene signal ( fig .
transcript levels of the csha3 gene in roots treated with 10 m cd , cu and ni were similar to the control , indicating that the metal effect on plasma membrane h - atpase in our short - term experiments did not involve the gene level ( fig .
also , higher concentrations ( 100 m ) of cu and ni did not affect the expression of the csha3 gene , whereas in roots treated with 100 m cd the level of the transcript markedly declined ( fig .
expression of the pm - h - atpase gene in cucumber roots treated with 10 m cd , cu or ni ( a , b ) and 100 m cd , cu or ni ( c , d ) .
total rna was isolated from cucumber roots untreated ( control , white bar ) or treated for 2 h with 10 or 100 m heavy metals ( grey bars ) . to evaluate the expression of pm - h - atpase and actin ( internal standard ) genes , semi - quantitative rt - pcr ( titan one tube rt - pcr system , roche ) with specific primers for each gene was performed .
ethidium bromide - stained bands for the atpase transcripts were quantified with respect to the band of actin .
rt - pcr results are presented as the gel image ( a , c ) and as a ratio of pm - h - atpase gene signal in the selected line to the actin gene signal ( b , d ) .
all experiments were repeated three times independently with comparable results ( sd ) . since the activity of h - atpase
can also be rapidly changed through reversible protein phosphorylation , it was tested if cantharidin , the specific inhibitor of protein phospatases ( pp2a , pp1 ) , could affect the inhibitory effect of metals on the enzyme activity .
when cucumber plants were exposed to 100 m cd , cu and ni in the presence of cantharidin the metal - dependent inhibition was partially diminished ( fig .
4 ) suggesting that the effect of metals could involve dephosphorylation of h - atpase protein .
effect of metals and cantharidin on the hydrolytic activity of h - atpase in plasma membranes of cucumber roots .
the plasma membranes were isolated from the control roots ( control ) , from roots treated for 2 h with 100 m cd , cu and ni , ( white bars ) or from roots treated for 2 h with metals and 50 m cantharidin ( grey bars ) . hydrolytic activity of h - atpase was measured as described in the materials and methods .
data are expressed as a percentage of the enzyme activity determined in plasma membranes of control roots .
the differences in phosphorylation of plasma membrane h - atpase after metal treatment of plants ( 2 h with 100 m cd , cu and ni ) were also confirmed with the antibody against phosphothreonine ( fig .
5b ) . to verify the specificity of the antibody against phosphoamino acids , a control with antibody against plasma membrane h - atpase , 46e5b11d , was used ( fig .
figure 5b shows that the phosphorylation of plasma membrane atpase decreased in plants treated with heavy metals compared with the control .
this result strongly indicates that , under heavy metal stress , dephosphorylation of plasma membrane h - atpase occurs .
western blot of plasma membrane protein ( obtained from control plants or plants treated with 100 m cd , cu , ni ) with antibodies raised against plasma membrane h - atpase -46e5b11d ( a ) , and phosphothreonine ( b ) .
therefore the amount of atp in tissues could be one of the factors limiting an active phospho - enzyme level .
for that reason , atp levels in cucumber roots treated for 2 h with heavy metals were also measured .
in general , atp contents in roots of plants stressed with every metal were lower than in the control ( fig . 6 ) .
plants were exposed for 2 h to the mes - naoh solution without ( control , white bar ) or with 10 m cd , cu or ni ( grey bars ) and 100 m cd , cu or ni ( black bars ) .
the data represent an average ( sd ) of three experiments ; each assay was performed three times .
plasma membrane functions are rapidly altered by heavy metals present in the environment at high concentrations .
the first diagnostic symptom of membrane damage by heavy metals is an increase in its permeability ( fodor et al . , 1995 ) with a subsequent disturbance in the ionic balance of the cell .
it is mainly caused by metal - induced changes in the composition of the membrane lipids and the saturation of fatty acids ( de vos et al . , 1993 ) .
however , the action of metals is much more complex and comprise various events including the oxidation and cross - linking of protein thiols resulting in an inhibition of the key membrane proteins ( harms et al . , 1994 ) .
one of the membrane enzymes which is altered in plants stressed with metals seems to be the h - atpase , the only proton pump operating in plasma membranes and playing a central role in the regulation of ion homeostasis .
it was shown that the hydrolytic activity of h - atpase in roots of different plants was inhibited by cd ( kennedy and gonsalves , 1989 ; fodor et al . , 1995 ) as well as cu ( burzyski and kolano , 2003 ) .
the short - term treatment of cucumber with cd or cu reduced the hydrolysis of atp in the plasma membranes of root cells ( fig .
moreover , the atp - dependent proton transport in membranes isolated from roots stressed with metals was also inhibited in a similar manner ( fig .
the effect of metals on both hydrolytic and transporting activities was dependent on their concentrations in the nutrient solution and correlated well with an accumulation in the symplast of the cells ( table 1 ) .
a similar effect was observed in roots treated with higher concentration of ni ( 100 m ) , whereas lower concentrations of the metal slightly elevated both activities of the plasma membrane proton pump .
comparable observations were made earlier by burzyski and buczek ( 1994 ) and by ros et al .
( 1992 ) for the plasma membrane atpase in cucumber and rice grown with nickel .
interestingly , the action of nickel on the proton pumps was parallel to its effect on plant growth : low concentration of ni stimulated the growth of various crop species , while higher concentrations of the metal inhibited the growth of plants ( brown et al .
since one of the main function of plasma membrane h - atpase is the regulation of growth ( rayle and cleland , 1992 ) , it could be suggested that the enhanced growth of plants subjected to low concentrations of ni and the lowered growth in the presence of higher concentrations of ni is , at least partially , a result of the modification of plasma membrane h - atpase activity by nickel .
( 2005 ) also observed the increase of plasma membrane h - atpase activity when plants were subjected to another metal aluminium .
results from real - time rt - pcr and immunodetection analysis indicated that the stimulation of the plasma membrane proton pump activity by al , in soybean roots , is caused by transcriptional and translational regulation .
total , symplastic and apoplastic metal levels in root tissues after 2 h of cucumber exposition to cd , cu or ni 10 seedlings were incubated in 50 cm of 10 or 100 mol dm of metal chloride solutions ( ph 5.5 ) .
cd , cu or ni levels were determined as described in the materials and methods .
as mentioned above , the effect of metals on the plasma membrane proton pump was correlated with their concentration in the nutrient solution . however ,
possible reasons for the differential action of the metals on enzyme activity , in spite of similarities in their uptake , is not clear . the strongest inhibition of h - atpase was obtained after treatment of plants with cadmium .
there is no correlation between enzyme protein inhibition and lewis acidity of metal ions or when the inhibition by metals is compared with the various ligands ( hydroxyl , carboxyl , phosphoryl , amino , sulphydryl ) present in cells ( filippis , 1979 ) .
heavy metal toxicity can be due to membrane distortion as shown earlier by the altered lipid content ( burzyski and kolano , 2003 ) .
1995 ) have indicated that saturation of fatty acid in the membrane of plants growing with metals increased , whereas the level of sterols significantly decreased . since lipid composition and membrane fluidity
are considered as significant factors regulating the plasma membrane h - atpase ( hernandez et al . , 2002 ) , an inactivation of the proton pump could be the result of the metal - induced changes in the plasma membrane .
inhibition of the plasma membrane proton pump in root cells under heavy metal stress could also result from alteration at the transcriptional as well as the post - transcriptional level .
the plasma membrane h - atpase is encoded by a multigene family and it is generally accepted that the expression of at least some of them is differentially regulated in response to a variety of environmental stresses .
thus it could be assumed that heavy metals also affect the activity of the proton pump through an alteration of specific gene expression .
however , using genetic approaches , it has been proved that , among the metals used in these experiments , only cadmium markedly decreased the level of csha3 mrna .
those observations are in agreement with other findings that show that cd is a very effective modulator ( up- and down - regulation ) of plant gene expression ( fusco et al . , 2005 ; kovalchuk et al . ,
( 2005 ) analysed the global genome expression in plants exposed to cd and have shown that there are at least 65 up - regulated and 338 down - regulated genes .
also in brassica juncea l. , known as a hyperaccumulator plant , 73 transcript - derived fragments were identified as cd - responsive ( fusco et al . , 2005 ) .
among them 52 genes have been identified as encoding proteins with varied physiological functions ( transcriptional factors , expression regulators , stress - responsive proteins , and proteins involved in general metabolism ) .
such results indicate that the response of plants to cd stress at the genetic level is very broad .
the other metals used in our experiments ( cu and ni ) did not change the level of plasma membrane h - atpase transcripts in cucumber roots ( fig .
thus the alteration of the plasma membrane proton pump activity in cucumber roots stressed with those metals seem not to involve the gene expression level .
however , h - atpase is encoded by a gene family and only one isoform was analysed at the transcript level .
evidence has been presented that the plasma membrane h - atpase activity is rapidly modulated through the phosphorylation / dephosphorylation mechanism ( schaller and sussman , 1988 ; kobus and janicka - russak , 2004 , janicka - russak and kobus , 2006 ) .
the activation of the enzyme is due to its phosphorylation followed by binding of 14 - 3 - 3 protein , whereas the dephosphorylation of h - atpase by specific phosphatases belonging to the pp2 , and the release of the 14 - 3 - 3 protein from the complex , inactivates the enzyme . to determine if the metal - induced inhibition of the plasma membrane proton pump in cucumber roots was attributed to its post - translational modification , the effect on the membrane proton pump of cantharidin , a specific inhibitor of protein phosphatases ,
as shown , cantharidin applied together with 100 m cd , cu and ni distinctly diminished the inhibitory effect of the metals ( fig .
4 ) , suggesting that the metals altered the proton pump activity mainly via dephosphorylation of its protein .
the level of h - atpase phosporylation was distinctly lower in plasma membranes from cucumber roots treated with cd , cu or ni ( fig .
5 ) demonstrating that alteration of the plasma membrane proton pump under heavy metals is due to dephosphorylation of the enzyme protein . considering that our plants ( control plants and those treated with heavy metals ) were exposed for 2 h to mes - naoh without ca in solution , a set of supplementary experiments with plants exposed to metals in mes - naoh with or without 200 m ca ( caso4 )
nevertheless no differences were found in both the hydrolytic and the transporting activity of the h - atpase .
also western blot analysis of plasma membranes with the antibody against phosphothreonine showed that the presence of calcium in the solution did not affect the metal action on h - atpase phosphorylation in the short - term experiments presented in this paper ( data not shown ) .
as protein phosphorylation depends on the energetic status of the cell , the level of endogenous atp was measured .
each metal treatment decreased the amount of the adenosine triphosphate in root tissues , suggesting that , besides the dephosphorylation intensity of the proton pump , the atp level is also an important factor in enzyme deactivation under heavy metals stresses .
it is known that the main source of atp in root tissues is mitochondrial respiration . until now
, the effect of heavy metals on respiration and atp content in plant cells has not been sufficiently studied .
however , it was revealed that cd treatment of plants significantly reduced the oxygen consumption by roots ( reese and roberts , 1985 ) as well as disordering the electron transport chain ( kessler and brand , 1994 ) .
thus it is suspected that altered respiration could enhance the inhibition of plasma membrane proton pump activity in plants treated with metals .
there is the possible action of heavy metals on atpase activity through decreasing the energy charge of the tissue .
such a correlation is indicated since the endogenous amount of atp , the substrate for plasma membrane h - atpase , decreased under heavy metal stress .
in addition , it is well known that endogenous atp influences atpase activity , not only as the substrate but also through its function as an activator .
since protein phosphorylation depends on the endogenous atp level , its reduction upon the addition of heavy metals is in line with our hypothesis .
data of experiments with cantharidin and western blot with antibodies against phosphothreonine support this supposition .
we think that some changes in plant cells initiated under heavy metals lead to an increase in the activity of specific phosphatases , responsible for the dephosphorylation of h - atpase protein . taken together
, the data presented suggest that alterations of the plasma membrane proton pump under heavy metal stresses are mainly due to the post - translational modification of its protein . only in the case of cadmium action ,
|
the effect of heavy metals on the modification of plasma membrane h+-atpase ( ec 3.6.3.14 ) activity in cucumber roots was studied . in plants stressed for 2 h with 10 m or 100 m cd , cu or ni the hydrolytic as well as
the transporting activity of h+-atpase in the plasma membranes of root cells was decreased .
transcript levels of cucumis sativus plasma membrane h+-atpase in roots treated with 10 m cd , cu , or ni as well as with 100 m cu or ni were similar to the control , indicating that the action of metals did not involve the gene expression level . only in roots exposed to 100 m cd was the level of plasma membrane h+-atpase mrna markedly decreased .
the inhibition of the plasma membrane proton pump caused by 100 m cd , cu and ni was partially diminished in the presence of cantharidin , a specific inhibitor of protein phosphatases .
western blot analysis with the antibody against phosphothreonine confirmed that decreased activity of plasma membrane h+-atpase under heavy metals resulted from dephosphorylation of the enzyme protein .
taken together , these data strongly indicated that alteration of the enzyme under heavy metal stresses was mainly due to the post - translational modification of its proteins in short - term experiments .
|
Introduction
Materials and methods
Statistics
Results
Discussion
|
a sandwich or laminate technique is one of the methods proposed for composite resin restorations , which was introduced for the first time by mclean , et al . in 1985 . the basic idea behind this technique is to use two different restorative materials for a single restorative procedure so that the maximum physico - mechanical and esthetic properties of these two materials can be exploited simultaneously .
generally , the first component is a layer of conventional or resin - modified glass - ionomer cement which has drawn attention due to its capacity to form an inherent bond with tooth structures and the resultant better seal and decrease in microleakage ( particularly in dentinal walls ) ; it can also release fluoride and decrease the odds of carious lesions .
the second component is a layer of composite materials ( including composite resin , compomer , and ormocer ) , which is used to compensate for the limitations and disadvantages of glass - ionomer cements , including weak mechanical properties and inappropriate esthetic appearance .
it has been reported that the combination of these two materials have different property results in the clinical success of restorations .
the success of the laminate technique depends on the strength of the bond between the glass - ionomer and the resin composite materials in addition to the strength of the bond between the glass - ionomer cement and dentin . however , there is a relatively weak bond strength between conventional glass - ionomers and composite resin materials , mainly because of the lack of a chemical bond between these two materials and the low cohesive strength of glass - ionomer.[35 ] several studies have evaluated different surface preparation techniques for glass - ionomers , such as the use of different bonding systems and surface etching procedures in order to increase the bond strength between these two materials in the laminate technique .
etching the surface with phosphoric acid has yielded different results . in a study carried out by sheth , et al . etching the surface of glass - ionomer had no effect on bond strength increase ; however , an increase in bond strength after acid etching has been reported in another study .
it has also been reported that premature etching of glass - ionomer cement ( before its initial setting reaction ) and failure to use an adhesive resin between the glass - ionomer cement and composite resin increases the odds of restoration failure . in another study , it was demonstrated that the use of a self - etch adhesive system on half - set glass - ionomer cement ( before its initial setting ) increases the bond strength between the glass - ionomer cement and composite resin more than that observed with the use of total - etch systems ( its application after the initial setting of the cement ) .
it has also been reported that the use of a glass - ionomer - based adhesive system applied after the cement 's initial setting improves the bond strength between the glass - ionomer cement and composite resin compared to that with the use of total - etch adhesive systems under similar conditions .
a new group of composite resin materials , giomers , have been introduced in less than a decade , which consist of reacted glass - ionomer fillers in a resin matrix ; they are used in cavities in a manner similar to composite resins with the application of an adhesive system . in addition to appropriate esthetic results , easy polishing , fluoride recharging potential and strength , these materials release fluoride which may enhance their antibacterial effects .
however , no studies to date have evaluated the bond strength between glass - ionomers and giomers ; therefore , the aim of the present study was to evaluate the effect of three surface preparation methods on the shear bond strength of giomer to different surface treated conventional glass - ionomer .
sixty cylindrical specimens were used in the present in vitro study . in order to prepare the samples ,
a plastic mold ( with an inner diameter of 6 mm and a height of 4 mm ) was placed on a glass slab .
then conventional glass - ionomer ( gc fuji ii , gc corporation , tokyo , japan ) was packed into the plastic mold after mixing according to the manufacturer 's instructions .
another glass slide was used on the other side of the mold to make the free surface of the conventional glass - ionomer smooth .
then the samples were randomly divided into three groups of 20 . according to the manufacturer
, the initial setting of gc fuji ii conventional glass - ionomer lasts in 5 minutes and a half , and the net setting time is 2 minutes and a half .
three groups were compared ; a total - etch adhesive resin or a self - etch giomer - based adhesive resin on set glass - ionomer ; or the self - etch giomer - based adhesive resin on the unset glass - ionomer ( before completion of the initial setting ) . in group 1 the surfaces of the specimens were etched with 37% phosphoric acid ( 3 m espe dental products , st .
paul , mn , usa ) for 15 seconds after 5 minutes and 30 seconds from the mixing procedure ; the initial setting reaction was confirmed with the use of a sharp dental explorer .
it should be pointed out that a complete setting of conventional glass - ionomer takes 24 - 72 hours .
after surface etching , a single bond total - etch adhesive system ( 3 m espe dental products , st .
paul , mn , usa ) was applied according to the manufacturer 's instructions and light cured [ table 1 ] .
a second transparent mold , with a diameter of 3 mm and a height of 2 mm , was placed on the conventional glass - ionomer specimen and a giomer restorative , beautifil ii a3-shade ( shofu dental corporation , osaka , japan ) , was packed into the transparent mold and cured using a halogen light - curing unit ( astralis 7 ; ivoclar vivadent , fl-9494 schaan , liechtenstein ) .
the light - directing probe had a diameter of 8 mm and directed a light ray at an intensity of 700 mw / cm perpendicular to the surface , barely touching the surface , for 40 seconds . a light intensity of 700 mw / cm was confirmed using a radiometer before the start of each experimental session .
after transparent mold removal , the specimens were cured for another 20 seconds from each direction .
then the samples were kept in humidity chamber at 37c for 1 hour before being immersed in distilled water at 37c for the next 23 hours . in the next stage ,
a 500-cycle thermocycling procedure was carried out at 5c/55c 5c with a dwell time of 30 seconds and a transfer time of 10 seconds .
chemical composition and application mode of adhesives used in group 2 , the procedures were similar to those in group 1 except that fl - bond ii ( shofu dental corporation , osaka , japan ) self - etch adhesive resin was used on set glass - ionomer surface according to the manufacturer 's instructions and light cured [ table 1 ] , without acid etching . in group 3 ,
all the procedures were similar to those in group 2 except that fl - bond ii adhesive was applied according to the manufacturer 's instructions right after the initial setting and before hardening of glass - ionomer ( 2 minutes and a half after the initiation of mixing , which is equal to the cement 's net setting time ) .
it should be pointed out that during that time the surface hardness of the cement was sufficient to place the second mold without damaging the cement . in order to measure the shear bond strength ,
the samples were mounted in cold - cured acrylic resin from the glass - ionomer side and the shear bond strength values of the samples were measured in newton using a universal testing machine ( h5k - s model , hounsfield test equipment , surrey , uk ) at a crosshead speed of 1 mm / min using a 0.5 mm - wide chisel .
then the shear bond strength values were calculated in mpa by dividing the force ( in newton ) by the surface area ( mm ) of the samples .
data were analyzed by one - way analysis of variance ( anova ) and pairwise comparisons were made by a tukey test using spss/ win.15 .
the failure modes were determined under a stereomicroscope ( smz1500 , nikon , tokyo , japan ) at 20 .
the failure modes were classified as follows : adhesive failure : failure at giomer glass ionomer cement interface .
in addition , to evaluate the interface between the conventional glass - ionomer and giomer in the groups under study , two additional specimens were prepared in each group and subsequent to longitudinal sectioning , they were evaluated under an scanning electron microscope [ sem ] ( tescan , vega ii xmu , brno , czech republic ) [ figure 1 ] .
scanning electron micrographs of conventional glass - ionomer and giomer interface in the study groups ( mag 500 ) .
arrows indicate margins of adhesive resin between glass - ionomer and giomer . in the three scanning electron micrographs ,
the materials which are placed on the right and left sides of the adhesive resin indicate glass - ionomer and giomer , respectively
shear bond strengths in mpa ( means and standard deviations ) for the study groups are represented in table 2 .
there were statistically significant differences in shear bond strength values between the study groups ( p < 0.0005 ) .
pairwise comparisons by a tukey test revealed that there were statistically significant differences in shear bond strengths between group 2 and the two other groups ( p < 0.0005 ) , whereas the bond strength difference between groups 1 and 3 was not statistically significant ( p = 0.609 ) . means and standard deviations ( sd ) of the shear bond strengths ( mpa ) measured in study groups
moreover , all the cohesive failures were within glass - ionomer cement and there were no cohesive failures inside the giomer .
failure modes of the study groups sem photomicrographs of the glass - ionomer and giomer interface in the study groups are represented in figure 1 . in the sem photomicrograph in group 1
[ figure 1a ] the hybrid zone ( the area of adhesive resin penetration into glass - ionomer ) is almost homogeneous but has irregular borders on the glass - ionomer cement side . in the sem photomicrograph in group 2
[ figure 1b ] the hybrid zone is regular and homogenous and in the sem photomicrograph in group 3 [ figure 1c ] the hybrid zone is irregular and non - homogenous .
moreover , the use of self - etch adhesive resin ( fl - bond ii ) in groups 2 and 3 resulted in the thicker hybrid zone than total - etch adhesive resin ( single bond ) in group 1 .
an appropriate bond between glass - ionomer and the superficial resin materials is very important for the success of the sandwich technique . in the present study shear bond strength of conventional glass - ionomer to giomer
was evaluated using three different surface preparations for glass - ionomer ( use of self - etch adhesive on glass - ionomer with or without complete initial setting reaction and use of total - etch adhesive on glass - ionomer after completion of the initial setting reaction ) . in designing the present study ,
the use of total - etch adhesive on glass - ionomer without the completion of the initial setting reaction was not considered , because in total - etch adhesives , rinsing after etching and contamination with moisture before completion of the initial setting reaction of glass - ionomer influences the surface integrity of the cement .
the results of the present study showed that shear bond strength of glass - ionomer to giomer depends on surface preparation . in this context , in the group in which glass - ionomer was set and self - etch adhesive was used , the highest shear bond strength was recorded compared to two other groups , which is consistent with the results of a study carried out by gopikrishna , et al . on glass - ionomer - based adhesives ; in the case of complete setting reaction the bond strength was higher than that in incomplete setting reaction .
contrary to the results of the present study , knight , et al . reported no statistically significant differences in the bond strength before and after initial setting of glass - ionomer in the co - cure technique , where conventional glass - ionomer , resin - modified glass - ionomer ( rmgi ) , and composite resin were placed in consecutive layers before light - curing procedure .
the differences in the results of that study and the present study might be attributed to different etching times in the two studies .
the etching time was 15 seconds in the present study , while in the study done by knight , et al .
it appears that longer etching time paves the way for greater destruction of glass - ionomer surface by the acid and decreases the bond strength .
in addition , a different technique was used in the above - mentioned study , i.e. , a layer of rmgi was used over conventional glass - ionomer before placement of composite resin and the curing process was carried out for rmgi and composite resin simultaneously .
it is well - known that the ph of glass ionomer cements is strongly acidic upon mixing , and it will increase with time , with the most rapid increase during the first 5 - 10 minutes of setting , regardless of the composition of the glass ionomer material . in comparison of groups 2 and 3 , it appears that the acidic ph of unset glass - ionomer prevented complete polymerization of the giomer - based self - etch adhesive and therefore decrease the bond strength .
in addition , this acidic ph might have an adverse effect on the polymerization of giomer itself . in a study carried out by mohamed - tahir , et al .
another study has shown that surface hardness of composite resin placed on polyalkenoate glass ( set for 4 minutes ) significantly decreased . moreover ,
sem observation of the interface in group 3 revealed an inhomogeneous structure and occasionally breakdown of the glass - ionomer surface , which may have affected the bond strength .
polyacrylic acid prevents polymerization of composite resin and results in composite resin softening ; apparently this effect was intensified in incompletely set glass - ionomer . in comparison of groups 1 and 2
, it appears that etching the glass - ionomer surface immediately after the initial setting in group 1 in the total - etch system resulted in the destruction of cement surface , crack formation and decrease in bond strength .
this speculation was confirmed by the sem micrographs of the interface . the irregular interface in group 1 may be an indication of damage and weakening of the glass ionomer surface rather than improved micromechanical interlocking , when compared to group 2 which was treated by a comparatively mild acid , i.e. , the self - etching primer .
several studies have demonstrated a decrease in bond strength of glass - ionomer to composite resin as a result of etching the glass - ionomer surface .
it has been reported that etching during the initial setting reaction of glass - ionomer leads to dissolution of weak calcium - polyacrylate rings , with the resultant deterioration of its physical properties . in the total - etch procedure
however , another reason for a higher bond strength in the self - etch group compared to the total - etch group is the fact that the acidic monomers in the self - etch primer can chemically bond to the calcium in glass - ionomer and increase bond strength .
in addition , a more appropriate compatibility of giomer with the giomer - based self - etch adhesive might be another reason for a higher bond strength in group 2 ; it has been suggested that the use of adhesives compatible with resin - based restorative materials can decrease deleterious chemical interferences .
moreover in the self - etch adhesive group , resin penetration occurs simultaneous with the etching process and it is probable that the discrepancy between these two processes is eliminated or minimized .
another factor that needs to be taken into account is the difference in composition and mechanical properties of the two adhesives ; fl - bond ii is a two - step self - etching adhesive with filler particles while single bond is a mixture of hydrophilic monomers and solvents that contains no fillers .
it has been suggested that the two - step self - etching adhesives that incorporate a hydrophobic resin as a separate bonding agent may have enhance mechanical properties compared to simplified adhesives . moreover , apart from the composition of resin matrix , addition of filler particles enhances the mechanical strength of the bonding layer and contributes to bond strength . in the present study
, no differences were observed in bond strength between groups 1 and 3 , and bond strength in both groups was significantly less than that in group 2 .
regarding failure mode in the present study , the majority of failures were of the cohesive type in glass - ionomer , which is consistent with previous studies .
this phenomenon might be attributed to lower mechanical properties of glass ionomer cements when compared to resin - based materials and the presence of numerous air inclusion bodies inside glass - ionomer , which act as stress concentration points and probably increase the odds of cohesive failure .
a disadvantage of sandwich technique is the absence of a chemical bond between conventional glass - ionomer and superficial resin materials ; therefore , researchers have focused on the establishment of a chemical bond between them .
considering the results of the present study , use of giomer - based self - etch systems not only decreases the time needed for the clinical application but also can result in the establishment of a chemical bond between giomer and conventional glass - ionomer .
taking into account the limitation of this in vitro study , etching the surface of set glass - ionomer with a total - etch system or placement of self - etch adhesive on the surface of glass - ionomer with incomplete initial setting compromised bonding of giomer to glass - ionomer .
|
background : an appropriate bond between glass - ionomer and the superficial resin materials is very important for the success of sandwich technique . the aim of the present in vitro study was to evaluate the effect of three surface treatments of conventional glass - ionomer on its shear bond strength to giomer.materials and methods : sixty cylindrical specimens of a conventional glass - ionomer ( gc fuji ii ) were prepared and randomly divided into three groups ( n = 20 ) .
the specimens in groups 1 and 2 were treated with total - etch adhesive resin ( single bond ) along with acid etching , and self - etch adhesive resin ( fl - bond ii ) on the set glass - ionomer , respectively .
specimens in group 3 were treated with self - etch adhesive resin ( fl - bond ii ) before initial setting of the glass - ionomer was complete .
then a giomer restorative ( beautifil ii ) was added to the specimens .
subsequent to thermocycling , the specimens were subjected to shear bond strength test .
failure modes were evaluated under a stereomicroscope .
data were analyzed by one - way analysis of variance and a post hoc tukey test at a significance level of p < 0.05.results:there were statistically significant differences in bond strengths between the groups ( p < 0.0005 ) .
differences in bond strengths between group 2 and other groups were significant ( p < 0.0005 ) while the differences between groups 1 and 3 were not significant
. failures were predominantly of the cohesive type in all the groups.conclusion:based on the results of this study , the use of self - etch adhesive resin ( fl - bond ii ) on the set glass - ionomer yielded the highest bond strength in the glass - ionomer / giomer sandwich technique .
|
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSION
|
the introduction of acid - etch technique has opened a new era in orthodontic bonding .
the increased adhesion produced by acid pretreatment , using 85% phosphoric acid was demonstrated in 1955 by buonocore.1 in 1965 , with the advent of epoxy resin , newman2 began to apply this to directly bond brackets in orthodontics .
many factors affect the retention of the brackets during fixed orthodontic treatment.3 healthy enamel is also needed for the retention of the bracket , and an altered enamel surface may affect the retention.4 even though product development of adhesives is taking place at a rapid rate , decalcification of enamel around orthodontic brackets , seen clinically as white spot lesions , has remained a neglected part of orthodontic care .
decalcification is defined as loss of calcified tooth substance , and it occurs when the ph of the oral environment favors diffusion of calcium and phosphate ions out of enamel.4 preventing this decalcification that occurs during orthodontic treatment is an important concern because these lesions are unaesthetic , potentially irreversible , and cariogenic .
the most important factors affecting the development of erosion during orthodontic treatment are oral hygiene , nutrition , and orthodontic bonding techniques .
sweets , carbonated fruit drinks , and other dietary acids lower the intraoral ph value below 5.5.4 - 6 factors other than ph , such as type of acid , pka , titrable acidity , buffering capacity , and temperature influence the dental erosive capacity of acidic liquids.7 enamel demineralization can occur , when the ph value in the mouth falls below 5.5 . the ph values of many soft drinks in the market are well below this value.8 soft drinks with their acidic ph and increased adhesion have the potential to demineralize enamel.8 frequent consumption of soft drinks is increasing in children , because of easy availability , increasing promotion , and sales of various commercially available soft drinks . the term soft drinks refer to all drinks except alcohol , mineral water , fruit juice , tea , coffee , or milk - based drinks , which may or may not be carbonated .
they are damaging because they contain high levels of sugar and also due to low ph levels ( ph < 5.5 ) .
soft drinks consumed frequently have been shown to cause extreme dental erosion.9 dental erosion is defined as the acid - induced loss of hard tissue , a chemical process in which bacteria play no part ; and hence , dental erosion is not associated with dental plaque . in an in vivo study
, it was found that the prevalence of dental erosion increased as the ph levels of the studied drinks decreased and as consumption increased .
other studies using scanning electron microscope ( sem ) have shown that soft drinks produce large areas of enamel decalcification .
in general , it is concluded that after immersion in beverages with low ph , the surface microhardness of the teeth is reduced.9 the appearance of white spot lesions caused by the demineralization of tooth enamel is a clinical problem associated with orthodontic treatment .
its prevalence is between 2% and 96% in patients with fixed appliances and is the result of demineralization process occurring around and beneath the brackets due to a decrease in ph .
studies that use sem to evaluate the effect of soft drinks on enamel sealed with orthodontic adhesives have observed areas of enamel showing an adhesive loss after exposure to soft drinks .
this suggests that soft drinks consumption may provoke an increase in micro - leakage beneath brackets and also compromise bond strength.10 studies5,11,12 have demonstrated that saliva forms an important defense mechanism against erosion .
it has been shown that all samples exposed to an erosive solution that were stored in saliva showed less erosion .
hence , this study was undertaken to study the effects of two soft drinks ( coca - cola and mirinda orange ) on the shear bond strength ( sbs ) of brackets and their effect on intact and sealed enamel by means of sem .
this study was conducted to evaluate the effect of two soft drinks , coca - cola and mirinda orange on sbs of orthodontic brackets and on the enamel surface .
one hundred and twenty premolar teeth , freshly extracted for orthodontic purposes , and free from enamel cracks , caries , and fillings were used in this research .
they were further subdivided into three groups : ( table 1 ) sample grouping for testing .
group i ( sbs , n = 60)group ii ( sem analysis with intact enamel [ ie ] , n = 30)group iii ( sem analysis with etched and sealed enamel , n = 30 ) .
group i ( sbs , n = 60 ) group ii ( sem analysis with intact enamel [ ie ] , n = 30 ) group iii ( sem analysis with etched and sealed enamel , n = 30 ) .
the teeth were cleaned in water to remove any traces of blood and then they were placed in 0.1% thymol solution .
subsequently , they were stored in distilled water , which was changed at regular intervals to avoid deterioration
. then teeth in each group were mounted vertically on three different color - coded acrylic boxes for identification ( green - artificial saliva , purple - coca cola , and red - mirinda orange ) .
the brackets were bonded on buccal surfaces with transbond xt according to manufacturer s instructions .
the buccal surfaces were polished with a rubber cup and polishing paste , etched with 37% o - phosphoric acid gel for 30 s , and then washed with water .
a thin layer of transbond xt primer was applied to the tooth and light cured .
the paste was applied to the bracket base and then pressed firmly onto the tooth .
excess adhesive was removed with a probe from around the base of the bracket and then the adhesive was light - cured , positioning the light guide on each interproximal side for 10 s. the specimens were divided randomly into three groups :
subgroup - a : controlthe specimens were submerged in artificial saliva for 15 days , renewing saliva daily.subgroup-b : coca - colathe specimens were submerged in coca - cola for 15 days , for 15 min , 3 times a day , separated by intervals of 2 h. at other times , they were kept in artificial saliva.subgroup-c : mirinda orangethe teeth were submerged in mirinda orange following the same procedure as in subgroup - b .
subgroup - b : coca - cola the specimens were submerged in coca - cola for 15 days , for 15 min , 3 times a day , separated by intervals of 2 h. at other times , they were kept in artificial saliva .
subgroup - c : mirinda orange the teeth were submerged in mirinda orange following the same procedure as in subgroup - b .
while artificial saliva was kept at room temperature , both coca - cola and mirinda orange were stored at a temperature of 5c .
the immersion times and schedules used in previous studies vary widely . in general , specimens were submerged in the soft drinks continuously for long periods,13 - 15 whereas in the present research an immersion schedule was used that would reproduce as closely as possible the situation in vivo . in this way , assuming that these drinks are consumed 3 times a day and that it might take around 45 min to consume one drink , the specimens were submerged in the drinks for 15 min at a time and afterward in artificial saliva , a procedure that was repeated 3 times a day .
the teeth were kept in saliva between immersions in the drinks in order to reproduce normal oral environment conditions and also to allow the possible remineralizing effects of saliva on enamel to take place.16,17 group i : containing 60 specimens was subdivided into three groups , each having 20 teeth which were used to carry out sbs testing , with the universal testing machine .
group ia ( n = 20 ) : 20 samples were color - coded with greengroup ib ( n = 20 ) : 20 samples were color - coded with purplegroup ic ( n = 20 ) : 20 samples were color - coded with red .
group ia ( n = 20 ) : 20 samples were color - coded with green group ib ( n = 20 ) : 20 samples were color - coded with purple group ic ( n = 20 ) : 20 samples were color - coded with red . the percentage of the bracket base surface area covered by adhesive was determined by adhesive remnant index ( ari).18 the ari scale ranges from 1 to 5 :
all of the adhesive remaining on the enamel , with impression of the bracket basemore than 90% of the adhesive remaining on the enamel surface<90% but more than 10% of the adhesive remaining on the enamel surface<10% of the adhesive remaining on the enamel surfaceno adhesive remaining on the enamel surface .
all of the adhesive remaining on the enamel , with impression of the bracket base more than 90% of the adhesive remaining on the enamel surface < 90% but more than 10% of the adhesive remaining on the enamel surface < 10% of the adhesive remaining on the enamel surface no adhesive remaining on the enamel surface .
the 60 teeth for sem study were further divided into two groups :
group ii ie ( n = 30 ) , where the buccal surfaces were polished with rubber cup and polishing pastegroup iii enamel etched and sealed ( es ) ( n = 30 ) with transbond xt primer , where the buccal surfaces were polished with a rubber cup and polishing paste , etched with 37% o - phosphoric acid gel , and primed with transbond xt , which was later light cured for 20 s.
group ii ie ( n = 30 ) , where the buccal surfaces were polished with rubber cup and polishing paste group iii enamel etched and sealed ( es ) ( n = 30 ) with transbond xt primer , where the buccal surfaces were polished with a rubber cup and polishing paste , etched with 37% o - phosphoric acid gel , and primed with transbond xt , which was later light cured for 20 s. of the 30 specimens that made up each group , 10 specimens of each group were placed in artificial saliva , coca - cola , and mirinda orange , respectively .
all specimens were cleaned in distilled water with ultrasonic agitation for 30 min and gently air - dried .
they were then affixed to sem stubs ; sputter coated with gold , and the enamel surface was examined under sem , operating at 20 kv , at 100 magnification .
the specimens were divided randomly into three groups :
subgroup - a : controlthe specimens were submerged in artificial saliva for 15 days , renewing saliva daily.subgroup-b : coca - colathe specimens were submerged in coca - cola for 15 days , for 15 min , 3 times a day , separated by intervals of 2 h. at other times , they were kept in artificial saliva.subgroup-c : mirinda orangethe teeth were submerged in mirinda orange following the same procedure as in subgroup - b .
subgroup - b : coca - cola the specimens were submerged in coca - cola for 15 days , for 15 min , 3 times a day , separated by intervals of 2 h. at other times , they were kept in artificial saliva .
subgroup - c : mirinda orange the teeth were submerged in mirinda orange following the same procedure as in subgroup - b .
while artificial saliva was kept at room temperature , both coca - cola and mirinda orange were stored at a temperature of 5c .
the immersion times and schedules used in previous studies vary widely . in general , specimens were submerged in the soft drinks continuously for long periods,13 - 15 whereas in the present research an immersion schedule was used that would reproduce as closely as possible the situation in vivo . in this way , assuming that these drinks are consumed 3 times a day and that it might take around 45 min to consume one drink , the specimens were submerged in the drinks for 15 min at a time and afterward in artificial saliva , a procedure that was repeated 3 times a day .
the teeth were kept in saliva between immersions in the drinks in order to reproduce normal oral environment conditions and also to allow the possible remineralizing effects of saliva on enamel to take place.16,17
group i : containing 60 specimens was subdivided into three groups , each having 20 teeth which were used to carry out sbs testing , with the universal testing machine .
group ia ( n = 20 ) : 20 samples were color - coded with greengroup ib ( n = 20 ) : 20 samples were color - coded with purplegroup ic ( n = 20 ) : 20 samples were color - coded with red .
group ia ( n = 20 ) : 20 samples were color - coded with green group ib ( n = 20 ) : 20 samples were color - coded with purple group ic ( n = 20 ) : 20 samples were color - coded with red .
the percentage of the bracket base surface area covered by adhesive was determined by adhesive remnant index ( ari).18 the ari scale ranges from 1 to 5 :
all of the adhesive remaining on the enamel , with impression of the bracket basemore than 90% of the adhesive remaining on the enamel surface<90% but more than 10% of the adhesive remaining on the enamel surface<10% of the adhesive remaining on the enamel surfaceno adhesive remaining on the enamel surface .
all of the adhesive remaining on the enamel , with impression of the bracket base more than 90% of the adhesive remaining on the enamel surface < 90% but more than 10% of the adhesive remaining on the enamel surface <
10% of the adhesive remaining on the enamel surface no adhesive remaining on the enamel surface .
the 60 teeth for sem study were further divided into two groups :
group ii ie ( n = 30 ) , where the buccal surfaces were polished with rubber cup and polishing pastegroup iii enamel etched and sealed ( es ) ( n = 30 ) with transbond xt primer , where the buccal surfaces were polished with a rubber cup and polishing paste , etched with 37% o - phosphoric acid gel , and primed with transbond xt , which was later light cured for 20 s.
group ii ie ( n = 30 ) , where the buccal surfaces were polished with rubber cup and polishing paste group iii enamel etched and sealed ( es ) ( n = 30 ) with transbond xt primer , where the buccal surfaces were polished with a rubber cup and polishing paste , etched with 37% o - phosphoric acid gel , and primed with transbond xt , which was later light cured for 20 s. of the 30 specimens that made up each group , 10 specimens of each group were placed in artificial saliva , coca - cola , and mirinda orange , respectively .
all specimens were cleaned in distilled water with ultrasonic agitation for 30 min and gently air - dried .
they were then affixed to sem stubs ; sputter coated with gold , and the enamel surface was examined under sem , operating at 20 kv , at 100 magnification .
the brackets were debonded using the universal testing machine and the sbs was evaluated for all samples .
adhesive that remained after the bracket removal was assessed using magnifying glass and scored according to modified ari proposed by bishara et al.13 following this procedure a sem study was done to compare the surface changes seen on ie surface and etched and sealed enamel . when comparing the groups , higher mean sbs was recorded in group ia group followed by group ib and group ic , respectively .
the difference in mean sbs among the groups was found to be statistically significant ( p < 0.05 ) ( graphs 1 and 2 ) .
higher mean ari score was recorded in group ic followed by group ib and group ia , respectively .
the difference in mean ari among the groups was found to be statistically significant ( p < 0.001 ) ( graph 3 ) .
group ii ( ie ) intact enamel weibull survival analysis showed maximum probability failure of 75 % in group ib [ coca- cola ] and group ic [ mirinda orange ] and a minimum probability failure of 40% in group ia [ control ] .
the results confirm that probability failure of bracket bond strength is significantly less in the control group ia [ artificial saliva ] than the group ib[coca- cola ] and group ic [ mirinda orange ] .
this study further confirms statistically significant decrease in shear bond strength of group ib [ coca- cola ] and group ic [ mirinda orange ] and adequate bond strength in group ia [ control ] ( table 3 ) .
group iia ( control ) : the control group showed a healthier enamel surface ( figure 1 ) . scanning electron microscope : intact enamel ( artificial saliva ) .
group iib ( coca - cola ) : the enamel surface in the test specimens in coca - cola group showed increased surface irregularities , areas of crater - like depression , and extensive erosion pattern ( figure 2 ) . scanning electron microscope : intact enamel ( coca - cola ) .
group iic ( mirinda orange ) : the enamel surface in the test specimens in mirinda orange group showed milder surface irregularities when compared to that of the group iib ( coca - cola ) ( figure 3 ) .
group iii ( etched and sealed enamel ) group iiia ( control ) : the control group showed healthier enamel surface with etched and sealed enamel ( figure 4 ) .
group iiib ( coca - cola ) : the enamel surface showed a greater degree of demineralization and also erosion was seen on the sealed enamel surface ( figure 5 ) .
group iiic ( mirinda orange ) : the enamel surface of the test specimens in mirinda orange group also showed demineralization and mild erosion in the sealed area .
but , the degree of demineralization is less when compared to group iiib ( coca - cola ) ( figure 6 ) .
scanning electron microscope : etched and sealed enamel ( mirinda ) . when comparing group ii ( ie ) and group iii ( etched and sealed enamel ) , group ii ( ie ) showed greater enamel demineralization .
this was , especially more in the coca - cola group than in the mirinda orange group .
group ii ( ie ) intact enamel weibull survival analysis showed maximum probability failure of 75 % in group ib [ coca- cola ] and group ic [ mirinda orange ] and a minimum probability failure of 40% in group ia [ control ] .
the results confirm that probability failure of bracket bond strength is significantly less in the control group ia [ artificial saliva ] than the group ib[coca- cola ] and group ic [ mirinda orange ] .
this study further confirms statistically significant decrease in shear bond strength of group ib [ coca- cola ] and group ic [ mirinda orange ] and adequate bond strength in group ia [ control ] ( table 3 ) .
group iia ( control ) : the control group showed a healthier enamel surface ( figure 1 ) . scanning electron microscope : intact enamel ( artificial saliva ) .
group iib ( coca - cola ) : the enamel surface in the test specimens in coca - cola group showed increased surface irregularities , areas of crater - like depression , and extensive erosion pattern ( figure 2 ) .
group iic ( mirinda orange ) : the enamel surface in the test specimens in mirinda orange group showed milder surface irregularities when compared to that of the group iib ( coca - cola ) ( figure 3 ) .
group iii ( etched and sealed enamel ) group iiia ( control ) : the control group showed healthier enamel surface with etched and sealed enamel ( figure 4 ) .
group iiib ( coca - cola ) : the enamel surface showed a greater degree of demineralization and also erosion was seen on the sealed enamel surface ( figure 5 ) . scanning electron microscope : etched and sealed enamel ( coca - cola ) .
group iiic ( mirinda orange ) : the enamel surface of the test specimens in mirinda orange group also showed demineralization and mild erosion in the sealed area .
but , the degree of demineralization is less when compared to group iiib ( coca - cola ) ( figure 6 ) .
scanning electron microscope : etched and sealed enamel ( mirinda ) . when comparing group ii ( ie ) and group iii ( etched and sealed enamel ) , group ii ( ie ) showed greater enamel demineralization .
this was , especially more in the coca - cola group than in the mirinda orange group .
group ii ( ie ) intact enamel weibull survival analysis showed maximum probability failure of 75 % in group ib [ coca- cola ] and group ic [ mirinda orange ] and a minimum probability failure of 40% in group ia [ control ] .
the results confirm that probability failure of bracket bond strength is significantly less in the control group ia [ artificial saliva ] than the group ib[coca- cola ] and group ic [ mirinda orange ] .
this study further confirms statistically significant decrease in shear bond strength of group ib [ coca- cola ] and group ic [ mirinda orange ] and adequate bond strength in group ia [ control ] ( table 3 ) .
group iia ( control ) : the control group showed a healthier enamel surface ( figure 1 ) . scanning electron microscope : intact enamel ( artificial saliva ) .
group iib ( coca - cola ) : the enamel surface in the test specimens in coca - cola group showed increased surface irregularities , areas of crater - like depression , and extensive erosion pattern ( figure 2 ) .
group iic ( mirinda orange ) : the enamel surface in the test specimens in mirinda orange group showed milder surface irregularities when compared to that of the group iib ( coca - cola ) ( figure 3 ) .
group iii ( etched and sealed enamel ) group iiia ( control ) : the control group showed healthier enamel surface with etched and sealed enamel ( figure 4 ) .
group iiib ( coca - cola ) : the enamel surface showed a greater degree of demineralization and also erosion was seen on the sealed enamel surface ( figure 5 ) . scanning electron microscope : etched and sealed enamel ( coca - cola ) .
group iiic ( mirinda orange ) : the enamel surface of the test specimens in mirinda orange group also showed demineralization and mild erosion in the sealed area .
but , the degree of demineralization is less when compared to group iiib ( coca - cola ) ( figure 6 ) .
scanning electron microscope : etched and sealed enamel ( mirinda ) . when comparing group ii ( ie ) and group iii ( etched and sealed enamel ) , group ii ( ie ) showed greater enamel demineralization .
this was , especially more in the coca - cola group than in the mirinda orange group .
bonding of orthodontic brackets has become an accepted clinical technique since 1970 ( zachrisson , 1994 ) .
bonding has largely replaced banding and is superior to banding in terms of gingival and dental health and esthetics .
the bonding procedure is based on enamel alteration created by acid etching of enamel as developed by buonocore.1 the advantages of direct bonding are easy bracket placement , acceptable clinical success rate , and reduction in chairside time . significant bond failure is reported to vary between 0.5% and 16%.19 - 22 however , the fact is that damage caused to the enamel can not be ignored .
even though many efforts have been made to avoid enamel damage and demineralization , such as crystal growth technique for bonding,23 studies done by it was found that bond failures by other methods are higher when compared with the conventional method .
this irreversible surface enamel demineralization can be further aggravated by the frequent ingestion of soft drinks .
as soft drinks consumption is emerging as a social trend in urban india , asking the patient to avoid consumption of soft drinks is easier said than done .
the term soft drinks refers to all kinds of drinks except alcoholic ones , either carbonated or non - carbonated.9 it consists of acidic content such as phosphoric acid , which reduces the ph of the oral cavity.24 the increased consumption of soft drinks causes enamel erosion.25 the structure of enamel surface is crucial for bracket retention .
any modification of enamel surface may affect this retention.26 in dental caries , loss of minerals from the enamel subsurface occurs due to acids released from the microbial plaque .
in enamel erosion , exposure to extrinsic acids results in demineralization and the factors contributing to the progression of erosion are poor oral hygiene and inadequate bonding procedures.27,28 it is reported that consumption of acidic and alcoholic drinks causes softening of the enamel due to loss of minerals from enamel and can decrease bracket retention.29 - 31 it also causes an increase in the micro - leakage under brackets.28 sixty premolars were selected for this study , as it was easier to obtain an intact tooth of an orthodontic extraction .
the brackets were debonded using a universal testing machine and sbs was evaluated for all samples .
acidic soft drinks may have two concomitant effects on the bond strength of orthodontic brackets .
it can directly deteriorate the structure of adhesive materials , on the other hand , it can cause erosive lesions on the enamel surface around the brackets , and hence decrease the bond strength.21,32,33 the results indicating a minimal difference in sbs among the groups in the present research ( p > 0.05 ) were similar to another study.34 sbs of the coca - cola group was slightly greater than the mirinda orange group .
the results of the present study are contrary to the studies35,36 showing that phosphoric acid - based drinks have lower sbs potential than citric acid - based drinks .
low dose citrate increases the ph and decreases the acidogenicity of the dental plaque , and its use is recommended to reduce the cariogenicity of non - alcoholic soft drinks.37 a study38 showed that soft drinks with a low ph , a phosphoric acid base , and a citric acid base could decrease the sbs of the brackets .
the values obtained by sbs testing for all groups were statistically evaluated by analysis of variance to calculate the p value .
the mean sbs of group ia ( control ) sample was 7.33 1.72 mpa , group ib ( coca- cola ) was 6.24 1.59 mpa , and that of group ic ( mirinda orange ) was 5.30 2.74 mpa .
the lowest mean resistance to shearing forces was shown by mirinda orange group ( 5.30 2.74 mpa ) followed by coca - cola group ( 6.24 1.59 mpa ) and highest resistance to shearing forces was by the control group , i.e. , artificial saliva ( 7.33 1.72 mpa ) . in this study
conducted , ph was critically low for group ib ( 2.5 ) and group ic ( 2.7 ) .
these groups showed significantly lower values of sbs when compared to the control group , i.e. , the artificial saliva ( 6.7 ) . when evaluating sbs of group ib ( coca - cola ) and group ic ( mirinda orange ) it was found that group ib ( coca - cola ) had a greater sbs than group ic ( mirinda orange ) .
weibull survival analysis was done to predict the number of bonds likely to fail at a clinically acceptable strength of 7 mpa .
weibull analysis is a survival analysis , which has the ability to provide reasonably accurate failure analysis and failure forecasts with extremely small samples .
any adhesive that remained after the bracket removal was assessed and scored according to ari proposed by bishara.20 when a bonded bracket is removed , failure at one of the following interfaces must occur : between the bonding material and the bracket , within the bonding material itself , or between the bonding material and the enamel surface .
failure at the enamel surface is not desirable because the bonding material may tear the enamel surface as it pulls away from it .
the interface between the bonding material and the bracket is the usual and preferred site of bracket debonding.39 in this study , after evaluating the ari score , higher mean score was recorded in group ic followed by group ib and group ia , respectively .
the difference in mean ari among the groups was found to be statistically significant ( p < 0.001 ) . in order to find out among which pair of groups there existed a significant difference , a mann whitney
the results showed that difference in mean ari scores was found to be statistically significant between group ia and group ib ( p < 0.05 ) , group ia and group ic ( p < 0.05 ) as well as between group ib and group ic ( p < 0.001 ) .
chi - square test revealed that the association between ari scores and the groups was found to be statistically significant ( p < 0.01 ) .
higher number of samples in group ia had an ari score of 3 and majority of the samples in group ib had an ari score of 4 . in group ic , majority of the samples had an ari score of 5 or 4 .
the phosphoric acid - based coca - cola ( group ib ) and citric acid - based mirinda orange ( group ic ) with significantly low ph showed a greater number of samples with bracket bond failure between the bonding material and enamel surface . in the control ( group ia )
, most specimens showed bracket debonding showed bracket debonding occurring mostly at the interface of bonding material and the bracket which is the most desirable way of debonding .
a study23 compared the erosive capabilities of a citric acid - based orange juice drink and phosphoric acid - based diet cola drink .
it was found that the phosphoric acid - based diet cola had more erosive potential than the citric acid - based juice drink . following this procedure
all samples to be examined were gold - coated and subjected to sem under magnification of 100 . the sem results were interpreted and compared within the groups .
group ii with ie showed the highest level of enamel demineralization in group iib followed by group iic .
group iib , i.e. , the coca - cola drink group showed larger areas of demineralization when compared to group iic , i.e. , the mirinda drink group .
group iii with etched and sealed enamel showed a greater level of enamel demineralization in group iiib followed by group iiic . here , again enamel demineralization was more in coca - cola drink group , i.e. , group iiib when compared to the mirinda orange drink group , i.e. , group iiic .
when coca - cola drink group and mirinda orange drink group were compared with sem , the enamel defects in coca - cola drink group were more extensive and noticeable than the mirinda orange drink group .
this could be because the erosive effect of phosphoric acid in cola group was more than the citric acid in mirinda orange drink group . while mirinda orange drink group was found to be less erosive than coca - cola drink group , yet
the enamel surface of etched and sealed enamel was found to be less erosive with sem when compared to the enamel surface with ie .
thus , among the two soft drinks , coca - cola drink group showed greater erosive potential than the mirinda orange .
but , mirinda orange drink group had lower resistance to shearing forces when compared to coca - cola drink group .
therefore , patients should be advised to consume fewer soft drinks , the choice of drink for the prevention of dental erosion and the frequency of its consumption should be reduced as it decreases the strength of the brackets .
hence , the orthodontist should properly guide the patient and give instructions to improve the efficiency of orthodontic treatment .
the present in vitro study concluded that :
the maximum sbs was seen in the control group ( artificial saliva ) , followed by coca - cola group and the minimum bond strength was recorded in mirinda orange grouphigher mean ari score was recorded in mirinda orange group followed by coca - cola group and with lowest mean ari in control group ( artificial saliva ) , respectively .
the difference in mean ari among the groups was found to be statistically significantwhen comparing group ii ( ie ) and group iii ( etched and sealed enamel ) , group ii ( ie ) showed greater enamel demineralization , which was specially more in the coca - cola group than in the mirinda orange group .
the maximum sbs was seen in the control group ( artificial saliva ) , followed by coca - cola group and the minimum bond strength was recorded in mirinda orange group higher mean ari score was recorded in mirinda orange group followed by coca - cola group and with lowest mean ari in control group ( artificial saliva ) , respectively . the difference in mean ari among the groups
was found to be statistically significant when comparing group ii ( ie ) and group iii ( etched and sealed enamel ) , group ii ( ie ) showed greater enamel demineralization , which was specially more in the coca - cola group than in the mirinda orange group .
|
background and objectives : this study was conducted to evaluate the effect of two soft drinks , coca - cola and mirinda orange on bracket bond strength , on adhesive remnant on teeth after debonding the bracket , and to observe by means of scanning electron microscope ( sem ) the effect of these drinks on intact and sealed enamel.methods:120 non - carious maxillary premolar teeth already extracted for orthodontic purposes were taken and divided into three groups , i.e. , coca - cola drink , mirinda orange , and control ( artificial saliva ) group .
brackets were bonded using conventional methods .
teeth were kept in soft drinks for 15 days , for 15 min , 3 times a day , separated by intervals of 2 h. at other times , they were kept in artificial saliva .
the samples , thus obtained were evaluated for shear bond strength using the universal testing machine and subsequently subjected for adhesive remnant index ( ari ) scores .
sem study on all the three groups was done for evaluating enamel surface of the intact and sealed enamel.results:the lowest mean resistance to shearing forces was shown by mirinda orange group ( 5.30 2.74 mpa ) followed by coca - cola group ( 6.24 1.59 mpa ) and highest resistance to shearing forces by control group ( 7.33 1.72 mpa ) .
the ari scores revealed a cohesive failure in control samples and an adhesive failure in mirinda and cola samples .
sem results showed areas of defect due to erosion caused by acidic soft drinks on intact and sealed enamel surface.conclusion:mirinda group showed the lowest resistance to shearing forces , followed by coca - cola group and with the highest resistance to shearing forces by the control group .
there were significant differences between the control group and the study groups .
areas of defects , which were caused by erosion related to acidic soft drinks on the enamel surface around the adhesive , were seen .
areas of defects caused by coca - cola were more extensive when compared to mirinda orange drink .
|
Introduction
Methods
Storage of test specimens and experimental groups
Bond strength test
Evaluation of adhesive remnant on teeth after debonding
SEM
Results
SEM
Interpretation
Discussion
Conclusion
|
periodontal diseases ( pds ) constitute a series of infections caused by the
microorganisms that colonize sites at or below the gingival margin .
these infections
commonly lead to periodontal inflammation and often result in the destruction of the
supportive periodontal tissues .
the organisms that cause these diseases reside in
unique structures termed biofilms that offer partial protection to the colonizing
organisms from the defense mechanisms of the host , as well as from the
antimicrobials used for treatment .
the
primary goal of conventional periodontal therapy is the alteration of subgingival
biofilms present on periodontally diseased sites that could be associated with the
progressive destruction of the supportive periodontal tissues .
it is well documented that mechanical therapy combined with
oral hygiene instruction is effective in achieving this goal .
comparisons between
manual and power - driven approaches to plaque and calculus removal and their effects
on clinical outcomes show that both are equally effective . over the last 3 decades ,
locally delivered , anti - infective pharmacological agents
have been employed in attempts to treat local bacterial infections associated with
gingivitis and periodontitis .
techniques of the local anti - infective therapy with
pharmacological agents have varied widely , both in the methods of drug delivery and
in the pharmacological agents employed .
drug delivery methods initially included
oral rinses , then irrigation devices , followed by subgingival irrigation using
syringes or powered irrigation devices .
more recently , drug delivery has involved
the incorporation of anti - infective drugs in sustained - release vehicles enabling
subgingival administration of the drug .
these sustained - release vehicles overcome
many of the problems inherent in other drug delivery methods , such as rinsing or
irrigation .
these systems exhibit
shortcomings including inadequate or unpredictable penetration of the periodontal
pocket by the drug , rapid clearance of the drug from the pocket resulting in
inadequate time of the exposure of subgingival bacteria to the drug and poor patient
compliance .
local anti - infective therapy has also employed an eclectic variety
of pharmacological agents from topical antiseptics to broad - spectrum antimicrobials .
chlorhexidine , tetracycline , minocycline , metronidazole , doxycycline , as well as
other antimicrobials and antiseptics have been used in several forms .
two systematic
reviews with the meta - analysis of data from studies evaluating the
effect of locally delivered antimicrobials as an adjunctive therapy to scaling and
root planing ( srp ) revealed evidence of improved clinical outcomes in terms of the
greater pd reduction and/or clinical attachment gain compared to srp alone .
this was
particularly true with the use of tetracyclines , although the clinical significance
remains questionable .
the aim of the present study was to compare the clinical and microbiological effects
of scaling and root planing with hand instruments to a non - surgical treatment with
the use of an ultrasonic device combined with the application of locally delivered
doxycycline .
the subjects for the present cohort study were recruited from the postgraduate clinic
of the department of preventive dentistry , periodontology and implant biology ,
school of dentistry , aristotle university of thessaloniki , greece between september
2004 and march 2005 .
the ethical committee of the school of dentistry of aristotle
university of thessaloniki , greece approved the study protocol and all participating
patients signed an informed consent at the beginning of the study .
the patients ( n = 20 ) comprising the control group were the same as in our previous
study .
another twenty adult patients
with generalised advanced chronic periodontitis were recruited for the study following a screening examination of 28
patients by one examiner ( i.v . ) , which included full mouth probing and radiographic
examination , in order to comprise the control group .
the inclusion and exclusion
criteria were as follows : ( i ) subjects must be adults between 18 and 70 years of age .
( ii ) subjects must have at least 4 sites with initial probing pocket depth ( ppd )
5 mm in at least 2 quadrants , demonstrating bleeding on probing .
( iii ) subjects must not have received any periodontal treatment during the previous 6
months .
clinical examinations were performed at the screening examination , 3 and 6 months
after treatment and involved assessment of ppd , clinical attachment level ( cal ) ,
gingival bleeding index ( gbi ) and plaque
index .
the measurements of ppd and cal
were performed at six sites per tooth with a manual periodontal probe ( hu - friedy
pcp - unc 15 , hu - friedy , chicago , il , usa ) to the nearest millimetre .
prior to the
study , the intra - examiner variability test was carried out to assess the accuracy of
examiner 's measurements . in order to assure the reproducibility of
measurements , all recordings regarding ppd and cal were repeated after a period of
30 minutes . in the event of a difference of > 2 mm between the two measurements ,
the mean of pair of the
two closer measurements was evaluated for further analysis .
the patients fulfilling the necessary prerequisites were assigned into 2 groups of 20
patients each : control group ( scaling and root planing with hand instruments - srp )
and experimental group ( ultrasonic debridement + doxycycline - ud + doxy ) .
at all
time points , the outcomes of research were assessed blind and the examiner ( i.v . )
, k.p . ) who were
also unaware of the treatment that the patient had received ( coded samples ) .
experimental design and treatment procedures at the screening examination , a full mouth measurements of clinical parameters were
recorded and intraoral radiographs were taken . after a period of 1 week ( baseline
examination ) , subgingival plaque samples were taken from 6 preselected sites from
each patient .
the sites were selected according to their initial probing depth and
were divided in 3 categories : ( i ) 2 sites with ppd 4 mm ( shallow pockets ) ,
( ii ) 2 sites with ppd > 4 to 6 mm ( moderate pockets ) and ( iii ) 2 sites
with ppd > 6 mm ( deep pockets ) .
no furcation , endo - periodontic defects or third
molars were included in the study material .
microbiological sampling at the same
sites , as at the baseline examination , and a full mouth clinical recordings were
repeated at 3 and 6 months after the baseline . at the same session ,
supragingival scaling was performed with hand instruments and
ultrasonics , and oral hygiene instructions ( ohi ) were given by the examiner .
the ohi
included twice - daily tooth brushing , using the modified bass technique , and
once - daily inter - dental cleaning with inter - dental brushes .
at the next appointment ,
two weeks after the baseline examination , the allocated intervention was
initiated .
hand instrumentation of the whole
dentition was performed at weekly intervals in three to four sessions by using
gracey curettes under the local anaesthesia ( sg 3/4 , 11/12 , 13/14 and after five
curettes sas 3/4 , 11/12 , 13/14 , hu - friedy pcp - unc 15 , hu - friedy , chicago , il ,
usa ) .
patients of the experimental group received debridement of whole dentition in 3 - 4
sessions , under the local anaesthesia , at weekly intervals , utilizing a
piezoelectric ultrasonic device ( ems piezon , ems , nyon , switzerland ) with a and p
instruments ( swiss instrumentspm , ems , nyon , switzerland ) under water irrigation .
the
teeth were treated until a smooth , appropriately debrided surface was achieved . in
the four deeper , preselected pockets 8.8% doxycycline gel ( atridoxtm , atrix
laboratories , inc . ,
collins , co , usa ) was applied at the completion of therapy
and after the first re - examination at 3 months . in both groups
the endpoint of the smoothness was judged by the supervisor ( i.v . ) ,
who decided upon the completion of root instrumentation by using a periodontal probe
( hu - friedy pcp 11 , hu - friedy , chicago , il , usa ) and an explorer ( hu - friedy
wilkins - tufts 17/23 , hu - friedy , chicago , il , usa ) .
collins , co , usa ) is a
subgingival controlled - release product composed of a two syringe mixing system .
syringe a contains 450 mg of the atrigel delivery system , which is a bioabsorbable ,
low viscosity polymeric formulation composed of 36.7% poly(dl - lactide ) ( pla )
dissolved in a biocompatible carrier of 63.3% n - methyl-2-pyrrolidone ( nmp ) .
the
product , when mixed , is a pale yellow , viscous liquid with a concentration of 8.8%
doxycycline hyclate .
the two syringes are coupled together mixing the two components
for 100 cycles . by the use of 23-gauge cannula attached to the delivery system
the
test product was slowly introduced into the periodontal pocket , starting from the
base of pocket , until it reached the gingival margin .
following withdrawal of the
cannula tip , a curette was used to pack any overflow of the drug down into the
pocket .
no periodontal dressing or adhesive was used . upon contact with the
crevicular fluid , the liquid product solidifies and
the patients were instructed not to perform
oral hygiene measures at the treated areas for 1 week . microbiological evaluation after the isolation with cotton rolls , drying and removal of supragingival plaque ,
the subgingival samples were taken with a sterile gracey curette ( hu - friedy ,
chicago , il , usa ) , were subsequently placed individually in 200 l of te buffer
( tris hcl 10 mm , edta 1 mm , ph = 7.5 ) and stored after the treatment with an alkali
solution ( 0.5 m naoh ) at - 4 oc .
the microbiological samples were evaluated separately for 3 bacterial species using
the " checkerboard " dna - dna hybridisation technique as described by
socransky et al . .
the subgingival
species used for development of digoxigenin - labelled whole genomic probes were
porphyromonas gingivalis ( fdc 381 ) , tannerella forsythia ( fdc 338 ) and treponema
denticola ( td1 ) .
the primary analysis was " per protocol " and included all patients who attended the final
examination .
data were entered into an excel sheet database ( ms office excel 2000 ;
microsoft corporation , redmond , wa , usa ) .
mean and standard error of the mean ( sem )
were calculated for every parameter .
levene 's test for the quality of error
variance was applied in order to check the homogeneity of clinical parameters at the
baseline .
the analysis was made for plaque index , gbi , ppd and cal based on full
mouth measurements ( the third molars were not included ) .
a further analysis was
performed for ppd and cal for the three different categories , according to the
initial pocket depth .
the first category comprised pockets with initial pocket depth
lesser or equal to 4 mm , the second pockets with initial pocket depth > 4 to
6 mm and the third pockets with pocket depth greater than 6 mm .
bacterial species were quantified following the formation of reference curve , which
allowed the conversion of chemiluscent signals to total bacterial counts ( total lab
v2005 , nonlinear dynamics ltd , newcastle upon tyne , uk ) .
the homogeneity of two
groups at the baseline for microbiological parameters was checked using the
mann - whitney test .
averaged bacterial scores from each subject were averaged for
each group and compared at all time - points .
a further comparison at the three
examinations was made for the sites with initial ppd > 4 mm ( moderate and deep
pockets ) , which were those that received the drug in the control group . the differences over time within groups for both clinical and microbiological results
were analysed with the non - parametric test wilcoxon signed ranks .
the comparison
between control and control group was performed using the mann - whitney test .
all statistical analysis was carried
out with the aid of statistical software ( spss version 12.0 , spss inc . ,
at the baseline examination 40 patients entered the study ( 20 in the srp group , 20 in
the ud + doxy group ) , from which 33 subjects completed the 6 month protocol ( 16 in
the srp group , 17 in the ud + doxy group , mean age 50.46 , range 37 - 69 years ) .
four
patients did not return for any re - examination , while another three did not attend
the final examination .
one patient moved , three patients started work , preventing
visits to the clinic and the other three were unwilling to finish the study for
personal reasons .
the flowchart of the patients is illustrated in figure 1 and the characteristics of the patient
sample that completed the study are summarised in table 1 .
the initial statistical analysis revealed no statistical
differences between the two groups at the baseline examination .
the intra - examiner
variability test demonstrated that the reproducibility of the measurements of ppd
and cal within 1 mm was 90% .
epidemiological characteristics of the patient sample ( mean sem , per
protocol analysis ) ppd = probing pocket depth ; cal = clinical attachment level .
an average of 69.1% of all pockets in the srp group and 71.5% in the ud + doxy group
had initially ppd 4 mm .
at the 6
month examination in the srp group the shallow pockets represented 84.9% , the
moderate pockets 11.3% and the deep pockets only 3.8% of all pockets . in the ud +
doxy group
the oral hygiene status , as assessed by the plaque index , during the course of study
is shown in table 2 . at the baseline the mean
full - mouth plaque scores were 88% in the srp group and 81% in the ud + doxy group . a
statistically significant decrease to 26% in the control group and to 37% in the
control group
was recorded at the 6 month examination ( wilcoxon signed ranks test , p
< 0.05 ) .
no statistically significant differences were observed between the two
groups at any interval ( mann - whitney test , p > 0.05 ) .
mean plaque index and gingival bleeding index scores ( mean sem [ mm ] ) at
different examination intervals for control and experimental group
patients statistically significant difference from baseline ( wilcoxon
signed ranks test , p < 0.05 ) .
gingival bleeding index ( gbi ) a statistically significant reduction in gbi scores was observed in both treatment
groups following treatment .
gbi indices also the improved following a similar
pattern in both groups ; at the 6 month re - examination the gbi was reduced from 59%
to 33% in the srp group and from 61% to 32% in the ud + doxy group ( wilcoxon signed
ranks test , p < 0.05 , table 2 ) . no
statistically significant difference in gbi between the two groups was observed at
any examination interval ( mann - whitney test , p > 0.05 ) .
probing pocket depth ( ppd ) changes in the ppd are presented in table 3 .
a marked mean ppd reduction was observed
for both treatment modalities at the 3 month re - examination ( 0.88 mm for the srp and
0.94 mm for the ud + doxy group , wilcoxon signed ranks test , p < 0.05 ) . during
the following observation period ( 3 to 6 month examination )
no statistically significant differences were observed between the two groups
at any interval ( mann - whitney test , p > 0.05 ) .
the ppd measurements were further analysed for the three different categories of
initial pocket depth . for the shallow ( ppd 4 mm ) , the moderate ( > 4 to
6 mm ) and the deep pockets ( ppd > 6 mm )
the results are presented in
table 3 . in the deep pockets an
additional , statistically significant reduction was observed in ppd between 3 and 6
months in the experimental group ( wilcoxon signed ranks test , p < 0.05 , table 3 ) . probing pocket depth ( ppd ) scores for the various ppd categories at
different examination intervals for control and experimental group
patients statistically significant difference from baseline ( wilcoxon
signed ranks test , p < 0.05 ) . statistically significant difference from 3 months ( wilcoxon
signed ranks test , p < 0.05 ) .
no statistically significant differences were observed
between groups ( mann - whitney test , p > 0.05 ) . clinical attachment level ( cal ) a statistically significant improvement of cal was revealed for both groups at the 3
month examination ( 0.50 mm for the srp group and 0.46 mm for the ud + doxy group
respectively , wilcoxon signed ranks test , p < 0.05 , table 4 ) .
statistically significant differences between the two groups
were not recorded at any interval ( mann - whitney test , p > 0.05 ) . in the srp group
the 6 month result remained significantly better compared to the baseline score
( wilcoxon signed ranks test , p < 0.05 ) , whereas in the ud + doxy group the mean
cal score did not demonstrate a significant difference in comparison with the
baseline value ( wilcoxon signed ranks test , p > 0.05 ) .
clinical attachment level scores for the various probing pocket depth
categories at different examination intervals for control and experimental
group patients statistically significant difference from baseline ( wilcoxon
signed ranks test , p < 0.05 ) .
no statistically significant differences were observed
between groups ( mann - whitney test , p > 0.05 )
. a similar analysis as for the ppd measurements was made for the cal measurements as
well .
the results for the shallow , moderate and deep pockets are shown in table 4 .
the differences between 3 and 6 month
re - examinations and the baseline examination were statistically significant for the
two groups ( wilcoxon signed ranks test , p < 0.05 ) , whereas no statistically
significant , inter - group differences were observed ( mann - whitney test , p >
0.05 ) .
the results for all the
investigated species and for all of the six sites per patient are summarised in
table 5 . at the 3 month of examination ,
this decrease was statistically
significant only for porphyromonas gingivalis ( wilcoxon signed ranks test , p <
0.05 ) . at the 6 month re - examination , a statistically significant difference with
baseline was observed in the experimental group for the treponema denticola
( wilcoxon signed ranks test , p < 0.05 ) , attributable to a further reduction in
numbers of this species between the re - examinations .
no statistically significant
differences were found between the two groups at any time interval ( mann - whitney
test , p > 0.05 ) .
mean numbers ( x105 , mean sem ) of the 3 microorganisms ' species
tested in various initial probing pocket depths at different examination
intervals statistically significant difference from baseline ( wilcoxon
signed ranks test , p < 0.05 ) .
pg = porphyromonas gingivalis ; tf = tannerella forsythia ; td =
treponema denticola .
the frequency distribution revealed an increase in the percentage of sites with less
or equal to 105 microorganisms for porphyromonas gingivalis , tannerella forsythia
and treponema denticola and a subsequent decrease in the percentage of sites with
more than 105 microorganisms ( table 6 ) .
frequency distribution ( % ) of sites depending on various microorganisms
species concentration tested at different examination intervals pg = porphyromonas gingivalis ; tf = tannerella forsythia ; td =
treponema denticola .
the results of further analysis that was performed for the sites that had initial ppd
> 4 mm , are presented in table 5 .
a
statistically significant decrease was found for porphyromonas gingivalis in both
groups at the 3 month examination ( wilcoxon signed ranks test , p < 0.05 ) .
no
statistically significant differences were found between the two groups at any time
interval ( mann - whitney test , p > 0.05 ) .
the findings of the present study indicate that the adjunctive use of doxycycline
failed to significantly improve the therapeutic outcome of mechanical treatment . in
a previous study ,
the hand instruments
were compared to the ultrasonics in the treatment of chronic periodontitis , but no
difference was found between the two treatment modalities .
the addition of locally
delivered doxycycline in the treatment protocol of present study did not seem to
offer any beneficial effect to the patients treated with ultrasonic debridement in
comparison to the conventional hand instrumentation alone . a marked reduction in every clinical parameter was observed for both treatment
modalities in this study .
overall a statistically significant mean reduction in ppd
of 0.88 mm and a mean cal gain of 0.59 mm was recorded for the srp group .
the
corresponding values for the ud + doxy group were 0.92 mm and 0.31 mm .
cal scores at
the 6 month re - examination did not show a statistically significant difference
compared to baseline scores in the experimental group . in the deep pockets an
additional , statistically significant , reduction was observed in ppd between 3 and 6
months .
this could be explained by the fact that the test drug was applied to
only 4 pre - selected pockets and therefore it would be very difficult to provide a
true clinical benefit as the measurements were performed for the whole
dentition .
the question of whether the adjunctive use of locally delivered antimicrobials to
mechanical treatment offers an improved clinical and microbiological outcome over
srp alone remains unanswered .
a number of studies have reported only limited
improvement of cal and ppd recordings when comparing the combined treatment protocol
( srp plus locally delivered antimicrobials ) to conventional mechanical treatment
alone [ 17 - 20 ] .
generally a ppd reduction of 1 - 1.5 mm in moderate pockets ( 4 - 6 mm of initial ppd )
and of 2 - 2.5 mm in deeper sites has to be expected after mechanical root
instrumentation [ 21 - 26 ] .
this occurs concomitantly with a cal gain of approximately
0.5 mm and 1.5 mm in the moderate and the deep pockets respectively .
any additional
pocket reduction or cal gain , following the administration of the drug , would
therefore represent a true clinical benefit and hence may reduce the need for
additional periodontal surgery .
previously published studies referring on the utilization of locally delivered
doxycycline polymer focused on its effects when used as a monotherapy .
comparable
efficacy of the 2 treatments in the terms of reduction in pocket depth and gain in
clinical attachment level was reported in two parallel multicenter studies comparing
the locally delivered doxycycline to the srp alone . in another multicenter study ,
heijl et al . using a split mouth design ,
observed changes in probing depth comparable to this study for each treatment .
the
difference between the responses to scaling alone and scaling combined with
tetracycline fibers were small and not statistically significant . in a systematic
review
, the reviewers generally found
modest differences for ppd favouring the combined treatment . these , even when
statistically significant , ranged from 0.1 mm to 0.5 mm , and were of little clinical
importance .
effects for cal gains were smaller and statistical significance was less
common . in very accurately designed study
two
different approaches were evaluated for the non - surgical treatment of moderate
periodontal pockets .
traditional srp was compared to ultrasonic debridement followed
by 8.5% doxycycline hyclate administration in sites deeper than 5 mm . at the 3 month
examination
better results regarding ppd and cal were observed in the ultrasonic
debridement group than in the srp group .
a retreatment of non - responding sites ( ppd
> 5 mm ) consisting of ultrasonic debridement combined with doxycycline
application in the srp group at this time point lead to a similar clinical outcome
for both groups 6 months after baseline . taking into account the reduced time needed
for ultrasonic treatment
the authors concluded that simplified , subgingival
debridement in conjuction with doxycycline application could be a justified approach
for deeper periodontal pockets .
although , in the present study the time required for the instrumentation was not
specifically recorded , the therapists felt that ultrasonic debridement was less time
consuming than hand instrumentation .
the patient - centered outcome variables , like
reduced treatment time , are considered of importance in the evaluation of treatment
outcome today .
simplified , less time and effort demanding procedures like ultrasonic
debridement combined with locally delivered antimicrobials , i.e. doxycycline
hyclate , could represent a reliable alternative in treating moderate periodontal
pockets or can be an option for patients where a surgical intervention is not
indicated . in this respect the findings of the present study are in accordance with
those of the wennstrom et al .
adjunct use of the locally delivered drugs seems to have a beneficial effect in
smokers and in retreatment of the patients with persisting periodontal pockets .
the
adjunctive use of locally delivered doxycycline in smokers may offer some benefit
for the active and supportive periodontal treatment in both clinical
and microbiological findings .
initial studies on maintenance patients
have shown that the local application of tetracyclines seems to offer improved
results [ 19 - 34 ] , although more recent data failed to support those findings .
in accordance with that ,
tomasi et al .
concluded that locally delivered doxycycline failed to improve the healing outcome
following the re - instrumentation with a piezo - ceramic ultrasonic device of
periodontal pockets with post - therapeutic depth greater or equal to 5 mm . at the 3 month re - examination
both therapeutic approaches resulted in a statistically
significant reduction of the number of porphyromonas gingivalis only . a profound ,
yet not statistically significant , reduction was observed for tannerella forsythia
and treponema denticola as well , but in no case was eradication of the
periopathogenic species found . following the administration of drug , an adjunct
effect of doxycycline on the presence of treponema denticola
no statistically significant differences were observed between
the two groups regarding the microbiological parameters .
the findings are in
accordance with earlier studies comparing scaling and root planing alone with
scaling and root planing followed by the application of locally delivered drugs in
initial and supportive periodontal treatment . in contrast , goodson et al . in a large
, multicenter trial showed a greater reduction in
the numbers and proportions of red complex bacteria following the adjunctive use of
minocycline microspheres . in order to be effective
antimicrobials must reach their target site and be
maintained there in sufficient concentrations long enough for their antimicrobial
effect to occur .
the concentration
required for efficacy is often estimated from the minimum inhibitory concentration
( mic ) , although this has substantial shortcomings ; in particular , the fact that mic
is assessed in vitro whereas the drug is active in vivo in a constantly changing
host environment .
other problems related to estimating effective drug concentrations
in vivo are the large number of periodontal subgingival organisms and the large
variation in the mics of isolates .
defining the minimally effective concentration
is complex and most dosages are based
on in vitro experiments in which bacteria were grown under planktonic conditions .
it
is known that bacteria are organized into biofilms when present in the periodontal
pocket .
these biofilms will probably require a significantly higher concentration of
antimicrobial to kill those bacteria where cargill et al . found that legionellae in biofilms were 135 times more
resistant to iodination when compared to microorganisms growing in non - organised or
planktonic fashion [ 42 - 44 ] .
this is a strong indication that locally delivered
antimicrobials should be used adjunctively with mechanical instrumentation to
disrupt the biofilms .
a concern in the use of antimicrobials in the treatment of chronic periodontitis is
the risk for the emergence of an antibiotic - resistant complex of species .
this issue
in relation to the use of locally applied , sustained - release doxycycline was
evaluated in a recent study of walker et al . .
it was concluded that doxycycline treatment did not result in a
change of actual number of resistant bacteria or in the acquisition of antibiotic
resistance .
according to the findings of present study the adjunctive use of locally delivered
doxycycline did not seem to add any beneficial effect to the active treatment of
chronic periodontitis . only in deep pockets an additional improvement
was observed
between 3 and 6 months in the experimental group , which however was not strong
enough to provide a statistically significant difference between the two groups . in
conclusion , it is suggested that more long - term studies focusing on the outcome of
the combined therapy during supportive periodontal treatment are needed , where more
favourable results could be expected , especially in the case of deep pockets .
|
abstractobjectivesto evaluate the efficacy of locally delivered doxycycline as an adjunct to
non - surgical treatment with the use of an ultrasonic device compared to
scaling and root planing using hand instruments , by means of clinical and
microbiological criteria.material and methodsthirty three patients with chronic periodontitis participated in this cohort
study and were divided into two groups .
patients in control group received
scaling and root planing using hand instruments , whereas patients in control
group received ultrasonic debridement and 8.8% doxycycline gel was applied
after initial therapy and at 3 months at preselected sites .
clinical
recordings concerning probing pocket depth , clinical attachment level ,
plaque index and gingival bleeding index were performed at baseline , 3 and 6
months after baseline .
subgingival samples were analysed using the
" checkerboard " dna - dna hybridisation technique for porphyromonas
gingivalis , tannerella forsythia and treponema denticola.resultsboth treatments resulted in significant improvement in all clinical
recordings .
six months after the treatment a statistically significant
decrease was observed for porphyromonas gingivalis in both of groups and
treponema denticola in the control group ( p < 0.05 ) .
no inter - group
differences were observed ( p < 0.05).conclusionsboth treatment modalities provided comparable clinical and microbiological
results in the treatment of chronic periodontitis .
|
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
ACKNOWLEDGMENTS AND DISCLOSURE STATEMENTS
|
it is known that 9095% of all cancers are caused by or closely associated with environmental factors and lifestyle .
this includes diet ( 3035% ) , cigarette smoking ( 2530% ) , and alcohol consumption ( 46% ) .
cigarette smoking is an important risk factor for heart disease , chronic obstructive pulmonary disease , stroke , and acute respiratory diseases .
in addition to all these noncancer diseases , it is also highly associated with human cancer development .
the international agency for research on cancer ( iarc ) identified cigarette smoking as the cause of cancer in more organ sites than any other human carcinogens .
these include cancers of the lungs , oral cavity , larynx , nasal cavity , esophagus , stomach , pancreas , liver , kidney , urinary bladder , uterine cervix , and bone marrow .
there are over 5000 chemical compounds identified in tobacco and 62 of these have been evaluated by iarc as showing
sufficient evidence for carcinogenicity in either animals or in humans [ 2 , 3 ] .
the major carcinogenic compounds include , but not limited to , radioactive polonium , n - nitrosamines such as 4-(methylnitrosaminao)-1-(3-pyridyl)-1-butanone ( nnk ) , polycyclic aromatic hydrocarbons ( pahs ) ( e.g. , benzo[a]pyrene ( bap ) ) , and benzene .
multistep processes of genetic and molecular defects have taken place before the manifestation of cancer . traditionally , there are three basic stages of carcinogenesis : initiation , promotion , and progression
. carcinogenesis process is usually accompanied by changes in structure and function of central genomic information coded in the dna leading to various oncogene activations and tumor suppressor gene inactivations .
in addition , multiple signaling pathways may also be deregulated during the process of cancer development . cancer growth also requires molecular changes that either affect the tumor cells themselves or alter the interaction between tumor cells and their surrounding stromal environment or the immune system .
cigarette smoke components have been reported to promote tumorigenesis by several mechanisms involving all three stages of carcinogenesis .
genotoxic agents in cigarette smoke induce dna damage through several mechanisms including gene point mutation , deletions , insertions , recombinations , rearrangements , and chromosomal aberrations .
in addition to genotoxic effects , nongenotoxic effects of cigarette smoke are also extremely important .
these effects can also act as modulators which alter cellular functions including cell proliferation and cell death .
while synergistic effects of genotoxic carcinogens are known to occur , interaction between non - genotoxic ( epigenetic ) factors and genotoxic agents may also synergistically increase the risk for carcinogenesis .
the genotoxicity leading to carcinogenesis has been extensively reviewed in recent years [ 911 ] . in this present review , aside from a brief overview on the genotoxic effects of cigarette smoke components , we will provide a more extensive review on the non - genotoxic mechanisms of carcinogenesis by cigarette smoke or its components .
step 1 ( initiation of carcinogenesis)carcinogenesis may be the result of chemical or biological insults to normal cells through multistep processes that involves genomic changes ( initiation of cancer development ) .
some of the cigarette smoke components can act directly on dna , but many require enzyme conversion before becoming carcinogenic [ 10 , 11 ] .
most of such conversions involve metabolic changes via cytochrome p450s ( p450s ) such as p450s 1a2 , 2a13 , 2e1 , and 3a4 to form the electrophilic entities that can bind to dna to form dna adducts .
such adduct formation is usually at the adenine or guanine sites of the dna and lead to mutations such as those observed in the kras oncogene in lung cancer or those in the tp53 gene in a variety of cigarette smoke - induced cancers [ 13 , 14 ] .
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) and n-nitrosonornicotine ( nnn ) are the most potent tobacco - specific nitrosamines in tobacco products and cigarette smoke . these compounds are formed from tobacco alkaloids like nicotine during the curing process of tobacco and are important tobacco carcinogens that can affect different tissues depending on the specific nitrosamines or their metabolites involved [ 5 , 10 ] .
nnn has been shown to be carcinogenic to esophagus , nasal cavity , and respiratory tract in laboratory animals . in humans , metabolites derived from nnk and the metabolites of nnk
can also be identified in the smoker 's urine .benzo[a]pyrene ( bap ) , one of the pahs , is classified as a group 1 carcinogen to humans .
it has been shown to have strong association and tumor - induction potentials in lungs , trachea , and mammary glands .
the carcinogenic potency of bap has been demonstrated to be related to its metabolites which form dna adducts with site - specific hotspot mutation in the p53 tumor suppressor gene .
positive correlations of such adduct formation and tumor are indeed found in the lung cancer tissues of cigarette smokers .
these findings indicate that dna mutations are increased in both tumor and nontumor bearing tissues of smokers .
however , it must be pointed out that dna adduct formations induced by cigarette smoke still can not fully represent all the risk factors for cancer development in cigarette smokers .
for example , while there is higher incidence of pancreatic cancer in cigarette smokers than nonsmokers .
assays for nnk metabolites in pancreatic cancer tissues in humans showed no significant difference between smokers and nonsmokers .
thus , it is apparent that nnk - induced dna adducts alone are not solely responsible for the pancreatic cancers in cigarette smokers .
nevertheless , nnk and its metabolite , nnal ( 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol ) , are the only environmental carcinogens known to induce pancreatic cancer in animal models .
thus , the contribution of nnk to pancreatic cancer in cigarette smokers still can not be ignored .
furthermore , it is suggested that , in addition to dna damage , synergistic interactions between dna reactivity and epigenetic actions such as increased cell proliferation induced by nnk or by other chemicals in cigarette smoke may be needed for actual cancer development in such patients [ 23 , 24 ] .
there is indication that cigarette smoke carcinogens or cocarcinogen , such as nicotine , may also play a direct role to enhance cancer promotion and progression in human cancers after cancer development .
such genotoxic mechanisms for cancer initiation and carcinogenesis by cigarette smoke components are well covered and discussed in several excellent reviews [ 5 , 10 , 11 , 2628 ] .
, we will provide more information on the non - genotoxic ( epigenetic ) mechanisms involved in cancer promotion and progression via cigarette smoke .
carcinogenesis may be the result of chemical or biological insults to normal cells through multistep processes that involves genomic changes ( initiation of cancer development ) .
some of the cigarette smoke components can act directly on dna , but many require enzyme conversion before becoming carcinogenic [ 10 , 11 ] .
most of such conversions involve metabolic changes via cytochrome p450s ( p450s ) such as p450s 1a2 , 2a13 , 2e1 , and 3a4 to form the electrophilic entities that can bind to dna to form dna adducts .
such adduct formation is usually at the adenine or guanine sites of the dna and lead to mutations such as those observed in the kras oncogene in lung cancer or those in the tp53 gene in a variety of cigarette smoke - induced cancers [ 13 , 14 ] .
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone ( nnk ) and n-nitrosonornicotine ( nnn ) are the most potent tobacco - specific nitrosamines in tobacco products and cigarette smoke .
these compounds are formed from tobacco alkaloids like nicotine during the curing process of tobacco and are important tobacco carcinogens that can affect different tissues depending on the specific nitrosamines or their metabolites involved [ 5 , 10 ] .
nnn has been shown to be carcinogenic to esophagus , nasal cavity , and respiratory tract in laboratory animals . in humans , metabolites derived from nnk and the metabolites of nnk
benzo[a]pyrene ( bap ) , one of the pahs , is classified as a group 1 carcinogen to humans .
it has been shown to have strong association and tumor - induction potentials in lungs , trachea , and mammary glands .
the carcinogenic potency of bap has been demonstrated to be related to its metabolites which form dna adducts with site - specific hotspot mutation in the p53 tumor suppressor gene .
positive correlations of such adduct formation and tumor are indeed found in the lung cancer tissues of cigarette smokers .
these findings indicate that dna mutations are increased in both tumor and nontumor bearing tissues of smokers .
however , it must be pointed out that dna adduct formations induced by cigarette smoke still can not fully represent all the risk factors for cancer development in cigarette smokers .
for example , while there is higher incidence of pancreatic cancer in cigarette smokers than nonsmokers .
assays for nnk metabolites in pancreatic cancer tissues in humans showed no significant difference between smokers and nonsmokers .
thus , it is apparent that nnk - induced dna adducts alone are not solely responsible for the pancreatic cancers in cigarette smokers . nevertheless , nnk and its metabolite , nnal ( 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol ) , are the only environmental carcinogens known to induce pancreatic cancer in animal models .
thus , the contribution of nnk to pancreatic cancer in cigarette smokers still can not be ignored .
furthermore , it is suggested that , in addition to dna damage , synergistic interactions between dna reactivity and epigenetic actions such as increased cell proliferation induced by nnk or by other chemicals in cigarette smoke may be needed for actual cancer development in such patients [ 23 , 24 ] .
there is indication that cigarette smoke carcinogens or cocarcinogen , such as nicotine , may also play a direct role to enhance cancer promotion and progression in human cancers after cancer development .
such genotoxic mechanisms for cancer initiation and carcinogenesis by cigarette smoke components are well covered and discussed in several excellent reviews [ 5 , 10 , 11 , 2628 ] .
, we will provide more information on the non - genotoxic ( epigenetic ) mechanisms involved in cancer promotion and progression via cigarette smoke .
step 2 ( cancer promotion)cancer promotion is characterized by deregulation of signaling pathways which control cell proliferation , apoptosis , and so forth , . it is believed that although there are various genetic pathways which may lead to cancer development or cancer behaviors , there are certain hallmark capabilities or mechanisms which are commonly shared by all tumors . in the following discussion
cancer promotion is characterized by deregulation of signaling pathways which control cell proliferation , apoptosis , and so forth , .
it is believed that although there are various genetic pathways which may lead to cancer development or cancer behaviors , there are certain hallmark capabilities or mechanisms which are commonly shared by all tumors . in the following discussion
these signals are transmitted into cells by receptors that bind distinct signaling molecules . in cancer cells ,
receptor overexpression allows cancer cells to become hyper - responsive to low levels of growth factors that generally are not sufficient to trigger proliferation in normal cells .
nicotine , a major component of cigarette smoke , is known to be a chemical that plays an important role in carcinogenesis in cigarette smokers .
nicotine behaves like those growth factors which exert their biological functions mainly through the nicotinic acetylcholine receptors ( nachr ) , -adrenoceptors ( -ar ) or epidermal growth factor receptor ( egfr ) .
the functions of these receptors are cell - type specific and the expression level and receptor sensitivity can be modified by nicotine . obviously , alterations in either the receptor expressions or sensitivity play an important role in cigarette smoke - induced carcinogenesis [ 3436 ] .
recent study by lee et al . reported that 9 nachr expression in human breast tumors is elevated in advanced stages of breast cancer and plays important roles in human breast carcinogenesis .
nicotine has been shown to mediate 9 nachr signaling and upregulate cyclin d3 expression in breast caner cells and breast cancer tissues .
furthermore , it is also found that activation of the expression of 9 nachr by nicotine is through akt signaling and activation of 9 nachr signaling would elevate the phosphorylation status of adhesion molecule which plays a role in cancer metastasis .
this enhancement has been shown to be via 7 nachr with activation of erk1/2 cascade as well as induction of nf-b and bad phosphorylation .
all these events eventually lead to inhibition of apoptosis and increase of cancer risk .
these findings were further supported by wada et al . who observed that nicotine promoted cell proliferation via 7 nachr mediated p44/p42-mapk activation .
moreover , in our own study , we also reported that nicotine induced human bladder cells proliferation through erk1/2 and stat3 signaling downstream of 7 nachr and -adrenoceptors ( -ar ) . in sum , all these studies indicate that nicotine , an important ingredient of cigarette smoke , promotes cellular proliferation which plays a critical role in carcinogenesis .
other than nicotine , nitrosamines , such as nnk and nnn , also induced cancer cells growth through nachr .
nnk induced carcinogenesis by binding to nachr especially for 7 nachr , whereas the biological impact of nnn is mainly modulated by 4/2 nachr [ 8 , 4446 ] .
it has been demonstrated that nicotine or nnk stimulated lung cancer cell proliferation via 7 nachr with activations of pkc , raf1 , akt , erk1/2 , and transcription factors such as jun , fos , and myc [ 4749 ] .
question has been raised concerning the possibility that specific nachr subunit upregulated by nicotine or nnk may be tissue specific or dependent .
for instance , with nicotine or nnk , 7 nachr is the primary nachr subunit which mediates tumorigenesis in lungs giving rise to pulmonary squamous cell carcinoma and mesothelioma . on the other hand ,
thus , the specific types of nachr expressed in cancer cells may be considered as useful molecular targets for potential clinical therapy .
however , most of the nachr present in cancer cells are still not functionally characterized yet .
future study will be needed to understand the functions of different nachr subtype in cancer cells and the downstream signal pathways involved in tumorigenesis .
in addition to nachr , a number of studies indicated that nicotine and nnk might also exert their biological activities through activation of receptors such as -adrenoceptors ( -ar ) , egfr , or insulin - like growth factor receptor ( igfr ) or transactivation by nachr signaling .
for example , nnk can stimulate ht-29 cell proliferation through -ar followed by cyclin amp elevation and cox-2 expression .
consistently , nnk stimulates the growth of pulmonary adenocarcinoma in vitro and in vivo via the release of arachidonic acid through cox-2 and 5-lipoxygenase ( 5-lox ) pathways that are mainly regulated by -ar . in another study by schuller and cekanova ,
nnk is reported to stimulate 2-ar receptor pathway ( including pka , camp , creb ) and transactivate egfr pathway ( such as raf-1/erk1/2 signaling ) in the development of lung cancer .
it has also been reported that antagonists of -ar can inhibit the development of nnk - induced lung adenocarcinoma .
such antagonists are also found to be effective in reducing the stimulatory effects of nicotine on pkc , erk1/2 activations , cox-2 expression , and gastric cancer cell proliferation .
elevation of noradrenaline by nicotine via 7 nachr up - regulation leading to significantly enhanced growth and angiogenesis in both gastric cancer and colon cancer has also been demonstrated .
various investigators have also shown increases in neurotransmitters lead to -ar activation , transactivation of egfr , and the release of egf [ 32 , 54 , 56 ] .
indeed , our recent investigation provided compelling evidence that chronic nicotine exposure induced release of noradrenaline via 4/2 nachr activation followed by -ar transactivation .
our study further demonstrated that blocking of -ar with antagonist reversed the nicotine - induced cellular proliferative and chemoresistance .
al - wadei et al . first reported that nicotine contributes to the development of smoking - related pancreatic ductal adenocarcinoma ( pdac ) with elevated levels of stress neurotransmitters ( adrenaline and noradrenaline ) and induction of camp , pcreb , and perk1/2 , and inhibition of -aminobutyric acid ( gaba ) .
gaba has been reported to possess tumor suppressor function suppressing both -ar stimulated pdac growth and migration in vitro . however , while gaba is suppressed in pdacs , noradrenaline , pka , p - creb , and perk1/2 in these tissues are overexpressed .
these authors suggested that nicotine and nnk may contribute to the development of pdac in smokers by suppression of gaba with induction of stress neurotransmitters .
further proposed that nicotine induces the release of stress neurotransmitters through activation of 7 nachr and inhibits release of gaba via inhibition of 4/2 nachr .
it is now believed that the stress neurotransmitter released via nachr activation plays an important role in smoking - associated tumorigenesis . however , the precise mechanisms involved in the regulation and the function of neurotransmitter released by nicotine and nnk are still uncertain . future research on this area is encouraged . it has also been shown that nnk can promote -ar - mediated transactivation of egfr followed by erk1/2 phosphorylation leading to an increased proliferation in pancreatic cancer cells .
nnk is also reported to induce endogenous igfr which is associated with the development of lung tumors .
huang et al . also indicated that both activation of thromboxane a2 ( txa2 ) receptor and synthesis of txa2 play critical roles in nnk - promoted lung cancer cell proliferation .
txa2 activates the transcriptional factor creb through both erk and pi3k / akt pathways , which may also lead to pcna and bcl-2 overexpressions and cell proliferation .
these studies provide valuable information on the mechanisms which involve in proliferative signaling stimulated by nicotine and nnk through activation of nachr , -ar and other growth factor receptors in cancer cells .
triggering such receptors by cigarette smoke would further lead to rapid cell proliferation , cellular migration , invasion , and metastasis . in short , these investigations on the nachr , and nachr transactivated with other receptors represent the pivotal role in regulating multiple cellular cascades in general cell functions and in carcinogenesis .
nicotine is also known to influence signal transducers and activators of transcription 3 ( stat 3 ) which is an important signal transducer mediating signaling by numerous cytokines , growth factors , and oncoproteins .
findings from our own laboratories indicate that nicotine induces bladder cancer cells proliferation through 7 nachr , 42 nachr , and -ar followed by activation of erk1/2 and stat 3 .
stat3 signaling further enhanced nf-b activation , cyclin d1 overexpression , and cell cycle progression .
moreover , we also revealed that prolonged stimulation by nicotine upregulated 4/2 nachr and -ar followed with activation of stat 3 leading to significant increase in chemoresistance in cells from bladder cancers . in recent years
the biological effects of pahs are mainly mediated via aryl hydrocarbone receptor ( ahr ) . through ahr ,
other pahs , such as benz(a)anthracene ( baa ) , has also been found to increase dna synthesis and promote g1-s progression in serum deprived mcf-7 cells .
bap has been shown to increase incidence of tumors in estrogen - responsive rodents , suggesting that it may also affect er - mediated signaling .
some investigators believed that the estrogenic property of pahs may be responsible for the induction of cell proliferation .
bap and baa have been reported to act as estrogens that stimulate and initiate the er - mediated transcription and cell cycle progression and enhance er phosphorylation . on the other hand ,
there is also indication that the estradiol - dependent cell growth of mcf-7 cells can be inhibited by bap and baa [ 71 , 72 ] .
thus , the actions of pahs on estrogen - dependent cell proliferation are still controversial .
further studies are needed to elucidate more on the roles of pahs in carcinogenicity . in normal tissues ,
antigrowth signals can block proliferation by forcing the cell cycle progression into the quiescent ( g0 ) state .
the cell cycle transition from g1 to s phase is the key regulatory step in the cell cycle and is mainly regulated by cdk4/6-cyclin d and cdk2-cyclin e complexes .
nicotine has been reported to induce binding of raf-1 to rb with activation of cyclins and cdks as well as inactivation of rb . via activations of nachr and -ar ,
nicotine and nnk both exhibit mitogenic properties by inducing cyclin d1 overexpression leading to g1/s transition and increasing cell cycle progression [ 49 , 75 , 76 ] .
nnk can also stimulate normal human lung epithelial cells proliferation through nf-b and cyclin d1 upregulation in an erk1/2-dependent pathway . in our own laboratory
, we have also demonstrated that nicotine - induced cyclin d1 overexpression is regulated via stat3 , erk1/2 , and nf-b - dependent pathways in bladder cancer cells .
other study also shows that pi3k / akt - dependent cellular proliferation is also enhanced in response to nnk .
the pi3k / akt pathway is critical in cancer cells because it influences tumorigenesis , tumor growth , and therapeutic resistance .
the pi3k / akt activation is documented in both nnk - treated a / j mice and in human lung cancers from smokers .
it also plays a role in nnk - induced cell transformation , proliferation , and metastasis .
it has been suggested that akt and nf-b may serve as key targets for nicotine or nnk stimulation in the development of lung cancer .
west et al . also reported that beas2b cells treated with nnk for eight - week period increased cellular proliferation through activation of pi3k / akt pathways .
however , pi3k / akt activation does not always occur in all cancer cells induced by nicotine .
our previous study indicates that nicotine induced bladder cancer cell proliferation through stat3 and erk1/2 signalings instead of via akt pathway .
all these investigations suggest that nicotine or nnk can activate erk1/2 , stat3 , or akt signaling to interrupt the antigrowth signals leading to enhanced cell cycle progression and cancer promotion .
it is important to remember that cigarette smoke components other than nicotine or nnk may also impede on antigrowth mechanisms enhancing cancer development and promotion .
apoptosis plays an important role in controlling normal development , homeostasis , and immune defense via elimination of redundant or abnormal cells in the organism .
failure in cell elimination ( reduction of apoptosis ) may lead to undesirable cell survival and unchecked cell growths .
resistance to apoptosis is often seen in cancers where cancer cells tend to lose their proapoptotic potentials because of gene mutations .
nicotine has been shown to inhibit apoptosis induced by tumor necrosis factor ( tnf ) , by ultraviolet ( uv ) , radiation , or by chemotherapeutic drugs such as cisplatin , vinblastine , paclitaxel , and doxorubicin .
this antiapoptotic action has been shown to be via pi3k / akt , raf / mekk / erk1/2 , nf-b , bcl-2 , bax , bad , or surviving [ 23 , 8082 ] .
west et al . demonstrated inhibition of apoptosis and promotion of proliferation in human bronchial epithelium cells by nnk are induced via activation of 3/4 nachr followed by upregulation of akt , mitogen - activated protein kinase ( mapk ) , and pkc pathways .
similar results are also observed by xu and coworkers showing that both akt and survivin pathways are involved in anticisplatin - induced apoptosis by nicotine .
indeed , drug - induced enhancements of p53 and p21 expressions are shown to be suppressed by nicotine .
our recent study also indicated that long - term nicotine treatment activated 4/2 nachr and -ar leading to reduction of apoptosis induced by cisplatin or paclitaxol . consistently , zhao et al . also reported that nicotine induced up - regulation of mcl-1 phosphorylation though erk1/2 via -ar activation with increased chemoresistance ( anti - apoptosis ) of human lung cancer cells .
other investigators also indicate that nnk can prevent cell apoptosis by modulating the anti - apoptotic bcl-2 and c - myc proteins .
it is found to be associated with cellular proliferation and is elevated during the developments of certain malignant tumors such as gastric and thyroid cancers [ 1113 ] . comparing the ho-1 in lung tissues of smokers and nonsmokers , li et al . noticed that the expression of ho-1 is significantly increased in both tumor and nontumor tissues of smokers .
these studies further revealed that nnk or its metabolites probably induce oxidative stress in lung tissues with elevation on stimulates the expression of ho-1 .
such event is through erk and nf-b activation and bad phosphorylation induction leading to eventual apoptosis inhibition [ 11 , 85 ] .
cell proliferation and apoptosis can also be modulated by the peroxisome proliferator - activated receptors ( ppars ) .
ppar/ is expressed in most tissues and has been reported to be associated with cancer growths , especially those in liver , colon , breast and lungs [ 8789 ] .
sun et al . reported a novel mechanism that nicotine increases ppar/ expression through 7 nachr follow by pi3k / mtor activation leading to enhanced lung tumor cells proliferation .
in contrast to ppar/ , activation of ppar by its ligands induces apoptosis and inhibits cell proliferation .
thus , an intact ppar levels or its activation is needed to reduce cancer risk ( anti - apoptosis and cell proliferation ) .
furthermore , a significant reduction in the transcriptional activity of ppar and its endogenous ligands , including 15-s - hydroxyeicosatertraenoic acid ( 15(s)-hete ) and 3-s - hydroxyocatadecadienoic acid ( 13(s)-hode ) , are found reduced in lung tissues of nnk - treated mice . indeed , lung tumors developed in these mice later .
further suggested that the reduction of 15(s)-hete and 13(s)-hode may enable lung cells to be more resistant to apoptosis by nnk and facilitate tumor development in the animals .
in contrast to nicotine or nnk , pahs induce either apoptosis or antiapoptosis in mammalian cells [ 94 , 95 ] .
for instance , bap is known to induce signaling through igfr and increases cell survival through pi3k activation in human mammary epithelial cells .
reported that both akt and erk1/2 act as anti - apoptosis signals leading to bad phosphorylation .
however , bap can also induce apoptosis through p53 and p21 signaling in the same model .
the results suggest that bap is capable in stimulating both apoptosis and anti - apoptosis signals .
reported that light - irradiated bap ( lbap ) inhibited apoptosis through production of ros from degraded bap .
this anti - apoptotic signal induced by bap in combination with dna damage would increase the possibility of cell survival and producing mutations .
thus , while the apoptotic signal of bap induces cell death ( cytotoxicity ) , the anti - apoptotic signals of bap play an important role in cell proliferation and carcinogenesis .
the anti - apoptosis mechanisms induced by components of cigarette smoke are obviously quite complex .
it is evident that evading apoptosis plays a critical role in cigarette smoke - induced tumorigenesis and chemoresistance .
new understandings on the molecular target regulating the apoptotic and anti - apoptosis machineries by cigarette smoke could provide novel strategies for drug development with substantial therapeutic benefits .
when a cell population has progressed through a certain number of doublings ( replications ) , they would normally stop growing and enter into a process called senescence .
tumor cells , however , appeared to have limitless replicative potentials ( immortalization ) during tumor progression .
telomeres , which define the end segments of chromosomes , consist of short , tandemly repeated dna sequences ( ttaggg)n together with associated proteins .
small amount of these end dna sequences may be lost during each cell cycle as a result of incomplete dna replication .
however , de novo additions of ttaggg repeats by the enzyme telomerase may compensate for this loss .
thus , telomerase plays an important role in the maintenance of the telomere ends in normal cells .
ectopic expression of telomerase would immortalize the cells . by using human tissue samples , yim et al .
reported that there are different distributions of the telomerase activity between smokers or ever - smokers and non - smoker . a strong correlation between telomerase activity and the number of packs years smoked
can be established among these subjects indicating that there is an association between tobacco exposure and telomerase activity in the human bronchial epithelium .
increased telomerase activity would extend the lifespan of cells and put these cells to be at higher risks for malignant transformation and carcinogenesis .
similar finding is reported by targowski et al . that extensiveness of tobacco smoking correlated positively with increases in telomerase activity in tumor cells from patients with non small cell carcinoma of the lungs .
all these studies point to the fact that enhancement of the telomerase activity by cigarette smoke certainly underlies the cancer promotion potentials of cigarette smoke . however , which components in cigarette smoke altered telomerase activity are still not known .
indeed , there are indications that certain protein synthesis and mitochondria play central roles in neoplastic transformation .
it is well known that mtor and map kinase signaling pathways modulate the phosphorylation of transcriptional factors , stability of mrnas , and protein synthesis .
jin et al . reported that both nicotine and its metabolite nnk can induce survivin mrna expression through akt - mtor and mediated de novo synthesis of survivin protein in normal lung epithelial cell hbe cells .
this induced survivin expression has been claimed to play a role in the malignant transformation of hbe cells by stimulating the survival pathways .
cigarette smoke may damage respiratory chain function in mitochondria enhancing oxidative stress leading to mitochondria dysfunction [ 103 , 104 ] .
it has also been reported that nicotine exposure resulted in reduced pancreatic mitochondrial enzyme activity , degranulation of beta cells , elevated islet oxidative stress , and impaired glucose stimulated insulin secretion in rats . continued exposure to ros and free radicals from such mitochondrial stress may lead to mitochondria dna ( mtdna ) mutation which may play an important role in carcinogenesis . analyzing clinical samples ,
. also showed that tumor cells with mtdna mutations grow faster then cells without mitochondrial mutation .
hence , it is apparent that cigarette smoke would induce oxidative damage to the mtdna leading to more aggressive tumor growths .
step 3 ( cancer progression)the malignancy of a tumor is usually evaluated by its ability in invasion and metastasis as well as in the associated angiogenesis .
there are ample evidence which indicate that cigarette smoke participates in the processes of angiogenesis , invasion , and tumor metastasis .
the malignancy of a tumor is usually evaluated by its ability in invasion and metastasis as well as in the associated angiogenesis .
there are ample evidence which indicate that cigarette smoke participates in the processes of angiogenesis , invasion , and tumor metastasis .
angiogenesis , the development of new blood vessels from endothelial cells ( ecs ) , is a critical event which allows the cancer cells to receive adequate nutrients and oxygen .
angiogenesis involves mature vascular changes , including detachment of pericytes , degradation of extracellular matrix , endothelial cells remodeling , proliferation , migration , and formation of new endothelial cells into tubular structures .
survival and proliferation of vascular endothelial cells are often stimulated by tumor - derived mitogens , and vice versa .
tumor cells are known to activate angiogenesis by changing the balance of angiogenic inducers such as vegf ( vascular endothelial growth factor ) and bfgf ( basic fibroblast growth factor ) , and by countervailing inhibitors such as thrombospondin-1 .
it has been shown that nicotine can induce angiogenesis both in vitro and in vivo and contributes to the growth of tumors [ 30 , 110 ] .
similar to the fgf , nicotine is found to have the ability to promote migration , proliferation , tube formation and nitric oxide ( no ) production of endothelial cells .
no is a well - known vasodilator and angiogenesis mediator , and nicotine has been reported to enhance the expression of endothelial nitric oxide synthetase and promote no release .
nicotine is also found to induce expression of endothelial growth factors such as vegf , bfgf , pdgf , tgf- , and tgf- in endothelial cells and smooth muscle cells [ 112 , 113 ] .
enhanced bfgf release and increases in metalloproteinase expression with degradation of ecm have been demonstrated with nicotine [ 114 , 115 ] .
moreover , nicotine is found to induce secretion of prostacyclin which is a vasodilating molecule associated with endothelial cell proliferation , survival and migration .
these effects are believed to be associated with cigarette smoke - induced hyperplasia of the intima in the blood vessels and other vascular wall lesions .
7 nachr is important in both physiological and pathological angiogenesis [ 110 , 118 ] .
7 nachr in endothelial cells needs to be sensitized or activated by hypoxia or ischemia in order to induce angiogenesis .
indeed , specific antagonist of the 7 nachr ( -bungaratoxin ) is shown to inhibit nicotine - induced angiogenesis ( new vascular tube formation from endothelial cells ) [ 25 , 114 ] .
interestingly enough , it is apparent that the akt pathway is found to be not involved in either angiogenesis or vegf release induced by nicotine .
suggested that inhibition of erk1/2 , p38 mapk , and pi3k / akt can completely block and prevent endothelial tubule formation induced by nicotine - triggered 7 nachr activation .
consistent with heeschen 's study , zhang and coworkers reported that nicotine apparently increases angiogenesis and invasion by activating pkc , pi3k / akt , erk1/2 , mtor , and src in human nsclc .
excellent reviews on angiogenesis induced by nicotine were recently published [ 120 , 121 ] and will not be further discussed here .
interaction between nachr and the growth factor - mediated angiogenesis occurs at signaling and transcription levels .
nicotine - induced expression of vegf has been shown to be regulated by egfr transactivation and via the erk1/2 pathway in smooth muscle cells .
phosphorylation of the vegf receptor kdr by nicotine activates vegf and increases its activity .
additionally , nicotine can also upregulate the expression of vegf receptor vegfr2 during angiogenesis in certain cancer cells .
recent study further indicated that nicotine can synergistically promote the proangiogenic effect of estradiol in nonsmall lung cancer .
induction of angiogenesis in colon cancer by nicotine via -ar followed by arachidonic acid pathway has also been reported [ 32 , 125 ] . in sum
, 7 nachr subtype has been linked to angiogenic process induced by nicotine leading to tumor vascularity , inflammation , and ischemia . nevertheless , whether nicotine or nnk acts specifically via nachr or -ar receptors or both or whether it is controlled in a cell - specific manner needs further study .
other components present in cigarette smoke that may also contribute to angiogenesis remain to be identified .
several excellent reviews on the roles of nicotine and nachr in angiogenesis exist [ 117 , 120 , 121 , 126 ] .
the ability of invasion and metastasis allows cancer cells to escape from the primary tumor mass to new terrains in the body .
the genetic and biochemical determinants as well as the molecular mechanisms involved are still poorly understood .
many evidence indicate that cigarette smoking not only increases proliferation of cancer cells but also promotes metastasis .
clinical and epidemiological studies suggest that smokers have more rapidly progressing tumors and cancer metastasis than non - smokers .
these processes are now known to be dependent on cellular and stromal interactions and on extracellular matrix degradation .
e - cadherin is a cell - to - cell interaction molecule expressed on epithelial cells .
a loss of e - cadherin is seen in epithelial to mesenchymal transition ( emt ) , which is a major pathologic event in cancer metastasis .
chronic treatment of nicotine downregulated the expression of ecm proteins such as e - cadherin and -catenin with concomitant increases of fibronectin and vimentin in lung cancer cells .
wei et al . also indicated that nnk enhanced colon cancer cell migration with downregulation of e - cadherin .
this author also found that the expressions of snail and zeb1 , 2 major transcription repressors of e - cadherin , were also induced by nnk in colon cancer cell cultures .
its upregulation is significantly linked with tumor progression , metastasis and poor prognosis in lung cancer patients .
it has been shown that nnk can upregulate contactin-1 via 7 nachr / erk activation and enhances invasiveness of lung cancer cells .
breakdown of the extracellular matrix ( ecm ) through a family of enzyme called matrix metalloproteinases ( mmps ) is needed for tumor cells to invade adjacent tissue and to metastasize .
reported that nicotine enhanced the invasiveness of esophageal squamous carcinoma cells ( te-13 ) by up - regulating the expressions and activity of mmp-2 , and cox-2 .
nicotine is found to enhance the activity of mmp-2 , and mmp-9 as well as activation of plasminogen activators in a cox-2 and vegf - dependent manner .
it serves as a good marker for pdac ( pancreatic ductal adenocarcinoma ) metastasis especially in cigarette smoking population . in a recent investigation , lazar et al
. demonstrated that nicotine contributes to pdac metastasis through the induction of mmp-9 and vegf mediated by opn .
pahs , including bap , are also found to play a role in the promotion of cancer metastasis . through augmented cox-2 expression and pge2 production via activated ahr pathway , bap induces breast cancer cell invasions .
bap and pahs mixture has also been demonstrated to induce cancer cell invasions and metastasis through upregulating the expressions of mmps , proteinase - activated receptor-2 , fibronectin , migration stimulating factor , and bcl-2 protein in lung adenocarcinoma .
the importance of fgf-9 and its up - regulation by bap in lung cancer invasion and metastasis has been proposed .
indeed , recent study by ueng et al . demonstrated that bap increases the invasive potential of lung cancer cells in vitro .
such process involves the up - regulation of fgf-9 mrna expression via the p38 and erk1/2 pathways . during metastasis , the cancer cells co - opt signals that control leukocyte trafficking and chemokines - mediated cell migration .
among these chemokines , cxcr4 and its natural ligand cxcl12 serve as key mediators for tumor migration and metastasis .
nicotine has been shown to increase the expressions of several cxc chemokines receptors such as cxcr2 , cxcr3 , and cxcr4 as well as ccl12 in sclc cells suggesting the nicotine would stimulate cancer cell migration and eventual metastasis . although epidemiology studies have long demonstrated the relationship between smoking and cancer metastasis , the molecular mechanisms of metastasis influenced by cigarette smoke or its components remain very limited .
in this paper , we have reviewed the recent investigations concerning cigarette smoke and cancer development , promotion and progression . while chemicals with carcinogenic potentials in cigarette smoke are many ( over 62 ) , most research efforts have been devoted to three components of cigarette smoke : nicotine , nnk , and pahs . while pahs are common chemicals in the environment , nicotine and nnk are considered to be tobacco specific .
these three important components of cigarette smoke , especially nicotine and nnk , therefore , are targeted as the major compounds of focus in this review .
many previous reviews have devoted to the interrelationship between cigarette smoke and lung carcinogenesis or the genotoxicity of cigarette smoke or its components . in this review
, we are focused on the mechanistic information on tumorigenesis , especially those involving epigenetic or non - gentoxic effects . aside from lung cancer ,
it is our hope that this review will summarize the vast information cumulated in the literature and provide valuable reference resource for those who are interested in tobacco - related carcinogenesis .
the overall mechanisms on carcinogenesis cancer promotion and progression are complex involving many molecular targets which include receptors , cell cycle regulators , mitogen - activated protein kinases , apoptosis mediators , angiogenic factors , and invasion , and metastasis mediators . among the receptors ,
nachr , -ar , and ahr probably are the most important and have the closest association with cigarette smoke - induced carcinogenesis .
overexpression or activation of these receptors may result in the release of neurotransmitters and growth factors that participate in apoptosis inhibition , cell proliferation , angiogenesis , cancer cell invasion and metastasis .
it should be noted that the importance of nachr in cancer may be cell - type - dependent or specific and their sensitivity and expression can be also be modified by various environmental factors such as insecticide organophosphates . as shown in figure 1 , signaling pathways , pi3k / akt , stat3 , and erk1/2 play important roles in the carcinogenesis processes .
they are also common paths affected by the cigarette smoke components , including nicotine , nnk , and pahs .
in addition , pkc , akt , erk , and cox-2 signaling pathways are involved in both promotion and progression stages by cigarette smoke .
it is suggested that these molecules could be utilized the potential targets for future developments in cancer diagnoses or therapies .
avoidance of cigarette smoke remains to be the best way of prevention for cigarette - related cancer .
however , in view that tobacco smoke is legalized and smokers are still abundant , understanding on the health impacts by tobacco smoking still constitutes important public health concern . understanding the disease process and the mechanisms involved is the first step to solution . the emerging understanding on the molecular mechanisms in the development and progression of caners induced by cigarette smoke
provides novel inspirations and approaches for potential measures on early diagnosis , reduction in progression and metastasis , and therapy of cancers .
many dietary supplements , foods , or herbal medicines might significantly attenuate the proliferative effects by cigarette smoke .
, the natural compound pterostilbene could induce apoptosis and autophagy in chemoresistant bladder cancer cells derived from nicotine exposure .
future research on natural compounds may help to provide additional novel chemopreventive or chemotherapeutic possibilities in reducing cancer risks or other health impacts of cigarette smoke .
this review has also discussed the various molecular mechanisms and paths involved in carcinogenesis induced by cigarette smoke . however , there are still many mysteries in the carcinogenic process by cigarette smoke .
, most research efforts were focused on the proliferative and antiapoptosis mechanisms induced by cigarette smoking .
as tumors are the results of multiple and interactive genetic abnormalities , study of cancers induced by cigarette smoke should assess more than one or two acquired alterations or paths .
explorations of other paths or mechanism other than those popular ones are needed .
those molecular pathways which are significantly activated by cigarette smoke are probably the most important ones involved in cigarette smoke - induced tumorigenesis .
these pathways include nachr signaling ( such as 7 nachr , 9 nachr , or 4/2 nachr ) , -ar signaling , pi3k / akt signaling , erk1/2 signaling , stat3 signaling , vegf , and mmps pathways , and so on . targeting to modulate these pathways via dietary factors or therapeutic drugs may reduce cigarette smoking induced tumorigenesis significantly .
studies on the non - genotoxic ( epigenetic ) effects of cigarette smoke components are few and need more efforts .
the epigenetic effects of cigarette component must be evaluated to include both upstream and downstream pathways .
carcinogenesis is often species or cell - type specific and can be influenced by many factors or cofactors .
the same factor which is highly oncogenic to certain cell type or individuals may not be oncogenic to others . moreover , some cell type may become susceptible to a carcinogen only in the presence of certain factor(s ) , co - factor(s ) , genetic predisposition , or immune depression .
specific mechanism for carcinogenesis for the same carcinogen may also vary in different tissues .
synergistic interaction between cigarette smoke components and other environmental toxicants or carcinogens , such as arsenic or dioxin , on cancer development has been demonstrated both epidemiologically and in animal studies [ 143145 ] .
traditionally , most past investigations focused only on single compound or one cigarette smoke component . the synergistic interaction between otherwise
safe level of environmental chemical and low level of cigarette smoke or its component ( via either active or secondhand smoking ) for carcinogenesis raised novel public health concerns and challenging questions .
we have provided an overview on the major concepts and insights on the molecular mechanisms involved in cigarette smoke - induced cancers .
it is hoped that these mechanistic insights can be translated into practical applications for the prevention and treatment of cigarette smoke - related cancers .
we also hope that these suggestions will be helpful to those who are interested in this area of cancer research .
|
cigarette smoking is one of the major causes of carcinogenesis .
direct genotoxicity induced by cigarette smoke leads to initiation of carcinogenesis .
nongenotoxic ( epigenetic ) effects of cigarette smoke also act as modulators altering cellular functions .
these two effects underlie the mechanisms of tumor promotion and progression .
while there is no lack of general reviews on the genotoxic and carcinogenic potentials of cigarette smoke in lung carcinogenesis , updated review on the epigenetic effects and molecular mechanisms of cigarette smoke and carcinogenesis , not limited to lung , is lacking .
we are presenting a comprehensive review of recent investigations on cigarette smoke , with special attentions to nicotine , nnk , and pahs .
the current understanding on their molecular mechanisms include ( 1 ) receptors , ( 2 ) cell cycle regulators , ( 3 ) signaling pathways , ( 4 ) apoptosis mediators , ( 5 ) angiogenic factors , and ( 6 ) invasive and metastasis mediators .
this review highlighted the complexity biological responses to cigarette smoke components and their involvements in tumorigenesis .
|
1. Introduction
2. The Three Carcinogensis Steps Affected by Cigarette Smoke
3. Effects of Cigarette Smoke on Sustained Angiogenesis
4. Effects of Cigarette Smoke on Cancer Invasion and Metastasis
5. Summary, Conclusive Remarks, and Future Perspectives
|
population aging is the process by which older individuals become a proportionally larger share of the total population .
it was the most distinctive global demographic event that the world witnessed in the past century and is an important event for the twenty - first century too .
it was initially experienced by the more developed countries and has recently become apparent in the developing countries .
population aging will be faced globally by all countries in this century , although at varying levels of intensity and time . according to the world health organization ( who )
, world 's elderly population i.e. , people 60 years of age and older is approximately 650 million at present and by 2050 , it is forecast to reach 2 billion . in 2008 , five out of the top ten causes of mortality worldwide , other than injuries , were non - communicable diseases ( ncds ) and expected to go up to seven out of ten by the year 2030 . by then about 76% of the deaths in the world will be due to ncds .
government and the who have already recognized the huge burden of preventable disease , disability , death and distress caused by the ncds . the present international health agenda guided by who focuses on four conditions ( cardiovascular disease , diabetes , cancer and chronic respiratory disease ) responsible for most of the premature mortality and four lifestyle risk factors ( smoking , harmful alcohol use , lack of physical activity and high salt , high fat diets ) leading to above conditions .
medications play crucial role in geriatric health care as they treat chronic diseases , alleviate pain and improve quality of life .
age - related changes in drug disposition and pharmacodynamic responses have significant clinical implications , and increased use of a number of medications in elderly raises the risk of medicine - related problems that may occur . as medication use and the incidence of adverse drug outcomes increase with advancing age , it is important to ensure quality use of medicines in older people towards attaining a higher goal of healthy and active aging .
but , use of drugs by the elderly and their clinical outcomes especially adverse drug reactions ( adrs ) have not been prominent research topic and continues to be given low priority in national and international public health arena , at least in a developing country like india . however , we feel that this probably needs more attention in india because we have not completely dealt with the scourge of communicable diseases yet .
in addition , 70% of all older people now live in low- or middle - income countries , including india where sustainable pharmacovigilance systems has not developed as yet .
the relationship between increased use of drugs and elderly is well established . consequently , increased use of medications in elderly increases the risk of adrs .
studies from around the world have shown a definite correlation between increasing age and adr rate , at least for some medical conditions .
although very few such studies are available in indian settings , harugeri et al . in a hospital setting found that the prevalence of adr - related hospital admissions was 5.9% , while in another such study in india , it was observed to be 6.7% . for india ,
harugeri et al . predicted that 18000 bed days in a given time would be due to adrs in elderly and total cost of hospital stay due to adrs is estimated to be us $ 4350 ( inr 200100 ) , that is us $ 80.5 per patient ( 108.7% of per capita per year expenditure on health ) .
there has been much debate on whether advancing age by itself is a cause of increased risk of adrs but merely a marker for comorbidity , altered pharmacokinetics , and polypharmacy .
gurwitz and avorn concluded that patient - specific physiological and functional characteristics are probably more important than any chronological measure in predicting both adverse and beneficial outcomes associated with specific drug therapies , while studies around the world have clearly shown that the risks of adrs ( including interactions ) is related to the number of medicines taken and sometimes due to inappropriate use of medicines .
( 1991 ) observed an exponential rather than the linear relation between the risks of adr and the number of medicines taken in 9000 elderly italian patients receiving 10 medicines .
review of several studies by steward rb et al . found that the patients aged above 65 years use an average of two to six prescribed medications , and 1 - 3.4 non - prescribed medications .
out of all the factors that are most consistently associated with adverse drug reactions , polypharmacy is considered to be the most important .
thus , studies have correlated the integral association between old age and increased rate of adrs arising out of confounding association between age and polypharmacy contributed by age - related changes in pharmacodynamics and pharmacokinetics at least for some medical conditions .
the classical pharmacological classification of adrs by rawlins and thompson divides adr into two major subtypes : type a reactions , which are dose - dependent and predictable , and unpredictable type b ( bizarre or idiosyncratic ) reactions .
majority of adrs ( 80% ) causing admission or occurring in hospital setting are type a reactions .
they are predictable and potentially avoidable in nature as they are related to accentuation of known pharmacological effects of the drug .
drugs associated with type a reactions are generally with low therapeutic index and commonly used among elderly .
the lists of medicines most likely to be used in the elderly include antibiotics , anticoagulants , digoxin , diuretics , hypoglycemic agents , antineoplastic agents and non - steroidal anti - inflammatory drugs ( nsaids ) and these are responsible for 60% of adrs leading to hospital admission and 70% of adrs occurring in hospital .
type b adrs are usually uncommon , but rarely may sometimes cause serious toxicities . therefore , adrs in elderly are largely contributed by prescribing error e.g. , large doses of drugs without taking into account , the effect of age and frailty on drug disposition , especially renal and hepatic clearance
. the other contributing factor may be because of not considering the increased pharmacodynamic sensitivity of the elderly to several commonly used drugs , e.g. , central nervous system and cardiovascular drugs . since only around 3000 subjects receive a medicine prior to marketing
, it is not surprising that less frequent ( particularly type b ) adrs are often recognized only after marketing .
the capacity of premarketing studies to recognize adrs is further reduced by the limited numbers of patients in the age group of 65 or older in trials and that even smaller numbers of the oldest old .
in addition , long latency diseases like cancer are difficult to detect on account of the short duration of study .
the latter has been defined in the literature in relative terms ( for example , the administration of an excessive number of drugs ) and in absolute terms , ranging from two to more than six simultaneous medications .
in contrast to general population , the incidence of combination therapy is found to be greatest in the elderly .
a drug combination may sometimes cause synergistic toxicity , which is greater than the sum of the risks of toxicity of either agent used alone .
for example , the combination of corticosteroids and nsaids : the risks of development of nsaid - induced peptic ulcer in older patients may increases by 10% among elderly .
however , concurrent use of corticosteroids and nsaids had shown a risk of peptic ulcer disease that was 15 times greater than that of non - users of either drug .
similarly , the relative risk of hospitalization for hemorrhagic peptic ulcer disease in elderly patients ( above 65 years ) has been observed to be increased by manifolds on concurrent use of oral anticoagulants and nsaids , while the risks were lower when used alone .
this suggests that there is certainly a need to recognize and consider the aspects of synergism of toxicity while prescribing medications in elderly .
however , polypharmacy at times can effectively control certain disease condition e.g. , hypertension and epilepsy .
respecting the individual needs of every individual patient is the preferred approach of prescribing in elderly .
some of the steps that may be helpful in minimizing adrs in elderly may be :
maintaining accurate record of all medications in use : may include asking patients to bring all medications to clinic including the use of over - the - counter and complementary medicines.number of medications ( prescribed and non - prescribed ) : monitoring to balance the need and avoid polypharmacy while minimizing under - use of vital drugs.individual doses : reducing the doses wherever appropriate and titrating them carefully from a low starting dose , if pharmacodynamic sensitivity is likely to be the problem.simple regimens of medication : choose the preparation suitable for the patient and minimize the dose whenever needed , but avoid advising the patient to break a single tablet into two or three equal pieces.ensuring safe management of medications by patients : involving patients in decisions on their therapy by educating the patient about important side effects and what to do if they occur .
minimize hoarding of previously used and expired drugs.therapeutic drug monitoring is encouraged , but should not replace clinical observation .
considering an adr as a possible cause for any new problem.utilizing available strategies and inter - disciplinary collaboration to enhance the quality use of medicines .
maintaining accurate record of all medications in use
: may include asking patients to bring all medications to clinic including the use of over - the - counter and complementary medicines .
number of medications ( prescribed and non - prescribed ) : monitoring to balance the need and avoid polypharmacy while minimizing under - use of vital drugs .
individual doses : reducing the doses wherever appropriate and titrating them carefully from a low starting dose , if pharmacodynamic sensitivity is likely to be the problem .
simple regimens of medication : choose the preparation suitable for the patient and minimize the dose whenever needed , but avoid advising the patient to break a single tablet into two or three equal pieces . ensuring safe management of medications by patients : involving patients in decisions on their therapy by educating the patient about important side effects and what to do if they occur .
considering an adr as a possible cause for any new problem . utilizing available strategies and inter - disciplinary collaboration to enhance the quality use of medicines .
drugs are double - edged weapons and increased use of drugs by elderly increases the risk of adverse drug reactions causing increased morbidity and mortality . in addition , economic consequences of adrs constitute a problem of considerable magnitude .
developing countries have rapidly aging population and the governments are seeking guidance in promoting healthy and active aging
. however , strategies to increase opportunities for identifying adrs and related problems have not been emphasized in current policy responses in india to meet the increase in elderly population and chronic conditions .
in addition , there is definite paucity of good quality research and data on adrs in india .
further , growing pharmacotherapy with increase in the number of diseases in the elderly will require more timely and accurate drug safety data .
pharmacoepidemiological studies that encompass large numbers of elderly drug users are needed to obtain this information as increased knowledge of the frequency and cost of adverse drug reactions is important in enabling more rational therapeutic decisions by individual clinicians and more optimal social policy . given quantitative information on medication risks , clinicians will be able to change the use of the drug ( prescribe for fewer patients , use safer alternatives when available , and use in lower doses for shorter duration ) or can take measures to minimize side effects ( prescribe prophylactic medications , increase monitoring for side effects , and intensify patient education ) .
policy changes that can result from better data on drug toxicities include withdrawal of drug from the market , change in drug labeling , educational programs to physician and changes in academic curriculum of pharmacology .
this will result in promoting and ensuring a good health of older people , which can be of great benefit to their families and communities .
in the long run , more precise estimates of the true costs associated with adrs could stimulate development of prophylaxis and of alternative therapies .
|
medications probably are the single most important health care technology in preventing illness , disability , and death in the geriatric population .
age - related changes in drug disposition and pharmacodynamic responses have significant clinical implications ; increased use of a number of medications raises the risk that medicine - related problems may occur .
the relationship between increased use of drugs including the prescription medication and elderly is well established .
majority of adrs ( 80% ) causing admission or occurring in hospital are type a reactions .
although less common occurring in elderly , type b adrs may sometimes cause serious toxicity .
studies have correlated the integral association between old age and increased rate of adverse drug reactions arising out of confounding association between age and polypharmacy contributed by age - related changes in pharmacodynamics and pharmacokinetics at least for some medical conditions .
a drug combination may sometimes cause synergistic toxicity which is greater than the sum of the risks of toxicity of either agent used alone .
but , strategies to increase opportunities for identifying adrs and related problems have not been emphasised in current international policy responses especially in india to the increase in elderly population and chronic conditions .
careful epidemiological studies that encompass large numbers of elderly drug users are required to obtain this information as increased knowledge of the frequency and cost of adverse drug reactions is important in enabling both more rational therapeutic decisions by individual clinicians and more optimal social policy .
|
BACKGROUND
EXTENT OF ADVERSE DRUG REACTION PROBLEM IN ELDERLY
NATURE OF THE PROBLEM
POLYPHARMACY AND ELDERLY
STEPS OF MINIMIZING ADR IN ELDERLY
CONCLUSION
|
bioluminescence imaging ( bli ) using luciferase reporters has been indispensible for noninvasive monitoring of different biological processes such as tumor volume and transcriptional activation during tumor development / therapy , as well as immune cell infiltration into the tumor environment . unlike end - point analysis
, bli provides real - time , noninvasive assessment of in situ biological events , thereby giving a better picture of the kinetics of an entire process .
for example , as few as 10 cells expressing firefly luciferase ( fluc ) can be detected in deep tissue in some animal models .
additional progress has been made by the discovery and molecular construction of different luciferases with a multitude of properties , including secreted reporters such as gaussia luciferase ( gluc ) , multicolor light emission spectra for better tissue penetrance in vivo and spectral deconvolution , increased thermostability , and light output .
one limitation to current bioluminescence imaging is that typically only one and at most two luciferase reporters are used to measure one or two parameters . as
tumor formation is a complex process , concurrent measurement of several events will be important for the development of novel therapeutics and their transition to the clinic . in this study , we have characterized a codon - optimized vargula hilgendorfii luciferase ( vluc ) for mammalian gene expression , and showed that this luciferase can be multiplexed with gluc and fluc for sequential imaging of three different biological processes in the same biological system .
we then applied this triple imaging system to monitor the effect of adeno - associated virus ( aav)-mediated soluble tumor necrosis factor - related apoptosis - inducing ligand ( strail ) therapy against intracranial glioma tumors .
we first cloned vluc cdna , codon - optimized for mammalian gene expression , into a lentivirus vector under the control of the cytomegalovirus promoter ( lenti - vluc ) .
this vector also expresses the mcherry fluorescent protein separated from vluc by an internal ribosomal entry site , used to monitor transduction efficiency .
since vluc cdna carries a natural signal sequence , it is secreted to the conditioned medium once expressed in mammalian cells .
we first observed the level of vluc secretion by transducing 293 t cells with lenti - vluc and evaluating the vluc levels in cell lysates and conditioned medium using the vargulin substrate .
we observed that the majority of vluc activity ( 78% ) was contained in the medium fraction showing efficient secretion ( figure 1a ) .
next , we evaluated the light emission kinetics of vluc over time and observed a slow decay in light emission , with 44% of the initial signal remaining 5 minutes after substrate addition ( figure 1b ) . to further characterize vluc as a mammalian cell reporter , we measured the stability of the enzyme over time at 37 c
. conditioned medium from cells expressing vluc were incubated at 37 c in a humidified cell incubator .
we observed that vluc levels retained full activity over 12 days indicating high enzyme stability in conditioned medium ( figure 1c ) , similar to that reported for the secreted gaussia luciferase .
we next evaluated vluc as a reporter to monitor cell viability and proliferation over time .
u87 glioma cells transduced with lenti - vluc were seeded in a culture well and aliquots of conditioned media were collected at different time points and analyzed for vluc activity . in parallel ,
we observed a high correlation ( r = 0.98 ) between the vluc assay and the established viability assay ( figure 1d ) . to validate vluc as a reporter for in vivo imaging
nude mice were injected subcutaneously with 2 10 u87 cells stably expressing vluc ( through transduction with lenti - vluc ) .
ten days later , mice were injected intravenously ( iv ; through retro - orbital route ) or intraperitoneally ( ip ) with vargulin ( 4 mg / kg body weight ) and imaged using a cooled charge - coupled device camera at different time points . for iv injected mice
, we observed the peak luminescent signal immediately upon injection of vargulin , which rapidly declined to 25% of initial signal by 6 minutes and down to 10% by 26 minutes ( figure 2a ) .
for ip injected vargulin , the peak occurred 14 minutes after substrate injection ( figure 2a ) .
we then evaluated the possibility of multiplexing vluc with gluc and fluc for triple bioluminescence imaging by measuring the specificity of each luciferase for its substrate in vivo .
u87 cells stably expressing vluc , gluc , or fluc ( under control of the cytomegalovirus promoter ) were implanted subcutaneously in nude mice at three different sites .
ten days later , mice were imaged first for fluc - mediated bioluminescence imaging after ip injection of d - luciferin ( 200 mg / kg body weight ) and acquiring photon counts 10 minutes after injection .
twenty - four hours later , mice were imaged immediately after iv injection of coelenterazine ( 5 mg / kg body weight ) , and again signal was obtained only from tumor - expressing gluc ( figure 2b ) . finally , after another 24 hours , mice were iv injected with vargulin ( 4 mg / kg body weight ) and imaged immediately , which showed a signal only in tumor - expressing vluc ( figure 2b ) .
quantification of the luciferase signal from each tumor revealed that fluc and gluc mediated similar bioluminescence signal , whereas vluc had a significantly lower signal ( ~100-fold lower ; n = 3 mice ; p = 0.016 ; figure 2c ) .
we applied the triple luciferase reporter system to monitor glioma response to a gene therapeutic approach using the secreted soluble variant of the anticancer agent trail .
we first cloned strail under control of the constitutively active chicken -actin ( cba ) promoter into an aav2 inverted terminal repeat - flanked transgene cassette and pseudotyped it with an aavrh.8 capsid ( aav - strail ; figure 3a ) . as a control , we packaged a similar vector expressing green fluorescent protein ( gfp ) driven by the cba promoter .
we next cloned vluc cdna under control of cba promoter in another aav2 vector pseudotyped with aavrh.8 capsid ( aav - vluc ; figure 3a ) .
we then engineered u87 glioma cells to stably express fluc ( u87-fluc ) under control of cytomegalovirus promoter using a lentivirus vector ( figure 3a ) .
since trail is known to activate a series of events including the nuclear factor-b ( nf-b ) pathway , we engineered a lentivirus vector expressing gluc under the control of five tandem repeats of nf-b responsive elements ( figure 3a ) as described , and used it to transduce the u87-fluc cells generating u87-fluc / nf - gluc cells . to determine the functionality of the aav - strail construct , we transduced u87 cells with 10 genome copy ( gc)/cell of aav - strail or aav - gfp control vector .
three days later , conditioned media from these cells were harvested and analyzed for trail expression using an elisa kit .
we observed a trail concentration of 124 ng / ml from conditioned medium of aav - strail infected cells , while it was undetectable in the media from aav - gfp control cells , displaying the proper expression and secretion of strail into the conditioned medium of cells ( data not shown ) .
transfer of conditioned media from u87 cells transduced with aav - strail onto fresh u87 cells provided a modest ( ~25% ) yet significant ( p = 0.0035 ) killing effect ( supplementary figure s1 ) .
we then stereotactically implanted 10 u87-fluc / nf - gluc cells into the striatum of nude mice and allowed tumor formation .
nineteen days later , mice were randomly divided into two groups ( n = 5/group ) .
the first group was infused into the same coordinates used for tumor implantation with 10 gc of both aav - vluc + aav - gfp ( aav - vluc / gfp ; serves as a negative control for therapy ) .
the second group of mice was infused with 10 gc of both aav - vluc ( to monitor successful gene delivery ) and aav - strail ( antitumor therapy ; aav - vluc / strail ) . as a control ,
ten days after vector injection , we monitored aav gene delivery by injecting mice ( iv ) with vargulin substrate and immediately imaging using a cooled charge - coupled device camera .
evident bioluminescent signal ( average radiance of 5 10 p / second / cm / sr 2.3 10 ) was seen at the injection site of all aav - vluc injected mice , and not the aav - gfp controls , showing successful gene delivery ( figure 3b ) .
mice in both groups were monitored for tumor growth ( fluc imaging ) at week 2 post - vector injection ( corresponding to week 4 posttumor injection ) , which showed a robust antitumor response in mice treated with aav - vluc / strail as compared with the control group ( aav - vluc / gfp ; figure 3c ) .
trail binding to its death receptors recruits tnfr1-associated death domain protein ( tradd ) leading to nf-b activation .
we therefore sought to detect nf-b induction and therefore trail binding to glioma cells in our model .
we first confirmed the functionality of the nf - gluc construct by incubating u87-fluc / nf - gluc cells with tumor necrosis factor- , a known activator of nf-b , and showed a specific induction of gluc expression 48 hours after treatment ( figure 3d ) .
next , mice in both the aav - vluc / gfp ( control ) and aav - vluc / strail groups were imaged 22 days and 23 days after aav injection for fluc and gluc , respectively .
an increase in fluc signal was observed at this time point , showing that tumors regained resistance to strail therapy ( data not shown ) .
as expected , specific fluc bioluminescence at the tumor location was observed in both groups of mice , but visible gluc signal was detected only in mice injected with aav - vluc / strail and not aav - vluc / gfp control animals , indicating binding of strail to glioma cells and induction of the downstream nf-b pathway ( figure 3e , f ) .
all together , these results show that the triple luciferase system developed here could be used to image three independent cellular processes sequentially in the same animal model .
we next performed an extensive analysis of aav - mediated strail therapy in mice bearing intracranial u87 tumors .
ten mice were injected with 5 10 u87-fluc / nf - gluc cells into the striatum as above , and 14 days later , mice were randomized into two groups ( n = 5/group ) where the first group was infused into the same tumor implanted site with 10 gc of a combination of aav - vluc / gfp and the second group with the same amount of aav - vluc / strail .
mice were imaged at day 2 , 7 , 14 , and 21 after vector injection for tumor - associated fluc signal .
similar to the first experiment , aav - mediated strail expression slowed the tumor growth as compared with control mice . at day 2 after vector injection ,
the aav - vluc / gfp control group had a twofold higher signal ( p = 0.46 ) as compared with the aav - vluc / strail - treated mice ( figure 4a , b ) .
this difference increased to 6.13- ( p = 0.048 ) and 33.7-fold ( p = 0.0008 ) on day 7 and 14 , respectively .
all mice in the aav - vluc / gfp group were sacrificed between day 14 and 21 after vector injection due to tumor burden , while the entire group in aav - vluc / strail remained alive .
interestingly , the aav - vluc / strail group showed a 193-fold increase in the fluc signal ( and therefore tumor growth ) between days 14 and 21 , presumably due to u87-gained resistance to strail therapy ( figure 4a , b ) .
we also performed an experiment with a higher input of u87 cells and obtained similar results ( supplementary figure s2 ) .
we confirmed the luminescence data with a survival analysis , which revealed a significant survival increase ( p = 0.0088 ) for mice injected with aav - vluc / strail compared with aav - vluc / gfp controls ( figure 4c ) .
the median survival time for the aav - vluc / gfp group was 35 days while it was 80 days for the aav - vluc / strail group .
we first cloned vluc cdna , codon - optimized for mammalian gene expression , into a lentivirus vector under the control of the cytomegalovirus promoter ( lenti - vluc ) .
this vector also expresses the mcherry fluorescent protein separated from vluc by an internal ribosomal entry site , used to monitor transduction efficiency .
since vluc cdna carries a natural signal sequence , it is secreted to the conditioned medium once expressed in mammalian cells .
we first observed the level of vluc secretion by transducing 293 t cells with lenti - vluc and evaluating the vluc levels in cell lysates and conditioned medium using the vargulin substrate .
we observed that the majority of vluc activity ( 78% ) was contained in the medium fraction showing efficient secretion ( figure 1a ) .
next , we evaluated the light emission kinetics of vluc over time and observed a slow decay in light emission , with 44% of the initial signal remaining 5 minutes after substrate addition ( figure 1b ) . to further characterize vluc as a mammalian cell reporter , we measured the stability of the enzyme over time at 37 c
. conditioned medium from cells expressing vluc were incubated at 37 c in a humidified cell incubator .
we observed that vluc levels retained full activity over 12 days indicating high enzyme stability in conditioned medium ( figure 1c ) , similar to that reported for the secreted gaussia luciferase .
we next evaluated vluc as a reporter to monitor cell viability and proliferation over time .
u87 glioma cells transduced with lenti - vluc were seeded in a culture well and aliquots of conditioned media were collected at different time points and analyzed for vluc activity . in parallel ,
we observed a high correlation ( r = 0.98 ) between the vluc assay and the established viability assay ( figure 1d ) .
to validate vluc as a reporter for in vivo imaging , we first characterized its bioluminescence reaction properties in a quantitative tumor model .
nude mice were injected subcutaneously with 2 10 u87 cells stably expressing vluc ( through transduction with lenti - vluc ) .
ten days later , mice were injected intravenously ( iv ; through retro - orbital route ) or intraperitoneally ( ip ) with vargulin ( 4 mg / kg body weight ) and imaged using a cooled charge - coupled device camera at different time points .
for iv injected mice , we observed the peak luminescent signal immediately upon injection of vargulin , which rapidly declined to 25% of initial signal by 6 minutes and down to 10% by 26 minutes ( figure 2a ) . for ip injected vargulin ,
we then evaluated the possibility of multiplexing vluc with gluc and fluc for triple bioluminescence imaging by measuring the specificity of each luciferase for its substrate in vivo .
u87 cells stably expressing vluc , gluc , or fluc ( under control of the cytomegalovirus promoter ) were implanted subcutaneously in nude mice at three different sites .
ten days later , mice were imaged first for fluc - mediated bioluminescence imaging after ip injection of d - luciferin ( 200 mg / kg body weight ) and acquiring photon counts 10 minutes after injection .
twenty - four hours later , mice were imaged immediately after iv injection of coelenterazine ( 5 mg / kg body weight ) , and again signal was obtained only from tumor - expressing gluc ( figure 2b ) . finally , after another 24 hours , mice were iv injected with vargulin ( 4 mg / kg body weight ) and imaged immediately , which showed a signal only in tumor - expressing vluc ( figure 2b ) .
quantification of the luciferase signal from each tumor revealed that fluc and gluc mediated similar bioluminescence signal , whereas vluc had a significantly lower signal ( ~100-fold lower ; n = 3 mice ; p = 0.016 ; figure 2c ) .
we applied the triple luciferase reporter system to monitor glioma response to a gene therapeutic approach using the secreted soluble variant of the anticancer agent trail .
we first cloned strail under control of the constitutively active chicken -actin ( cba ) promoter into an aav2 inverted terminal repeat - flanked transgene cassette and pseudotyped it with an aavrh.8 capsid ( aav - strail ; figure 3a ) .
as a control , we packaged a similar vector expressing green fluorescent protein ( gfp ) driven by the cba promoter .
we next cloned vluc cdna under control of cba promoter in another aav2 vector pseudotyped with aavrh.8 capsid ( aav - vluc ; figure 3a ) .
we then engineered u87 glioma cells to stably express fluc ( u87-fluc ) under control of cytomegalovirus promoter using a lentivirus vector ( figure 3a ) .
fluc was used as a marker for tumor volume . since trail is known to activate a series of events including the nuclear factor-b ( nf-b ) pathway , we engineered a lentivirus vector expressing gluc under the control of five tandem repeats of nf-b responsive elements ( figure 3a ) as described , and used it to transduce the u87-fluc cells generating u87-fluc / nf - gluc cells
. to determine the functionality of the aav - strail construct , we transduced u87 cells with 10 genome copy ( gc)/cell of aav - strail or aav - gfp control vector .
three days later , conditioned media from these cells were harvested and analyzed for trail expression using an elisa kit .
we observed a trail concentration of 124 ng / ml from conditioned medium of aav - strail infected cells , while it was undetectable in the media from aav - gfp control cells , displaying the proper expression and secretion of strail into the conditioned medium of cells ( data not shown ) .
transfer of conditioned media from u87 cells transduced with aav - strail onto fresh u87 cells provided a modest ( ~25% ) yet significant ( p = 0.0035 ) killing effect ( supplementary figure s1 ) .
we then stereotactically implanted 10 u87-fluc / nf - gluc cells into the striatum of nude mice and allowed tumor formation .
nineteen days later , mice were randomly divided into two groups ( n = 5/group ) .
the first group was infused into the same coordinates used for tumor implantation with 10 gc of both aav - vluc + aav - gfp ( aav - vluc / gfp ; serves as a negative control for therapy ) .
the second group of mice was infused with 10 gc of both aav - vluc ( to monitor successful gene delivery ) and aav - strail ( antitumor therapy ; aav - vluc / strail ) . as a control ,
ten days after vector injection , we monitored aav gene delivery by injecting mice ( iv ) with vargulin substrate and immediately imaging using a cooled charge - coupled device camera .
evident bioluminescent signal ( average radiance of 5 10 p / second / cm / sr 2.3 10 ) was seen at the injection site of all aav - vluc injected mice , and not the aav - gfp controls , showing successful gene delivery ( figure 3b ) .
mice in both groups were monitored for tumor growth ( fluc imaging ) at week 2 post - vector injection ( corresponding to week 4 posttumor injection ) , which showed a robust antitumor response in mice treated with aav - vluc / strail as compared with the control group ( aav - vluc / gfp ; figure 3c ) .
trail binding to its death receptors recruits tnfr1-associated death domain protein ( tradd ) leading to nf-b activation .
we therefore sought to detect nf-b induction and therefore trail binding to glioma cells in our model .
we first confirmed the functionality of the nf - gluc construct by incubating u87-fluc / nf - gluc cells with tumor necrosis factor- , a known activator of nf-b , and showed a specific induction of gluc expression 48 hours after treatment ( figure 3d ) .
next , mice in both the aav - vluc / gfp ( control ) and aav - vluc / strail groups were imaged 22 days and 23 days after aav injection for fluc and gluc , respectively .
an increase in fluc signal was observed at this time point , showing that tumors regained resistance to strail therapy ( data not shown ) .
as expected , specific fluc bioluminescence at the tumor location was observed in both groups of mice , but visible gluc signal was detected only in mice injected with aav - vluc / strail and not aav - vluc / gfp control animals , indicating binding of strail to glioma cells and induction of the downstream nf-b pathway ( figure 3e , f ) .
all together , these results show that the triple luciferase system developed here could be used to image three independent cellular processes sequentially in the same animal model .
we next performed an extensive analysis of aav - mediated strail therapy in mice bearing intracranial u87 tumors .
ten mice were injected with 5 10 u87-fluc / nf - gluc cells into the striatum as above , and 14 days later , mice were randomized into two groups ( n = 5/group ) where the first group was infused into the same tumor implanted site with 10 gc of a combination of aav - vluc / gfp and the second group with the same amount of aav - vluc / strail .
mice were imaged at day 2 , 7 , 14 , and 21 after vector injection for tumor - associated fluc signal .
similar to the first experiment , aav - mediated strail expression slowed the tumor growth as compared with control mice . at day 2 after vector injection ,
the aav - vluc / gfp control group had a twofold higher signal ( p = 0.46 ) as compared with the aav - vluc / strail - treated mice ( figure 4a , b ) .
this difference increased to 6.13- ( p = 0.048 ) and 33.7-fold ( p = 0.0008 ) on day 7 and 14 , respectively .
all mice in the aav - vluc / gfp group were sacrificed between day 14 and 21 after vector injection due to tumor burden , while the entire group in aav - vluc / strail remained alive .
interestingly , the aav - vluc / strail group showed a 193-fold increase in the fluc signal ( and therefore tumor growth ) between days 14 and 21 , presumably due to u87-gained resistance to strail therapy ( figure 4a , b ) .
we also performed an experiment with a higher input of u87 cells and obtained similar results ( supplementary figure s2 ) .
we confirmed the luminescence data with a survival analysis , which revealed a significant survival increase ( p = 0.0088 ) for mice injected with aav - vluc / strail compared with aav - vluc / gfp controls ( figure 4c ) .
the median survival time for the aav - vluc / gfp group was 35 days while it was 80 days for the aav - vluc / strail group .
luciferases have played a major role in advancing our understanding of biological processes . a broader array of biocompatible , nontoxic , and efficiently expressed reporters that can be used together with existing luciferases can serve to expand this potential .
the present study demonstrates for the first time a triple luciferase reporter system for in vivo bioluminescence imaging .
we showed that vargula luciferase could be multiplexed with gaussia and firefly luciferases for sequential monitoring of three distinct biological phenomena .
we also showed that this triple bioluminescence imaging system yield specific , and detectable bioluminescence signal in deep tissues such as the brain ( with intact skull , ~3.5 mm ) of mice .
finally , we applied these reporters to monitor three cellular processes in an orthotopic brain tumor model in response to aav - strail therapy , thus validating the system for different applications . since the cloning and sequencing of vargula ( formerly cypridina ) hilgendorfii cdna in 1989
however , to our knowledge , no reports have applied it for in vivo imaging in mammals , probably owing to the unavailability of its substrate vargulin , which is now commercially available .
vluc cdna possess a signal sequence and therefore it is naturally secreted from cells allowing real time , multi time point analysis from the same well . in this study
, we characterized a codon - optimized vluc cdna for mammalian gene expression and showed vluc to be very stable with no significant decline in activity over 12 days in cell - free conditioned media at 37 c . despite
that the majority of vluc was found in the conditioned media of cells due to its native signal sequence , the intracellular vluc level was efficient for in vivo imaging . in applications where higher sensitivity is required ,
a membrane - bound variant of vluc could be used , which should yield higher cellular activity similar to recent reports for gaussia luciferase .
several systems for multimodal imaging exist , which incorporate different technologies such as fluorescence , bioluminescence , positron emission tomography , and magnetic resonance imaging .
while substantial multiparameter information can be gained by these systems , they have several drawbacks , including higher cost ( e.g. , magnetic resonance imaging ) , logistical concerns such as short half - life of some positron emission tomography probes , and broad technical expertise .
while fluorescent reporter - based imaging as well as luciferases emitting at different wavelengths can be used for multimodal applications , they require expensive and complicated instrumentation with spectral deconvolution to visualize each biological parameter . furthermore , fluorescence - based imaging is limited by a high background noise due to tissue autofluorescence as well as single animal analysis . on the other hand
, bli has low - to - no background and is thus more sensitive for deep tissue applications .
the triple in vivo bioluminescence imaging system described here yields multiparameter information distinguished by sequential imaging using different specific substrates , while remaining both cost - effective and highly sensitive , as well as being user friendly ; only a simple charge - coupled device camera is required with no need for sophisticated instrument hardware / software .
moreover , this same system can be extended and applied to any field to monitor three distinct biological phenomena in small animals .
it is important to note , however , that we have tested vluc expression in the brain of mice bearing intracranial tumors . since tumors may have comprised the integrity of the blood
brain barrier , it would be important to evaluate vargulin in a model with intact blood brain barrier to validate the ability of this substrate in crossing this biological barrier .
another drawback of the triple - reporter system is the need of methanol to dissolve the vargulin and coelenterazine substrates , limiting imaging frequency due to potential toxicity of the alcohol .
the use of water - soluble coelenterazine or the development of similar vargulin substrate would alleviate this issue .
trail has been regarded as an anticancer agent ; however , significant cancer types , including gliomas , are resistant to trail - induced cell death . mechanisms of resistance include downregulation of death receptors , decoy receptor expression , as well as overexpression of the caspase-8 inhibitor , c - flip , due to deregulation of the mtor signaling pathway .
in addition , nf-b induction by trail has been reported as a tumor cell resistance mechanism .
another disadvantage of using trail for brain tumor therapy is its inability in crossing the blood brain barrier . in this study
, we circumvented this problem by delivering strail directly to brain tumor environment using aav - mediated gene delivery .
we chose to use aavrh.8 as we have previously shown that this serotype yields excellent transduction efficiency of murine normal brain .
injection of aavrh.8 into the tumor results in transduction of primarily neuronal cells surrounding the tumor .
the transduced neurons serve as a therapeutic reservoir surrounding the tumor by synthesizing and secreting strail , which in turn will find and bind its death receptor present specifically on glioma cells . despite this continuous release of the anticancer agent by the normal brain
, we observed that tumors acquired resistance to strail therapy ; however , a significant increase in survival was observed using aav - mediated expression of strail compared with the control group .
the exact mechanism of trail resistance in this model is currently not known , but may involve nf-b signaling , as mice injected with aav - vluc / strail showed activation of the nf-b transcription factor , as monitored by gluc imaging .
it is noteworthy to mention that u87 cells showed low sensitivity to strail - mediated death in culture , as compared with the initial high sensitivity in vivo .
the culture experiment used conditioned medium from cells secreting strail ( 124 ng / ml as determined by elisa ) and therefore the cells will only get a
one - shot delivery , given that toxicity of trail to glioma cells is dose - dependent .
( 25% killing ) using a dose of 100 ng / ml of strail in culture .
although the level of aav - mediated strail expression in the brain is unknown , the transduction efficiency of the vector serotype used ( aavrh.8 ) is very high in the murine brain .
reported that despite this apparent low trail - sensitivity in vitro , an increase in survival rate in vivo was observed when using mesenchymal stem cell to deliver strail to u87 gliomas .
finally , resistance factors that allow glioma cell survival in culture may not suffice for in vivo tumor growth in a complex multicellular environment . in conclusion ,
we showed that each of these luciferases ( vluc , gluc , and fluc ) is specific to its own substrate and can be multiplexed together to monitor three distinct biological events in the same biological system .
this reporter system could be extended to different fields where simultaneous monitoring of multiple parameters is required .
u87 human glioblastoma cell line and 293 t human kidney fibroblast cells were obtained from the american type culture collection ( manassas , va ) .
both cell lines were cultured in high glucose dulbecco 's modified eagle 's medium ( invitrogen , carlsbad , ca ) supplemented with 10% fetal bovine serum ( sigma , st louis , mo ) and 100 u / ml penicillin , 100 g / ml streptomycin ( invitrogen ) in a humidified atmosphere supplemented with 5% co2 at 37 c .
the aav - cba - vluc vector was constructed by replacing egfp in aav - cba - egfp with the human codon - optimized vluc cdna ( a kind gift from dr rampyari walia ; targeting systems , el cajon , ca ) .
aav - cba - strail vector consists of a transgene cassette for soluble , secreted trail carrying amino acid ( aa ) 1150 from human flt3l , an isoleucine zipper domain , and the extracellular domain ( aa 114281 ) of the human trail designed based on previously reported h - flex - zipper - trail . in all aav vectors ,
all vectors carry a woodchuck hepatitis virus post - trancriptional regulatory element ( wpre ) downstream of the transgene .
aav vector stocks were produced by cotransfection of 293 t cells by calcium phosphate precipitation of vector plasmid , a mini - adenovirus helper plasmid pf6 ( from dr weidong xiao , university of pennsylvania medical center , philadelphia , pa ) , and aavrh.8 helper plasmid par8 as described .
aav2/rh.8 vectors were purified and titered as described , yielding typical titers of 10 genome copies ( gc ) per milliliter .
for strail expression in culture , aav vectors were packaged as aav2 since this serotype is known to transduce cells in culture much more efficiently as compared with aav2/rh.8 .
u87fluc - mcherry cells stably and constitutively expressing both firefly luciferase ( fluc ) and mcherry were generated by transduction with lentiviral vector cscw2-fluc - imcherry , which has been previously described .
u87fluc - mcherry cells were subsequently transduced with a lentivirus previously described as lenti - nf - gluc , which encodes a gluc transgene cassette driven by five tandem repeats of nf-b responsive elements .
this double - transduced u87 cells is referred to as u87fluc / nf - gluc cells .
the lentivirus vector encoding vluc was constructed by replacing the fluc insert in cscw2-fluc - imcherry with the human codon - optimized vluc cdna .
we achieve > 95% transduction efficiency of u87 cells at a multiplicity of infection of 10 with the different lentivirus constructs .
we do not find it necessary to sort the cells owing to this high transduction rate . in vivo tumor models .
all animal experiments were approved by the massachusetts general hospital subcommittee on research animal care following guidelines set forth by the national institutes of health guide for the care and use of laboratory animals .
six to 8 weeks old athymic nude mice were anesthetized with a mixture of ketamine ( 100 mg / kg ) and xylazine ( 5 mg / kg ) in 0.9% sterile saline . for subcutaneous tumors ,
mice were injected with 100 l of a 50:50 mixture of matrigel basement membrane matrix ( bd biosciences , franklin lakes , nj ) and 12 million u87 cells expressing gluc , vluc , or fluc resuspended in opti - mem . for the brain tumor model , 10 u87fluc/5nf - gluc cells ( in 2 l opti - mem )
were intracranially injected in the left midstriatum of nude mice using the following coordinates from bregma in millimeters : anteroposterior + 0.5 , mediolateral + 2.0 , and dorsoventral 2.5 .
these injections were performed using a micro 4 microsyringe pump controller ( world precision instruments , sarasota , fl ) attached to a hamilton syringe with a 33-gauge needle ( hamilton , reno , nv ) at a rate of 0.2 l / min . before aav vector injection
, mice were randomized into separate groups by placing all mice into a single cage by one operator and selected for each group by a different operator . for aav vector injections ,
mice were anesthetized as above and injected intracranially with 10 gc of each vector using the same coordinates as for tumor injections .
aav vectors were infused at a rate of 0.2 l / min using a micro 4 microsyringe pump controller attached to a hamilton syringe with a 33-g needle . in vitro and in vivo luciferase imaging .
d - luciferin was purchased from gold biotechnology ( st louis , mo ) and resuspended at 25 mg / ml in phosphate - buffered saline . for fluc imaging , mice were injected ip with 200 mg / kg body weight of d - luciferin solution , and imaging was performed 10 minutes later .
coelenterazine was obtained from nanolight technology ( pinetop , az ) and resuspended at 5 mg / ml in acidified methanol . for gluc imaging
, mice were injected iv ( through retro - orbital route ) with 5 mg / kg body weight of coelenterazine solution diluted in phosphate - buffered saline and imaging was performed immediately .
vargulin substrate was obtained from nanolight technology or from targeting systems and was resuspended at 5 mg / ml in acidified methanol .
for monitoring cell proliferation with vluc , we refreshed the media of cells 4 hours before measurements to avoid accumulation of the reporter .
for vluc in vivo imaging , mice were injected iv ( unless otherwise noted ) with 4 mg / kg body weight ( diluted in phosphate - buffered saline ) , and imaging was performed immediately . for sequential imaging of all three reporters , we imaged fluc on day 1 , gluc on day 2 , and vluc on day 3 , allowing 24 hours waiting period between the different imaging sessions .
imaging was performed using an ivis spectrum optical imaging system fitted with an xgi-8 gas anesthesia system ( caliper life sciences , hopkinton , ma ) .
data analysis for signal intensities , and image comparisons were performed using living image software ( caliper life sciences ) . to calculate radiance for each animal ,
regions of interest were carefully drawn around each signal , which is expressed as radiance ( photons / second / cm / steradian ) .
the functionality of the aav - strail vector was tested by transducing u87 cells with 10 gc / cell with aav2-strail or a negative control vector aav2-gfp .
three days later , media was harvested from all wells and a quantikine human trail elisa ( r&d systems , minneapolis , mn ) was performed as per the manufacturer 's instructions .
data presentation and calculations . for experiment in figure 1a , we calculated total relative light unit ( rlu ) in media and cell lysate as follows : ( rlu / volume assayed ) total volume in cell lysate or media . to calculate the percentage of secreted vluc
, we used the following equation : ( total rlu in media)/(total rlu in media + total rlu in cell lysate ) 100 . to calculate gluc flux in figure 3f
, we first divided fluc values ( tumor size ) for each mouse to the lowest fluc value of the three mice ( arbitrarily set to one ) .
next the gluc values were divided by each of the adjusted values to get the gluc flux , which was adjusted for tumor size . statistical analysis .
statistical analysis was performed using graphpad prism version 5.01 software ( la jolla , ca ) . for comparisons between two samples ,
an unpaired two - tailed t - test was performed . a p value of < 0.05 was considered to be statistically significant .
for analysis between multiple groups , a one - way analysis of variance was performed followed by a bonferroni 's multiple comparison test to compare two groups .
u87 glioma cells are moderately sensitive to killing by conditioned media from donor cells transduced with aav - strail
u87 glioma cells are moderately sensitive to killing by conditioned media from donor cells transduced with aav - strail . click here for additional data file .
|
bioluminescence imaging ( bli ) has shown to be crucial for monitoring in vivo biological processes .
so far , only dual bioluminescence imaging using firefly ( fluc ) and renilla or gaussia ( gluc ) luciferase has been achieved due to the lack of availability of other efficiently expressed luciferases using different substrates . here , we characterized a codon - optimized luciferase from vargula hilgendorfii ( vluc ) as a reporter for mammalian gene expression .
we showed that vluc can be multiplexed with gluc and fluc for sequential imaging of three distinct cellular phenomena in the same biological system using vargulin , coelenterazine , and d - luciferin substrates , respectively .
we applied this triple imaging system to monitor the effect of soluble tumor necrosis factor - related apoptosis - inducing ligand ( strail ) delivered using an adeno - associated viral vector ( aav ) on brain tumors in mice .
vluc imaging showed efficient strail gene delivery to the brain , while fluc imaging revealed a robust antiglioma therapy .
further , nuclear factor-b ( nf-b ) activation in response to strail binding to glioma cells death receptors was monitored by gluc imaging .
this work is the first demonstration of trimodal in vivo bioluminescence imaging and will have a broad applicability in many different fields including immunology , oncology , virology , and neuroscience .
|
Introduction
Results
Characterization of a codon-optimized Vluc for mammalian gene expression
Optimization of the triple
Sequential monitoring of three biological events in response to glioma therapy
Discussion
Materials and methods
Supplementary Material
|
the prevalence of pancreatic cystic neoplasms ( pcns ) is around 2.5%.1,2 pancreatic cysts are increasingly discovered probably because of the ubiquitous presence of multi - detector computed tomography ( ct ) scans and their increased use to evaluate patients with abdominal complaints .
endoscopic ultrasound ( eus ) plays a pivotal role in the evaluation of patients with pancreatic cysts but eus and cross - sectional imaging alone have proven to be inaccurate in identifying the exact nature of the cysts .
there is inadequate information on the natural history of pancreatic cysts but our knowledge base can not be expanded without being able to determine the exact nature of the cyst noninvasively . toward this end ,
eus with fine needle aspiration ( fna ) along with cyst fluid analysis has been advocated to improve the utility of eus in the diagnosis of pancreatic cysts .
size > 3 cm and/or the presence of mural nodules appear to be the best indicators of malignant change in patients with the side branch form of intraductal papillary mucinous neoplasm ( sb - ipmn ) and mucinous cystic neoplasms ( mcn).3 eus with cyst fluid aspiration and/or fna can play a role in differentiating pcns .
this paper will review the role of eus - fna in the evaluation of pcns .
if the cysts are incidental ( the patient has no symptoms referable to the pancreas ) , the great majority are determined to be neoplastic.4 in one study , the majority of incidentally detected pancreatic cysts ( 58% ) were mucinous and therefore had some malignant potential.4 these data suggest that all incidentally found pancreatic cysts should undergo further evaluation .
serous cyst adenomas consist of multiple " micro cysts " with thin septae coursing through the lesion .
however , 20% have a dominant macrocystic or even solid component which can result in confusion with mucinous cysts.5,6 mcns are almost exclusively located in the tail of the pancreas and are either unilocular or have only a small number of discrete compartments .
rarely they will have peripheral ( eggshell ) calcification which is highly predictive of cancer.7 mcn almost never communicate with the pancreatic ductal system .
the cystic component of the side branch form of ipmn is essentially a dilated side branch(s ) and by definition , is part of the pancreatic duct .
magnetic resonance imaging ( mri ) is superior in imaging sb - ipmn by virtue of its ability to visualize the pancreatic ductal system .
secretin stimulated mri / magnetic resonance cholangiopancreatography can be used to highlight the ductal anatomy8 and is commonly used in pancreatic centers but its superiority to non - secretin studies in the evaluation of ipmn has not been proven .
identification of sb - ipmn with eus can be made by visualizing a dilated side branch(s ) with connection to the main pancreatic duct .
depending on the plane of imaging , they may appear as a chain of lakes - separate cysts that become confluent when scanning back and forth across the cyst .
the primary problem with imaging alone in the evaluation of pancreatic cysts is that there are no clearly differentiating features that allow a high degree of diagnostic accuracy .
studies have shown that imaging alone is inaccurate in differentiating each of the types of cystic neoplasms.9 in a recent study from the group at the academic medical center in amsterdam , the accuracy in identifying the nature of a pancreatic cyst using eus imaging alone was only 23% to 46% amongst a group of expert endosonographers.10 this study used 4 criteria to differentiate cysts : 1 ) septations , 2 ) mural nodules , 3 ) a solid component , and 4 ) communication with the pancreatic duct . as a result of these discouraging reports , eus - guided cyst aspiration and analysis along with cytology have been used to improve diagnostic accuracy ; especially the differentiation between macrocystic serous cyst adenoma , sb - ipmn and mcn .
difficulties in establishing an accurate diagnosis with imaging alone have prompted investigators to pursue adjunctive measures .
the idea of using analysis of cyst aspirates to diagnose pcn dates back to 1991.11 the massachusetts general hospital surgical group is credited with advancing the clinical application of this concept12 and brugge13 then applied eus - fna techniques to obtain cyst fluid noninvasively .
1 to 2 ml of fluid is needed to perform carcinoembryonic antigen ( cea ) analysis . as a result
, the cyst should be 1 to 2 cm in size to obtain sufficient fluid for cea analysis .
recently , it has been possible to perform cea on as little as 500 l of fluid ( redpath integrated pathology , pittsburgh , pa , usa ) . because the fluid may be quite viscous , a 19 or 22 gauge needle is preferred .
intravenous antibiotics are recommended during the procedure with oral antibiotics given for 3 to 5 days afterwards .
it is recommended that the fluid be sent for cea , amylase and/or lipase , and cytology .
the rationale for analyzing for amylase or lipase is to aid in determining if there is ductal communication .
the cooperative pancreatic cyst study group then reported that cyst fluid analysis for cea was the best test to differentiate a mucinous from a nonmucinous cyst.14 however , the sensitivity and specificity for cea is 73% and 84% , respectively , and 25% of mucinous cysts will have a cea level less than 192 ng / ml.14 it may be possible to improve the analysis of cyst fluid by combining cea and molecular analysis ( dna quantity , k - ras mutations and allelic imbalance mutations ) .
sawhney et al.15 reported a 100% sensitivity of discrimination between mucinous and nonmucinous cysts using a combination of cea and molecular analysis .
it has also been reported that combining an extracellular mucin stain with cea analysis can provide an accurate discrimination between mucinous and nonmucinous cyst.16 problems with eus - guided cyst aspiration were delineated in a paper by de jong.17 one hundred forty - three consecutive patients with indeterminate pancreatic cysts underwent eus - guided cyst aspiration and the fluid was sent for cea , ca19 - 9 , amylase and cytology .
only 90% could be punctured ( cyst in inaccessible location or too small ) . of the remaining 128 patients ,
only 31% had adequate cellularity for analysis and there was sufficient fluid for cea in only 68 ( 49% ) .
fluid specimens from pancreatic cysts seldom yield cells because few viable cells are shed from the lining .
the lining epithelium can be patchy which also contributes to the difficulty in obtaining adequate samples .
the lining of serous cyst adenomas is a glycogen - rich cuboidal epithelium whereas mucinous cysts have a mucin containing columnar epithelium ( mcn is differentiated from ipmn by having ovariantype stroma ) .
a cytology brush within a 19 gauge needle has been developed in an attempt to improve cytologic yield during eus - guided cyst aspiration ( echobrush ; cook endoscopy , winston - salem , nc , usa ) .
the needle is passed into the cyst and then the brush is advanced against the inner wall of the cyst . the 1st report of using the cytology brush for pancreatic cyst cytology came from al - haddad et al.18 they studied 10 patients with pancreatic cysts and compared the cellular yield for standard fna compared to brush cytology of the cyst wall . in 7 of 10 patients , the cellular yield and detection of diagnostic cells was superior for the echobrush .
sendino et al.19 reported results in 30 patients with pancreatic cysts evaluated with the echobrush ( cook endoscopy ) . in 8/30 patients ( 27% )
however , the brush cytology provided a specific diagnosis in 20/22 ( 91% ) patients in which the technique succeeded .
the brush technique was superior to fluid aspiration ( 73% vs. 36% ; p=0.08 ) .
three patients experienced complications ( 10% ) : 1 self limited pancreatitis , 1 self limited bleed , and 1 retroperitoneal bleed resulting in patient death at 30 days .
a recent report presents better results in obtaining cytology by performing cyst wall puncture ( cwp).20 the technique reported was to advance a 22 gauge needle into the cyst and aspirate all the fluid contents .
then , without withdrawing the needle , the needle tip is moved back and forth across the residual hypoechoic cyst wall .
if the cyst wall was not visualized , the needle was moved 2 to 3 mm from the needle tip location where the aspiration was completed . using this technique , the authors reported obtaining material adequate for cytologic assessment in 81% of cysts ( 60/66 ) .
thirty percent of cysts with cea < 192 ng / ml were proven to be mucinous by cytology . in 67% of cases where the cyst fluid volume was insufficient for cea analysis , cytology demonstrated mucinous epithelium
the complication rate was 1.45% ( 1 episode of pancreatitis which was graded as mild and self - limited ) .
an alternative or adjunct to cyst wall cytology might be to directly examine the cyst wall .
there have been recent reports of using a small confocal microscopy probe ( mauna kea technologies , paris , france ) .
the cellvizio system is a probe based confocal microscopy system and a very small probe has been developed which can be passed down a 19 gauge needle . like any other optical biopsy system ,
konda et al.21 reported use of this technology on 18 patients ; 12 of whom had pancreatic cysts .
imaging succeeded in 17/18 patients and in 10/17 , the images were considered to be high quality .
future studies will need to address safety issues and determine if a specific diagnosis can be made easily and safely .
there have also been reports using the optical catheter used in the spyglass system ( boston scientific , natick , ma , usa ) to directly visualize the cyst lining .
direct visualization of both cysts revealed smooth lining and biopsies revealed a mucinous cystadenoma in both cases . there were no immediate complications in either case but one developed severe pancreatitis 1 month after the procedure .
identification of masses or nodules emanating from the cyst wall can indicate the presence of cancer or high grade dysplasia.3 however , it is important to make a differentiation between a mural nodule and an aggregate of mucin .
this distinction was studied by zhong et al.23 who reviewed pathology , eus and ct examinations from 57 patients who had undergone surgical resection of mucinous cysts .
cancer or high grade dysplasia was found in 23% of cysts with mural nodules verses 3% without nodules ( p=0.02 ) .
mucin balls accounted for 65% of the intracystic lesions detected by eus and were characterized by being round , having smooth edges and being anechoic in the center with an echogenic rim .
eus had sensitivity of 75% for the detection of mural nodules compared to 24% for ct .
aspiration of cyst contents is indicated if identification of the cyst can not be made with imaging alone . however ,
if there is an associated mass or mural nodule , then the mass itself should be targeted for cytologic examination . while mucinous cysts are the most common indication , cystic degeneration of neuroendocrine tumors and adenocarcinoma as well as solid pseudopapillary neoplasm and acinar - cell cystadenocarcinoma represent cystic neoplasms of the pancreas that present with a solid component . in these cases
any of the 3 gauges of needles ( 25 , 22 , or 19 ) could be used . in the instance of a mass
multiple punctures into a cyst is the main risk factor for infection . in the case of a mural nodule , it is best if the cyst fluid can be removed 1st leaving a solid mass to target .
our practice is to combine fluid aspiration ( performed 1st ) and then the needle is re - directed to the mass .
we prefer to have the 1st pass stained and interpreted before considering a 2nd pass to minimize the number of passes needed to establish the diagnosis and decrease the rate of complications .
there has been concern in performing eus - fna of a cyst in the body or tail of the pancreas if malignant transformation is suspected .
our japanese colleagues have been most vocal in expressing concerns about the potential of needle tract seeding of cancer cells.24,25 a resectable cystic mass in the body of the pancreas should be considered for surgical resection without fna in most circumstances .
pancreatic cysts are being increasingly recognized and when detected incidentally , they are likely neoplastic and should undergo further evaluation which should include eus .
the cyst fluid should be sent for cea , amylase and cytology . in the future
, safe techniques should be developed to improve the cytologic yield because it appears that the exact nature of the lining in mucinous cysts is predictive of their malignant potential.26,27
|
incidental pancreatic cysts are being increasingly recognized recently with incremented concern about health and frequent health check - up .
endoscopic ultrasound ( eus ) has emerged as the principal modality for imaging pancreas for various pancreatic diseases including pancreatic cyst .
but imaging alone can not accurately identify the exact nature of the pancreatic cyst .
eus - guided fine needle aspiration is a useful adjunctive procedure to differentiate pancreatic cystic lesions .
cystic fluid analysis with cytologic evaluation is important to diagnose etiology of pancreatic cystic lesions , helping the clinician to more accurately assess the presence or potential for malignancy .
|
INTRODUCTION
IMAGING OF PCN
EUS-FNA FOR PANCREATIC CYSTIC FLUID ASPIRATION
EUS-FNA FOR CYST WALL MASS
CONCLUSIONS
|
the treatment of lymphoma depends on the type , location and stage of the disease .
the majority ( 50%90% ) of oal is low - grade mucosa associated lymphoid tissue ( malt ) lymphoma . according to the world health organization modification of the real classification ,
the other 4 main types of oal are follicular cell , mantle cell , diffuse large b cell ( dlbcl ) and lymphoplasmacytic lymphoma .
they are all b cell , non - hodgkin s lymphoma , the subtype derived from various stages of b cell maturation .
the new classification of lymphoma is dependent on histological , immunophenotypic and molecular genetic analysis .
hodgkin s lymphoma does not affect the ocular adnexal structures unless there is widespread systemic involvement or a history of previous systemic hodgkin s lymphoma . according to the ann - arbor staging system lymphoma confined to the orbit
is designated as stage i , involvement of adjacent structures ( sinuses , tonsil and nose ) is stage ii .
stage iii is abdominal nodal disease below the diaphragm and iv by definition refers to disseminated involvement of one or more extra - nodal sites ( eg , liver , bone ) and e is used when there is a local extra - nodal extent ( eg , ie , iie , iiie and i ve ) .
the grading system is similar to nodal lymphomas . broadly speaking , oal with systemic involvement
requires systemic treatments whereas low - grade ( emzl or follicular cell ) lymphoma localized to the eye can be very successfully managed with local treatments such as cryotherapy , surgical excision or external beam radiotherapy . when considering local treatment it is important to exclude bilateral ocular involvement . even in the absence of clinical signs
staging investigations should include full blood count , hepatic enzymes including ldh , ct abdomen , chest and pelvis , mri or ct of the orbits , and a bone marrow biopsy and aspirate .
this is only appropriate for low - grade lymphoma localized to the conjunctiva or orbit with no systemic involvement .
surgical excision or cryotherapy is most appropriate for localized stage 1 conjunctival emzl / malt lymphoma.1 this is not curative treatment but can control local disease and relieve local symptoms .
tanimoto et al2 reported very good local control of stage 1 malt lymphoma following biopsy or surgical resection alone .
patients were followed for a median of 7.1 years , 69% of patients did not require any further treatment .
the estimated overall survival rates were 94% at 10 years and 71% at 15 years.2 although , patients should be closely followed for recurrence , histological transformation of the tumor and long - term systemic manifestations of lymphoma .
this has been the mainstay of treatment for localized oal for many years with excellent control rates .
central nervous system prophylactic radiotherapy is not required as metastatic intraocular or brain involvement is rare.3 the dose of radiation is tapered according to the grade of the lymphoma , 28 to 36 gy for low - grade lymphoma and 30 to 40 gy for high - grade .
emzl , lymphoplasmacytic and follicular cell lymphoma are considered low grade , whereas dlbcl and mantle cell are high - grade lymphomas .
five - year local control rates for emzl and follicular lymphoma after a dose of more than 30 gy reach 100%.4 however , high rates of delayed systemic recurrence suggest long - term follow up studies are required to assess the true benefit of radiotherapy.5 there is ongoing debate about use of lens shielding radiotherapy to protect the lens from cataract formation .
some studies show no effect on local recurrence , whereas in others all recurrences have occurred in patients whose lenses were shielded.6,7 partial orbit volume irradiation leads to unacceptable incidence of intraorbital recurrence and is presently not recommended .
ocular complications , including dry eye and cataract are frequently reported in patients that have long - term follow up.8 these localized complications need to be kept in mind by the treating oncologist .
ifn - alpha has been used for the treatment of systemic lymphoma for more than 20 years .
one study treated 5 patients with intralesional ifn - alpha using an intensive protocol.9 an initial complete response was seen in all patients with stage i oal , one patient with more advanced disease at presentation ( stage iia ) died from systemic lymphoma .
long - term follow up studies are required to assess the value of this treatment .
rituximab is a monoclonal antibiotic against cd20 positive b cells , which is being considered as an alternative first line treatment for localized cd20 positive oal to avoid the ocular complications of radiotherapy .
antibody - induced destruction of cd20 positive b cells can occur by apoptosis , complement mediated cytolysis and antibody - dependent , cell - mediated cytoxicity .
rituximab has also been used as monotherapy for localized emzl / malt , in some cases complete regression can be achieved .
initial responses are encouraging but the efficacy of rituximab is lower than that reported for gastric malt lymphoma,10 as long - term distal relapse is seen in 50% of cases . very few case series are published on the effect of single agent rituximab on ocular malt / emzl .
ferreri and associates achieved regression of malt lymphoma using first line single agent rituximab in all 5 newly diagnosed patients ; however early relapse was seen in 4 of the 5 patients.10 therefore , close follow up intervals are required to detect early relapse when rituximab is used alone .
rituximab was not successful for the treatment of local relapse.10 greater success is achieved when rituximab is used in combination with chemotherapy .
rituximab and chlorambucil were used as first - line therapy for 9 patients with newly diagnosed emzl .
treatment achieved complete remission in 89% with a median follow up of 2 years.11 first - line chemotherapy is often treatment of choice for mantle cell oal , as a high proportion of patients have systemic involvement at presentation .
encouragingly , rituximab containing chemotherapy for mantle cell oal had a significantly better 5-year survival than patients receiving chemotherapy alone ( 83% overall 5-year survival verses 8%).12 combination treatment with rituximab and chemotherapy or radioimmunotherapy may achieve a more durable local response and improve patient survival.1113
chemotherapy is reserved for treatment of oal when there is systemic involvement , except for the high - grade dlbcl when systemic chemotherapy is first - line treatment even for stage i disease .
more recently chlorambucil and the purine analogs fludarabine , clardarabine and pentostatin have also been used .
the extranodal lymphoma study group ( ielsg ) is currently recruiting patients to a clinical trial investigating the role of rituximab alone or in combination with chlorambucil for malt lymphoma ( ielsg # 19).14
novel mechanisms of tumor pathogenesis have related lymphoma to infection leading to new approaches to diagnosis and treatment .
chronic antigen stimulation may result in an abnormal lymphoproliferative response that ultimately develops into lymphoma .
this association is most profound for gastric malt cell lymphoma , which is related to chronic gastritis from helicobacter pylori infection in more than 90% of cases.15 h. pylori has been isolated from ocular lymphoma specimens .
however , h. pylori eradication is not effective treatment for oa malt lymphoma.16 chlamydia psittaci infection has also been linked to oal .
ferreri and associates17 identified c. psittaci in 80% of italian oa malt lymphoma specimens . in korea , c. psittaci was isolated in 78% of oa malt lymphoma specimens.18
subsequent reports from south florida , new york state , chicago , netherlands , japan and france failed to identify dna for c. psittaci in any of their oa malt lymphoma specimens.1924 there is wide geographical variation in the prevalence of c. psittaci infection in oa malt lymphoma specimens.25 of the european countries studied , the lowest rate of infection ( 12% ) was present in the uk .
results following first line antibiotic treatment are variable.26 abramson and coworkers27 documented a clinical response following antibiotic treatment in 3 patients with oa malt lymphoma .
ferreri and associates28 recorded an objective response following oral doxycycline in 4 out of 9 patients with c. psittaci positive oa malt lymphoma .
although , grunberger and associates29 were unable to replicate these results . in a multicenter prospective trial ,
27 patients with ocular adnexal malt lymphoma were treated with oral doxycycline . c. psittaci dna positive cases had an overall response rate of 64% , the response was of short duration.30 a clinical response
was also seen in 38% in c. psittaci dna negative cases.30 lack of response to antibiotic therapy may be a reflection of the biological and genetic heterogeneity of this disease .
the extranodal lymphoma study group ( ielsg ) has activated a prospective trial ( ielsg # 27 ) into oal to explore the role of antibiotic treatment and to identify potential infectious agents.14 in summary , advances in our knowledge of the immunophenotypic and genetic alterations in lymphoma cell lines have led to a better understanding of the pathogenesis of the disease .
the interplay between a possible infective agent and the immune response has allowed a new wave of treatment possibilities .
staging the patient at diagnosis guides subsequent management and local treatments should be reserved for localized disease .
however , the practical approach to the patient remains first do no harm and with this in mind ocular oncologists are tempted to administer an initial trial of oral antibiotics for stage 1 malt lymphoma .
although , general blanket treatment with antibiotics should be discouraged unless there is proof of an infective agent .
we recommend a diagnostic biopsy with immunophenotyping and genetic analysis to determine the grade of the lymphoma combined with pcr analysis to determine the presence of an infective agent . if h. pylori or c. psittaci infection is found a trial of oral doxycycline ( 100 mg twice daily for 3 weeks )
we have also seen some early impressive responses to systemic rituximab ( weekly intravenous infusion over 6 weeks ) . however , close follow up is required as others have reported early local relapse with rituximab alone.10 at present , despite the ocular side effects , localized radiotherapy still remains the standard first - line treatment for stage i low - grade oal .
|
most lymphomas that involve the ocular adnexal structure are low grade , b cell , non - hodgkin s lymphomas .
the treatment depends upon the grade and stage of the disease .
high grade lymhoma requires treatment with systemic chemotherapy whereas the localized low grade ( extranodal marginal zone lymphoma ) can be successfully managed with local radiotherapy .
chlamydia psittaci infection is associated with low grade ocular lymphoma ; however there is wide geographic variation in the strength of this association .
blanket antibiotic therapy is not advised unless there is proof of an infective agent .
the monoclonal antibody , rituximab , may be successful for cd20 positive lymphoma , although it is likely that rituximab will have better long - term results when used in combination with systemic chemotherapy .
|
Treatment options for ocular adnexal lymphoma (OAL)
Observation
Surgery and cryotherapy
Radiotherapy
Immunotherapy: interferon (IFN-alpha)
Immunotherapy: monoclonal antibody
Chemotherapy
Systemic antibiotics
|
uncontrolled bleeding remains major challenge , responsible for 40% trauma - related deaths , which could be prevented by timely intervention . in blunt trauma , extent of hemorrhage
can not be determined by physical examination , as vital signs may not give a clear picture in all the patients , especially young healthy once .
although abnormal vital signs does indicate shock but the absence of abnormality does not exclude hypoperfusion in trauma patients .
decision - making is difficult , regarding surgical intervention to treat injuries hidden from physical examination .
these patients are intensely vasoconstricted and may suffer end organ ischemia even with normal systolic blood pressure ( sbp ) .
although shock index ( si ) can be a useful parameter in acute hemorrhage , but it still needs more study in its support to be better than simple vital sign analysis .
blood ammonia has been shown to be elevated in hemorrhagic shock in animals and humans .
intestinal bacterial enzymes acting on it produces ammonia which is freely diffusible and moreover liver in not fit for detoxification of ammonia .
we investigated the significance of ammonia levels estimation in blunt trauma abdomen patients to predict internal hemorrhage , complications and correlated with need of intervention .
one hundred blunt trauma abdomen patients ( > 12 years old ) presented in trauma ward / emergency department of tertiary care hospital were included in the study .
group i required blood transfusion 2 units and/or intervention to control bleeding within 24 h following admission .
it was analyzed within 20 min of sampling on autoanalyser cobas 6000 ( roche diagnostics india pvt ltd ) .
routine investigations such as blood sugar , kidney function test , hemogram , x - ray chest , and ultrasound abdomen were done at admission .
group i comprised 62 patients , forty patients needed intervention and blood transfusion both , whereas 22 patients needed only 2 units of blood .
average age was 31.84 14.8 and 32.37 13.6 years , respectively , almost same in both the groups .
most of the patients had more than one organ system involved and most frequently injured organ was liver ( 40% ) followed by spleen ( 29% ) .
mean si and arterial ammonia levels were significantly higher in group i compared to group ii patients [ table 1 ] .
about 88.7% patient of group i had si > 0.9 compared to only 13.1% patients in group ii [ table 2 ] .
significantly higher number of patients developed complications in group i compared to group ii ( 38 vs. 7 ) and 8% mortality rate was noted in group i only .
patients , who developed complications or died , had higher levels of ammonia at admission [ table 3 and figure 1 ] .
mean shock index and ammonia levels percentage of patients showing shock index 0.9/0.9 ammonia levels in patients with complications and mortality in group i receiver operating characteristic analysis of hospital stay , shock index , and ammonia levels the cutoff point for ammonia levels is > 58.85 mol / l ( sensitivity 77.42% and specificity 93.37% ) .
positive predictive value and negative predictive value at this point are 96.7% and 78.9% , respectively .
the cutoff point for ammonia levels is > 58.85 mol / l ( sensitivity 77.42% and specificity 93.37% ) .
positive predictive value and negative predictive value at this point are 96.7% and 78.9% , respectively .
roadside accidents were a major cause of trauma ( motor vehicle injury ) in our study probably due to increase population and number of vehicles on the road . similar cause has been reported by other studies also .
a maximum number of patients ( 78% ) were below the age of 40 years suggesting this age group people are more outgoing and rash drivers making them more prone to accidents .
male preponderance ( 93% ) noted in our study is higher than earlier studies , may be reflecting indian culture where male member of the family being breadwinner , needs to travel more than females .
the routine evaluation of blunt trauma abdomen patients includes various clinical parameters , physical examination , radiological and laboratory findings .
increased ammonia levels as observed in our study could itself be danger signal in these patients as it is sign of blood in abdominal cavity due to blunt trauma . intestinal bacterial enzymes acting on it produces ammonia which is freely diffusible and moreover liver in not fit for its detoxification as hemorrhage decreases hepatic blood flow through portal vein causing dysoxia of cells
. increased ammonia levels have been reported in animal hemorrhagic shock and in few human studies as well .
cutoff value for ammonia was also calculated to maximize sensitivity and specificity in identifying patients requiring intervention .
the purpose of this study was to augment the diagnostic accuracy of routine clinical assessment of such patient .
high ammonia levels in bleeding patients ( group i ) correlated well with si as 88.7% of these patients had si > 0.9 , i.e. , having potentially severe trauma requiring immediate treatment .
other workers have also reported similar si in bleeding patients . although si can be useful clinical parameter in acute hemorrhage which depicts severity of situation in heterogeneous patients presented in emergency but it still needs more study in its support to be single reliable parameter .
five patients died in group i had high ammonia levels ( 87.4 mol / l ) at admission .
high ammonia itself is toxic and can cause various complications , so it must be diagnosed and treated as early as possible .
moreover , trauma patient loses lots of valuable time in reaching trauma center , but once reached the hospital , they need to be diagnosed correctly and quickly .
biochemical marker which is time and cost effective such as ammonia estimation can predict need of intervention in these patients and best treatment can be provided in the golden hour .
|
background : in blunt trauma , extent of hemorrhage can not be determined by physical examination , and vital signs may also not give clear picture in all the patients , especially young healthy ones .
hemorrhagic shock has been reported to increase blood ammonia levels .
arterial ammonia was analyzed in blunt trauma abdomen patients and correlated with shock index ( si ) .
its predictive value was determined for timely decision of intervention.materials and methods : hundred blunt trauma abdomen patients presented in the emergency ward of tertiary care hospital were included in the study .
group i comprised 62 patients requiring either blood transfusion 2 units and/or intervention to control bleeding within 24 h following admission .
group ii had 38 patients : not requiring transfusion / intervention during hospital stay .
arterial blood sample was taken immediately after admission ; ammonia was analyzed within 20 min of sampling on cobas 6000 ( roche ) .
si was calculated .
predictive value of ammonia was determined using receiver operating characteristic curve.results:ammonia levels and si were significantly ( p < 0.001 ) higher in group i compared to group ii patients ( 68.55 14.36
umol / l vs. 37.55 7.41 umol / l and 1.28 0.5 vs. 0.74 0.12 , respectively ) .
significantly higher number of patients in group i ( 88.7% vs. 13% ) had si > 0.9 .
ammonia levels were significantly higher in patients with complications and in those expired.conclusions:ammonia levels were significantly higher in patients requiring blood transfusion / intervention in 24 h of admission .
the best cutoff value to maximize sensitivity and specificity was ammonia > 58.85
mol / l .
ammonia estimation at admission can be clinically significant indicator of traumatic hemorrhage needing intervention .
|
I
M
Statistical analysis
R
Receiver operating characteristics analysis
D
Financial support and sponsorship
Conflicts of interest
|
the proportion of adults over the age of 65 is expected to increase over the next 40 years .
an anticipated rise in the number of older adults is expected to lead to an increase in the prevalence of age - related diseases , which in turn , might result in escalating health care costs and heightened distress among family and caregivers . cognitive impairment , and
more specifically alzheimer 's disease , is one of the most threatening age - related diseases , but even so - called normal age - related cognitive decline can cause agonizing distress and loss of personal identity . unfortunately ,
pharmaceutical treatments or preventions for cognitive impairment are only modestly effective , resulting in the search for nonpharmaceutical approaches such as intellectually stimulating activities , dietary interventions , and physical activity , for preventing or treating cognitive decline .
a recent report estimated that modifiable risk factors including education , smoking , mid - life obesity , hypertension , diabetes , depression , and physical inactivity contribute significantly to the risk of alzheimer 's disease , and that a 10% to 25% reduction in these factors could prevent as many as 3 million cases worldwide . yet , despite the recognition of the importance of modifiable risk factors in the incidence and prevalence of cognitive impairment , there is often a misunderstanding of the research that has been conducted examining whether intervening on these modifiable risk factors would have any noticeable effect on brain or cognitive health .
in contrast , a good deal of research has been conducted to examine the effects of physical activity and cognitive stimulation on human brain morphology and function .
the aim of this review is to summarize recent research findings that examine the potential for physical activity , cardiorespiratory fitness , and exercise interventions to enhance brain health in late life .
the studies reviewed here support the position that physical activity influences the endogenous pharmacology of the brain and takes advantage of the brain 's natural capacity for plasticity , well into late adulthood .
physical activity increases the lifespan , reduces the risk for many cardiovascular diseases and cancers , and also reduces the risk for cognitive decline and depression in late adulthood . in short
, we argue that the influence of physical activity on brain plasticity might have consequences not only for memory and other cognitive functions , but also has implications for many different psychiatric and neurologic conditions through a set of common biological pathways .
first , studies using rodent models for exploring the ways in which physical activity influences the brain can control when and how much physical activity the animal receives .
hence , the nature of these systematic experiments allows for causal and directional conclusions about the effects of physical activity on learning and memory , neurotransmitter systems , metabolic and growth factors , and cell proliferation .
second , animal models allow for an examination of the cellular and molecular events resulting from physical activity that are simply impossible to study in humans . for these reasons , it is important to describe this literature since it provides a causal and low - level biological foundation to understand the effects observed in human neuroimaging and clinical studies .
one of the earliest studies found that animals that were provided access to a running wheel in their cage tended to outperform their more sedentary counterparts on several different learning and memory tasks such as the t - maze and morris water maze . in one version of the morris water maze
, rodents are made to swim in an opaque pool until they find the location of a submerged platform that sits just below the surface . by using cues
located around the room , the rodent learns to navigate to the submerged platform more quickly after successive trials . in this task ,
both older and younger animals engaging in exercise demonstrate faster learning of the location of the submerged platform compared with rodents not engaging in exercise .
importantly , performance on the morris water maze has been frequently linked to the hippocampus , a medial temporal lobe structure critical in memory formation .
in fact , other studies utilizing hippocampus - sensitive tasks have also reported that exercise enhances both acquisition and rtention , suggesting that the hippocampus might be especially sensitive to the effects of exercise .
there is now substantial support for robust and consistent effects of physical activity on the morphology and function of the hippocampus .
for example , one of the most consistent findings in this literature is that exercise has the capacity to increase cell prolifration in the dentate gyrus of the hippocampus , even in aged animals .
these studies indicate that the hippocampus remains highly modifiable throughout the lifespan and that exercise has the capacity to take advantage of the plasticity of this structure .
cell proliferation in the hippocampus leads to an increased demand for nutrients to support the new neural architecture .
after exercise , increased vascularization has been routinely found in several different brain regions including the cerebellum , motor cortex , hippocampus , and frontal cortex . increased proliferation of cells and capillaries in the hippocampus work in concert to enhance learning and memory in behavioral paradigms , but these effects can also be observed on the neurophysiological level .
for example , exercise increases the number of synapses in the hippocampus , enhances indices of long - term memory formation , and elevates the rate of gene expression for molecules associated with learning and memory such as brain - derived neurotrophic factor ( bdnf ) and serotonin .
it is clear from this literature that exercise influences the integrity of the hippocampus by influencing gene expression , cell proliferation and survival , vascularization , and synaptic plasticity .
however , this literature has identified many different brain regions influenced by exercise , indicating that exercise has widespread effects . in conclusion , there are many different molecular and cellular pathways mediating the effects of exercise on cognitive and behavioral outcomes , including increased neurogenesis , angiogenesis , and the production of growth factors important in memory and cognitive function .
greater amounts of physical activity and higher cardiorespiratory fitness levels are associated with better cognitive function in older adults .
for example , older adult athletes outperform their more sedentary peers on many different cognitive tasks , and fitter individuals are faster and more accurate on executive functioning and memory tasks .
longitudinal studies of physical activity have also found that engaging in a greater amount of physical activity earlier in life is associated with better cognitive function later in life , with larger effects for individuals engaging in more intense exercises .
however , cross - sectional and longitudinal observational studies are often plagued by confounding factors that make it challenging to make causal claims about the link between physical activity and cognitive function .
in other words , in the studies described above it is equally likely that individuals with better cognitive function choose to participate in more physical activities than individuals with poorer cognitive function . along these same lines ,
physical activity might instead be a proxy for better health habits more generally rather than being specific to physical activity per se .
randomized controlled trials reduce or eliminate some of the limitations of cross - sectional and observational studies .
these types of interventions in which older adults are randomized to either a moderate intensity physical activity group or to a non - active or less - active control group , routinely demonstrate that increasing physical activity for 3 to 6 months is effective at improving cognitive performance .
for example , in one study , inactive older adults were randomized to either a moderate intensity physical activity group or to a stretching and toning control group for 6 months .
both groups came into the laboratory 3 days per week and the exercise group participated in moderate - intensity exercises for 30 to 45 minutes per day while the stretching group participated in stretching exercises for the same amount of time . trained exercise physiologists monitored heart rates , intensity , and compliance in both groups for the duration of the exercise regimen .
this study found that participation in moderate - intensity physical activity ( eg , brisk walking ) was effective at enhancing performance on tasks that measured executive functions , but was less effective at improving performance on tasks that measured other cognitive domains .
in contrast , the stretching and toning group did not show significant improvements in performance over this same period .
meta - analyses of physical activity interventions have confirmed that the effects of exercise on cognitive function in late life are both general and specific .
general in the sense that many different cognitive domains are improved after several months of exercise , but specific in the sense that executive functions are enhanced more than other cognitive functions .
first , in terms of cognitive function , the effects of exercise appear to be widespread , but most strongly associated with executive domains .
this suggests that brain regions and networks that support executive functions might be more sensitive to the effects of exercise than other brain areas .
the rodent literature largely supports this claim , with the largest and most consistent effects of exercise appearing in regions that support higher - level cognitive functions including the hippocampus , frontal cortex , and basal ganglia .
the second key principle emerging from these studies is that the brain remains modifiable well into late adulthood , and physical activity has the capacity to take advantage of brain plasticity .
brain plasticity resulting from exercise can be detected at the molecular and cellular level in rodents and at the cognitive level in humans .
these two points , specificity and plasticity , provide the foundation for neuroimaging methods to examine whether physical activity , fitness , or exercise has any appreciable effect on the morphology or function of the human brain . given
the principles described above , neuroimaging studies exploring these associations have hypothesized that physical activity would influence the morphology and function of the human brain and that the effects would be widespread but most consistently associated with regions that support higher - level cognitive functions such as the prefrontal cortex and hippocampus .
one of the unfortunate characteristics of the brain is that it generally shrinks and atrophies with advancing age .
in fact , both the prefrontal cortex and hippocampus shrink at roughly 1% to 2% annually in individuals over the age of 55 , with more precipitous rates of atrophy when individuals begin experiencing cognitive impairment .
although the rate and trajectory of decline varies from region to region , the general finding is that regions that support memory and executive functions show the earliest and most rapid decline .
interestingly , the loss of brain volume is mirrored by age - related changes in cognitive function with the most significant losses occurring on memory and executive tasks .
yet , it is these cognitive domains and brain areas that appear the most sensitive to physical activity training .
would greater amounts of physical activity or higher cardiorespiratory fitness levels have any beneficial or positive associations with the morphology of the older adult brain ?
there have now been several studies finding that older adults who are more fit , more physically active , and who participate in exercise interventions have greater brain volumes than their less fit and less active counterparts . in one cross - sectional study ,
cardiorespiratory fitness levels were assessed in a sample of cognitively healthy older adults and voxel - based morphometry was used to assess gray matter volume . although increased age was associated with reductions in gray matter volume throughout the prefrontal , temporal , and parietal cortices , these same brain regions showed less atrophy in adults that were more fit .
these results demonstrated that remaining more aerobically fit could help to preserve brain tissue that would normally atrophy with age .
higher fitness levels have now been associated with greater gray matter volume in other populations , including postmenopausal women receiving hormone therapy , a higher educated older adult sample , a sample with multiple sclerosis , and older adults with mild cognitive impairment .
these studies have all derived a similar conclusion from these results : individuals with a higher level of fitness have greater gray matter volume than less fit individuals , and the associations are relatively specific to brain areas that support higher - level cognition , including the prefrontal cortex .
as described above , much of the animal literature has focused on the effects of exercise on hippocampal plasticity and memory functions supported by the hippocampus .
this question is important since the hippocampus shrinks with advancing age and contributes to agerelated memory loss . in 165
cognitively normal older adults , cardiorespiratory fitness levels were recorded in addition to high - resolution anatomical images of the brain .
the size of the hippocampus was assessed using an automated segmentation algorithm that uses a point distribution model to determine the location , size , and shape of the structure .
a clear association was found between higher fitness levels and greater hippocampal volume , but importantly , greater hippocampal volume also mediated the fitness - memory association .
this result suggests that greater hippocampal volume is not just a meaningless by - product of more vascularization , but rather has a meaningful impact on memory function in late life .
this general association between higher fitness levels and larger hippocampal volume has now been replicated in individuals with mild cognitive impairment .
cross - sectional research defines important associations between variables of interest , such as cardiorespiratory fitness levels and cortical volume .
demonstrating these associations is necessary before embarking on a lengthy and expensive longitudinal randomized trial .
however , there are inherent limitations to cross - sectional designs that prohibit the ability to draw conclusions about the causal nature of physical activity on brain plasticity . several studies
for example , in the cardiovascular health study at the pittsburgh , pennsylvania site , 1479 ambulatory adults over the age of 65 were enrolled into a longitudinal study on the incidence of cardiovascular diseases .
information about lifestyles and physical function were collected as part of this study including information on the frequency and duration of walking .
approximately 9 years after the original enrollment period these same participants were recruited to participate in a brain mri study in which high - resolution brain images were collected .
the brain images from 299 cognitively normal adults were selected from this sample and used in an analysis to examine whether greater amounts of self - reported walking 9 years earlier was predictive of gray matter volume later in life .
the analysis of this data confirmed that greater amounts of physical activity was associated with greater gray matter volume in several different brain regions including the frontal cortex , parietal cortex , and temporal cortex including the hippocampus .
interestingly , after a period of 4 more years , 116 of these 299 adults were diagnosed with either mild cognitive impairment or dementia , but greater graymatter volume associated with physical activity was associated with a two - fold reduced risk of developing cognitive impairment .
this study demonstrated for the first time the link between participation in physical activityearlier in life , greater gray matter volume , and the reduced risk for cognitive impairment later in life .
this study and others demonstrate that the effects of physical activity on brain plasticity might endure and influence the risk for cognitive impairment over a span of several years .
randomized interventions have also reported that assigning sedentary older adults to engage in more physical activity results in an increase in graymatter volume in several different brain areas .
for example , colcombe et al randomized a group of cognitively normal adults to either a moderate - intensity walking exercise program or to a stretching and toning control group .
similar to the study described above , this study required participants to report to the laboratory three times per week for a period of 6 months .
high - resolution brain mri scans were collected both before and after the intervention period .
interestingly , the walking exercise group showed a significant increase in the volume of prefrontal and temporal brain areas along with an increase in the volume of the frontal white matter tracts especially the genu of the corpus callosum .
another randomized intervention of physical activity examined whether participation in 1 year of a structured exercise regimen would increase the volume of the hippocampus in older adults . in this study , 120 cognitively normal older adults participated in a similar exercise design as that described previously .
high - resolution brain scans were collected before the intervention , after 6 months , and then at completion of the 1-year trial .
although the thalamus and caudate nucleus did not show significant changes in volume resulting from exercise , there was an effect of exercise on the size of the hippocampus . whereas the stretching and toning control group displayed about a 1.4% decline in the size of the hippocampus the exercising group showed an increase of about 2% over this same 1-year period .
this study demonstrated that the volume of the hippocampus remains modifiable into late adulthood , and participation in 1 year of consistent and moderate intensity exercise was sufficient for increasing the size of the structure .
furthermore , the changes in hippocampal volume for the exercising group were correlated with improvements in memory performance suggesting an important link between changes in volume induced by exercise and memory enhancement .
in fact , more recent studies have found that the volumetric differences observed as a function of cardiorespiratory fitness mediate improvements in memory and executive function , again supporting the claim that these volumetric effects are not meaningless by - products , but important factors in promoting better cognitive function .
this discussion summarizes the relatively well - established scientific literature using cross - sectional , longitudinal , observational , and randomized controlled trials examining the effect of physical activity or cardiorespiratory fitness on regional gray matter volume .
these studies have consistently reported that higher fitness levels are associated with larger brain volumes , and that participation in only modest amounts of physical activity is sufficient for increasing gray matter volume in select brain regions .
in addition , these results are in line with the animal literature and human cognitive literature described in preceding sections demonstrating the brain plasticity and specificity of the effects of greater amounts of physical activity .
volumetric data has proven useful in identifying how physical activity could alter the morphology of the adult brain . however , other neuroimaging methods including functional magnetic resonance imaging ( fmri ) and resting state connectivity ( rs ) mri approaches allow for an investigation of the effects of physical activity on brain network dynamics . in one of the earliest studies to examine this , colcombe et al employed a task measuring selective attention and executive control in a two - part experiment . in the first experiment ,
higher cardiorespiratory fitness levels were associated with better performance on the task and this was paralleled by increases in fmri activity in the dorsolateral prefrontal and parietal brain regions .
the second experiment was a randomized exercise intervention in which adults were assigned to either receive a structured exercise regimen for 6 months or to a stretching and toning control group for the same amount of time .
the participants performed the same selective attention task as the participants in the first experiment .
the results from the randomized trial were strikingly similar to the results from the crosssectional study .
that is , after 6 months of the intervention , the exercise group showed increased activity in the dorsolateral prefrontal cortex and parietal cortex and decreased activity in areas that support conflict monitoring such as the anterior cingulate cortex .
these results are important because they demonstrate that in addition to volumetric changes resulting from exercise there are also significant changes in task - evoked brain function .
hence , the brain processes task demands more efficiently after only 6 months of exercise .
although there are only several published studies using fmri paradigms , each of these studies has found increased fmri activity in prefrontal regions including during a semantic memory task , the digit symbol substitution task , and the stroop task as a function of either higher cardiorespiratory fitness levels or greater physical activity levels . yet
, each of these studies also recognizes the complex nature of cognitive function and the necessity of understanding the networks of regions that support cognition and how physical activity exerts its effects on these networks .
hence , it is important to identify not only which brain areas are associated with physical activity , but also to understand how the communication between regions is influenced by physical activity .
could the functional connectedness of the network improve after several months of exercise and would these effects mediate improvements in memory and executive function ?
regions of interest can be used as seeds to examine whether regions that are functionally connected with the seed region vary as a function of some variable of interest ( eg , cardiorespiratory fitness levels ) . using a seed - based approach to examine functional connectivity , voss
et al found that older adults that had higher cardiorespiratory fitness levels had greater connectivity in the so - called default mode network .
further , they found that increased connectivity mediated the fitness related enhancements of executive control . since the default mode network is reduced in older adults with mild cognitive impairment and dementia , increased functional connectivity indicates that physical activity might reduce the risk of impairment by elevating the cohesiveness of the default mode network .
in fact , results from two randomized interventions indicate that the functional connectivity of these networks can be modified after several months of physical activity .
the studies described above focus on three forms of brain health and integrity in late life : morphology , task - evoked functional dynamics , and connectivity . for each of these measures ,
cross - sectional , observational , and randomized interventions indicate that physical activity is capable of modifying age - related losses and that physical activity - induced changes in brain integrity and function mediate improvements in cognition . in summary
, the human neuroimaging literature on physical activity indicates that the brain remains modifiable into late adulthood , the effects are distributed throughout the brain , but are most robust in the prefrontal and medial temporal lobe regions .
the cochrane collaboration performed a systematic review of the effects of exercise on depression in adults of mixed ages .
they identified 32 trials ( 1858 participants ) that fulfilled their inclusion criteria , of which 30 ( 1101 participants ) provided data for meta - analyses .
based on these 30 trials , the authors concluded that exercise seems to improve depressive symptoms in individuals with depression when compared with no treatment or a control intervention . in comparison to no treatment , exercise had a moderate effect size ( standardized mean difference [ smd ] -0.67 , 95% confidence ( ci ) -0.90 to -0.43 ) .
there were no significant differences when comparing the effects of exercise with cognitive therapy or antidepressant treatment .
when the authors limited the analyses to the four high - quality trials ( 326 participants ) , the pooled smd was -0.31 ( 95% ci -0.63 to 0.01 ) indicating a small effect in favor of exercise . among the 32 trials identified that fulfilled the inclusion criteria , 8 studies were focused on or included adults older than 60 years
six of the studies involved aerobic exercise and two studies progressive resistance training . of the 6 studies that involved aerobic exercise , various exercise and comparator interventions
blumenthal and colleagues ( 1999 ) studied community volunteers with major depressive disorder ( mdd ) ( n=156 ) mean ( sd ) age of 57 ( 6.5 ) randomized to aerobic exercise ( group walking or jogging 3 times per week ) , antidepressant pharmacologic treatment ( sertraline ) , or the combination .
they found that all treatment groups had statistically significant improvement in depression scores , although participants receiving medication alone had the fastest initial response .
after 16 weeks of treatment , exercise was equally effective in reducing depression among older adults with mdd .
a limitation to this study was the absence of a placebo or control intervention . in a follow - up study , blumenthal and colleagues examined community - dwelling older adults with mdd ( n=202 ) , mean ( sd ) age 52 ( 8) , randomly assigned to home - based exercise , supervised exercise in a group setting , sertraline , or placebo for 16 weeks . while there was a high placebo response rate , the efficacy of exercise was comparable to antidepressant pharmacotherapy , and both were better than placebo .
brenes and colleagues studied 37 older adults with a mean ( sd ) age of 73.5 ( 7.8 ) with minor depression , randomized to exercise , antidepressant pharmacotherapy ( sertraline ) , or usual care over 16 weeks . in the 32 participants who completed the study , they found trends for exercise and sertraline to be superior to usual care in improving emotional and physical functioning .
mather and colleagues examined whether exercise is effective as an adjunct to antidepressant pharmacotherapy in older adults .
eighty - six older adults with depression ( mean age 65 ) were randomly assigned to attend exercise classes or health education talks for 10 weeks . at 10 weeks , a significantly higher proportion of the exercise group ( 55% versus 33% ) experienced a greater than 30% decline in depressive symptoms as measured with the hamilton rating scale for depression .
mcneil and colleagues ( 1991 ) randomly assigned 30 community dwelling , moderately depressed older adults with a mean ( sd ) age of 72.5 ( 6.9 ) to 1 of 3 interventions : experimenter - accompanied exercise ( walking ) , social contact control condition , and a wait - list control .
they found that exercise and social contact both resulted in reductions in the beck depression inventory .
lastly , williams and tappen examined the effects of exercise training for depressed older adults with alzheimer 's disease .
subjects were randomly assigned to 16 weeks of comprehensive exercise , supervised walking , or social conversation .
they found that all three groups had a reduction in depressive symptoms , with exercise showing a slightly greater benefit .
two studies found antidepressant effects of progressive resistance training in older adults with depression . singh and colleagues studied the effects of progressive resistance training ( prt ) on depressed adults 60 years and older with a mean ( sd ) age of 71.3 ( 1.2 ) . over 10 weeks compared with an attention - control group , prt was associated with an improvement in the measures of depressive symptoms , quality of life , social functioning , and strength . in a follow - up study ,
singh and colleagues found that higher - intensity prt was more effective than low - intensity prt on depression in older adults .
these studies support the argument that exercise has antidepressant effects in older adults , yet the mechanism of action remains unclear .
the studies are careful to note the potential for the antidepressant effects of expectation and attention in research participation as well as for socialization when engaging in group exercises .
further , investigators have also suggested the effects of increased self - efficacy , a sense of mastery , positive thoughts , distraction from negative thoughts , and enhanced self - concept . however , biological mechanisms related to overall brain health are also likely related to the mood elevating properties of exercise .
these mechanisms , described earlier , include enhanced gray matter volume in prefrontal cortex and hippocampal brain areas , elevated functioning of brain circuits involved in mood and emotional function such as subregions of the frontal cortex and medial temporal lobe , and improvements in functional connectivity of the default - mode network .
in addition , it is also likely that exercise is having a pleiotropic effect on molecular systems related to the hypothalamic - pituitary - adrenocortical axis , dopaminergic , noradrenergic , serotonergic neurotransmission , immune function , and bdnf ( figure 1 ) .
however , these biological mechanisms of exercise have not yet been carefully studied in older adults with depression .
the role that changes in morphology and function could have on mitigating depressive symptoms remains speculative at this time .
in this review we have briefly summarized the expansive and ever - growing literature on the effects of physical activity on brain health and plasticity .
we can conclude from this overview that physical activity has consistent and robust effects on the brain , which mediate improvements in cognitive performance and reduce the risk for neuropsychiatric disorders .
the beauty of this research is that the effects appear consistent across species and populations indicating an exceptional level of translation that is rare to find in other disciplines .
the research in this area has demonstrated that the effects of physical activity on brain plasticity in late life has a remarkable degree of specificity , such that some brain areas appear to be more commonly or easily influenced by physical activity than other areas
one explanation might be that the hippocampus , frontal cortex , and neighboring areas are more inherently plastic than other brain areas and that the degree of specificity is simply a characteristic of the brain regions examined rather than anything specific to the capabilities of physical activity per se . however , an alternative explanation is that these brain areas contain some molecular or cellular process that is influenced by participation in physical activity .
for example , bdnf has been described as one possible molecular pathway by which exercise improves cognitive function , but bdnf levels are found in different concentrations throughout the brain with higher levels in the hippocampus and cerebellum than in other areas .
a third possible explanation could be that the brain areas that show the most amount of atrophy with age , including the frontal cortex and hippocampus , are the most sensitive to the effects of physical activity .
thus , the specificity of physical activity on the frontal cortex and hippocampus might be related to the atrophying nature of these areas . according to this reasoning ,
since these brain areas are shrinking with age , there is more room for them to grow with an intervention like physical activity .
therefore , the specificity of physical activity has to do with relatively little variation in the size and function of other brain regions with advancing age .
whatever the explanation for specificity , the effects appear to be both robust and consistent across samples and populations .
this indicates that these effects are unlikely to be confounded by a particular sample characteristic ( eg , gender ) or comorbidity ( eg , depression ) and more likely reflects an adaptive biological importance of physical activity to enhance and maintain the body 's organs , including the brain . despite the well - established literature linking physical activity to brain health and plasticity in late life ,
first , although a number of studies described above have found effects with moderate intensity exercise for several months , the exact dose - response nature of the link between physical activity and mood , cognition , or brain health remains unknown .
in other words , there is a very poor understanding of how much physical activity is necessary to observe effects .
second , individuals stop exercising for a variety of different reasons including injuries , illness , and personal issues ( eg , mourning ) . because of
this it is important to examine whether the effects of a physically active lifestyle are retained or lost after some period of inactivity .
unfortunately we have a very poor understanding of the retention of the effects of physical activity .
third , we have a very poor understanding of the types of exercises that might be most useful to promote a healthier brain .
it is conceivable that competitive sports like tennis offer additional benefits beyond noncompetitive sports because of their dependence on physical coordination , cognitive effort , and social interaction . in sum , although we have a solid understanding of the potential for physical activity to enhance cognitive and brain health in late life there remain many unanswered questions for future research to pursue .
|
the human brain shrinks with advancing age , but recent research suggests that it is also capable of remarkable plasticity , even in late life . in this review
we summarize the research linking greater amounts of physical activity to less cortical atrophy , better brain function , and enhanced cognitive function , and argue that physical activity takes advantage of the brain 's natural capacity for plasticity .
further , although the effects of physical activity on the brain are relatively widespread , there is also some specificity , such that prefrontal and hippocampal areas appear to be more influenced than other areas of the brain .
the specificity of these effects , we argue , provides a biological basis for understanding the capacity for physical activity to influence neurocognitive and neuropsychiatric disorders such as depression .
we conclude that physical activity is a promising intervention that can influence the endogenous pharmacology of the brain to enhance cognitive and emotional function in late adulthood .
|
Introduction
Establishing the molecular basis of physical activity on brain health
Effects of physical activity on cognitive function in humans
Effects of physical activity and aerobic fitness on neuroimaging indices of brain health
Common biological pathways in depression
Conclusions and future directions
|
multilevel cervical spondylotic myelopathy ( mcsm ) is a serious disease which can lead to spinal cord dysfunction and a substantial decrease in quality of life . in addition , mcsm requiring multilevel cervical spine surgery can be accompanied by extensive blood loss . as spinal surgery has become increasingly complex , so control of perioperative bleeding has become an important clinical issue for spine surgeons .
excessive blood loss can lead to a range of comorbidities such as anemia , hypotension , hematoma formation , and inadequate oxygenation of organs , thereby affecting patient outcome .
excessive blood loss often requires allogeneic blood transfusion , the risks of which are numerous and among which immunological reactions and transmission of viruses are considered the most serious . in addition , hematoma formation within a few millimeters of the spinal canal can cause considerable neurological damage .
although occurrences of postoperative spinal hematoma formation requiring emergency surgery are rare , it is nevertheless important to control perioperative bleeding to decrease its incidence . tranexamic acid ( txa ) is a synthetic derivative of the amino acid lysine which operates through competitive inhibition of the activation of plasminogen to plasmin by binding specific sites on both plasminogen and plasmin , thereby retarding fibrinolysis , the degradation of blood clots .
it has been widely used in various medical fields such as cardiac surgery , gynecology , dentistry , urological surgery , and liver transplantation to reduce the perioperative blood loss , as it is relatively inexpensive and has not been cited in the literature as causing any significant untoward side effects . in addition , txa has been reported to reduce blood loss and transfusion requirements during orthopedic surgery , most commonly in knee and hip joint replacement . although the benefits of txa in spinal surgery have been reported
indeed , there are no studies of txa in cervical laminectomy with lateral mass screw fixation and bone grafting ( clf ) so far .
clf has been widely used for many years for treating mcsm caused by multilevel cervical spinal cord compression .
although some authors suggest that laminoplasty is superior to laminectomy with lateral mass screw fixation and bone grafting regarding preserved range of motion , indications for the use of clf are broader than that of laminoplasty and recent studies showed good outcomes for clf . thus , the aim of this study is to evaluate the efficacy and safety of txa in controlling blood loss during clf for the treatment of mcsm .
a retrospective comparative analysis was performed in patients with mcsm undergoing clf of vertebrae c3 to c6 .
patients with cirrhosis of the liver , serious cardiac disease , chronic renal failure , cancer , allergy to txa , a history of thromboembolic disease ( deep vein thrombosis , ischemic heart disease , pulmonary embolism , transient ischemic attack , strokes , or subarachnoid hemorrhage ) , bleeding disorders , hypercoagulation status , disseminated intravascular coagulation , pregnancy , combined anterior and posterior spinal fusions , patients receiving antiplatelet and/or anticoagulant therapy at the time of the study , and treatments of vertebrae outside of c3 to c6 were excluded from the study . between november 2014 and april 2016 , 119 clfs fitting the inclusion criteria were performed in our center . each surgeon ( hao , liu , he , wu , wang , zheng , and zhao ) in this study had more than 20 years of experience in spinal surgery .
the control group consisted of 46 patients who underwent clf for the treatment of mcsm in our institution without being given txa . in both groups
, we analyzed patient demographic trait ( age at surgery , gender , body weight , height , and body mass index ) and duration of surgery which was defined as the time from the initial incision to the completion of wound closure .
the quantity of intraoperative and postoperative blood loss and the preoperative and day 1 postoperative hematological data for each patient were obtained in the 2 groups .
total blood loss was calculated as the sum of intraoperative and postoperative blood loss ( the quantity during the 1st 16 hours ) .
the spinous processes , lamina , and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision .
laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side , prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae ( c3c6 ) .
the excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material .
lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique .
all patients in the txa group were given a dose of 15 mg / kg of txa ( transamin ; daiichi pharmaceutical , tokyo , japan ) before a skin incision was made , followed immediately with a maintenance dose of 100 mg / hour , and continued until wound closure .
no patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug .
continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value .
statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables .
all analyses were performed using ibm spss statistics for windows , version 19.0 ( ibm corp . , united states of america ) .
the spinous processes , lamina , and lateral mass facet complexes were exposed using a standard posterior midline opening after longitudinally dividing the nuchal fascia in line with a midline skin incision .
laminectomy was performed using a high speed matchstick burr to drill troughs in the bone at the lateral edge of the lamina on each side , prior to complete removal of the entire lamina and associated ligamentum flavum of the target vertebrae ( c3c6 ) .
the excised lamina and spinous processes were cleaned of soft tissue and cut into pieces for use as autograft material .
lateral mass screws were placed bilaterally on vertebrae c3 to c6 then fixed with rods using the margerl technique .
all patients in the txa group were given a dose of 15 mg / kg of txa ( transamin ; daiichi pharmaceutical , tokyo , japan ) before a skin incision was made , followed immediately with a maintenance dose of 100 mg / hour , and continued until wound closure .
no patient in the control group received intraoperative administration of txa or any other antifibrinolytic drug .
continuous variable data were presented as mean and standard deviation while categorical variable data were presented as a number and its specific value .
statistical differences between the 2 experimental groups were compared using a chi - square test or fisher exact test for categorical variables and student t test for continuous variables .
all analyses were performed using ibm spss statistics for windows , version 19.0 ( ibm corp . , united states of america ) .
there was no statistical difference in demographic values ( patients age , gender , body weight , height , and body mass index ) between the 2 groups ( table 1 ) .
no statistically significant difference in duration of surgery was observed between the 2 groups , with a mean time of 155.72 15.59 and 153.52 11.91 minutes in the control and txa groups , respectively ( p = 0.387 ) .
there was significantly less intraoperative blood loss in the txa group compared to the control ( fig .
1 ) , at 179.66 81.45 and 269.13 94.68 ml , respectively ( p < 0.001 ) .
the txa group had significantly less postoperative blood loss during the 1st 16 hours compared to the control group ( 108.08 44.31 vs 132.83 49.39 ml , p = 0.005 ) .
total blood loss in the control group ( 401.96 127.88 ml ) was significantly higher than that in the txa group ( 287.74 115.40 ml , p < 0.001 ) .
changes in blood hemoglobin ( hgb ) content and hematocrit ( hct ) were not statistically significantly different between the 2 groups : preoperative hgb :
138.28 9.12 g / l ( control ) and 140.42 10.05 g / l ( txa ) ; postoperative hgb : 122.35 10.74 g / l ( control ) and 125.21 11.29 g / l ( txa ) ; preoperative hct : 41.78 2.62% ( control ) and 42.07 2.08% ( txa ) ; and postoperative hct : 36.50 3.22% ( control ) and 37.32 2.41% ( txa ) .
although the hgb and hct values in the txa group were all higher than those of the control group , the differences were not statistically significant ( fig .
no patient required an allogeneic blood transfusion during or after the surgery in either group and no serious intra- or postoperative complications , for example , dural tear , infection , epidural hematoma formation , deep - vein thrombosis , pulmonary embolism , allergic reaction , renal failure , or cardiopulmonary complications were observed in either group .
furthermore , no minor side effects associated with the use of txa such as nausea , vomiting , headache , or diarrhea occurred in either of the groups . demographic parameters of patients in each group .
the txa ( tranexamic acid ) group had significantly less intraoperative , postoperative , and total blood loss compared to the control group . there were no significantly different in blood hgb hct between the control and txa groups .
hgb = hemoglobin , hct = content and hematocrit , txa = tranexamic acid .
numerous studies have supported the use of txa in orthopedic surgery . although it has been evaluated in many studies , the safety and efficacy of txa for blood loss reduction in spinal surgery has not been clearly demonstrated , especially for clf .
clf for the treatment of mcsm has proved to be a successful technique for restoring normal cervical lordotic alignment , recovering neurological function , and decreasing the morbidity of c5 palsy and axial pain .
the principal disadvantage of this approach is a significant decrease in motion due to the fixation of the target vertebrae .
laminoplasty allows a better range of neck motion although it becomes more restricted in flexion and extension over time . for patients with mcsm combined with simple instability or correctable kyphosis , clf is a more suitable method than laminoplasty , even if the consequence is neck stiffness .
fibrinolysis increases transiently when patients undergo surgery , and it has been shown that it contributes to perioperative blood loss during spinal surgery .
txa inhibits fibrinolysis by blocking the lysine - binding sites of plasminogen , plasmin , and tissue plasminogen activator .
since fibrinolysis is activated immediately during and after surgery , txa should be administered before surgery begins .
the half - life of txa is approximately 80 minutes in patients with normal renal function .
pharmacokinetic evidence suggests a loading dose of 10 to 15 mg / kg followed by a maintenance dose of 1 mg / kg / hour or repeated dosing .
li et al conducted a meta - analysis of 6 randomized controlled trials and believed that higher txa dosage ( 15
yang et al conducted a meta - analysis of 9 randomized controlled trials which had a similar sample to li 's study .
the 2 meta - analyses arrived at a similar conclusion although the quantity of blood loss and incidence of blood transfusion were different .
elwatidy et al used a high single dose ( 30 mg / kg ) of txa and found that it reduced total blood loss and incidence of blood transfusion .
raksakietisak et al reported that 2 doses of txa ( 15 mg / kg ) reduced perioperative blood loss and incidence of blood transfusion among low - risk adult patients undergoing elective complex thoracolumbar spine surgery .
, we evaluated the effects of txa with a dose of 15 mg / kg followed by a maintenance dose of 100 mg / hour .
the txa treatment group lost significantly less blood than that of the control group , including during the intra- and postoperative periods .
total blood loss in the txa group was 28% less than that of the control group . although the hgb levels and hct in the txa group were higher than that of the control group , the differences were not significant , possibly because the sample size was too small and the perioperative blood loss in clf relatively minor .
a theoretical anxiety associated with the use of txa is its potential for inducing thromboembolic complications .
however , many studies have shown that the administration of txa does not increase this risk . in our study , there were no clinical symptoms or signs of thromboembolic events , such as deep - vein thrombosis or symptomatic pulmonary embolism .
thus , the use of txa in patients undergoing clf should be regarded as safe .
additionally , we are unsure whether the complexity of the surgery has an effect on the benefit of txa .
a prospective randomized controlled study would better determine the safety and efficacy of txa during laminectomy with lateral mass screw fixation and bone grafting .
in this study , blood loss ( both intra- and postoperative ) in the txa group was significantly lower than that in the control group , and no major intraoperative complications occurred .
our results indicate that the use of intravenous txa is both safe and effective in reducing blood loss in clf .
prophylactic txa may provide the benefit of limiting excessive blood loss in posterior approach cervical spinal surgery .
a future prospective randomized controlled trial will provide superior evidence of the efficacy and safety of txa .
|
abstractto assess the safety and efficacy of tranexamic acid ( txa ) for decreasing perioperative blood loss in cervical laminectomy with lateral mass screw fixation and bone grafting ( clf ) , in which all surgical procedures are identical.from november 2014 to april 2016 , we performed a retrospective comparative analysis of 119 patients with multilevel cervical spondylotic myelopathy who had undergone a clf from c3 to c6 in our center .
all surgeries were performed on the patients using a consistent , standard procedure .
patients were divided into control ( 46 ) and txa ( 73 ) groups according to whether or not they had received txa treatment before and during surgery .
demographic profiles of patients such as gender , age , body weight , height , and body mass index were collated and differences between the 2 groups compared .
preoperative and postoperative hematological data in addition to intraoperative and postoperative blood loss were compared between the 2 groups .
additionally , any complications of txa were also evaluated to assess safety.there was no statistically significant difference in demographic traits between the 2 groups .
intraoperative blood loss in the txa group ( 179.66 81.45 ml ) was significantly lower than that of the control group ( 269.13 94.68 ml , p < 0.001 ) , as was postoperative blood loss ( 108.08 44.31 and 132.83 49.39 ml , respectively ; p = 0.005 ) .
total blood loss in the txa group ( 287.74 115.40 ml ) was also significantly lower than that of the control group ( 401.96 127.88 , p < 0.01 ) .
no major intraoperative complications occurred in any of the cases.txa significantly reduced perioperative blood loss in clf with no major side effects .
|
Introduction
Material and methods
Surgical procedure
Drug dose
Statistical analysis
Results
Discussion
Conclusions
|
musculoskeletal stiffness and limited range of motion ( rom ) can restrict functional joint
movement , particularly during ankle supination following neurological diseases1 .
ankle dorsiflexion rom restrictions impair
dynamic balance and gait , which may contribute to secondary injuries ; reduced ankle rom and
tight calf muscles can cause poor gait , inefficient energy use , impaired balance , and an
increased risk of falls2 .
musculoskeletal
transformations manifesting as abnormal joint stiffness and limited rom result in numerous
restrictions in the function and joint movements of patients3 .
limited passive ankle dorsiflexion rom during knee extension can
alter foot positioning and result in compensatory foot movements , leading to an abnormal
gait , which can result in ankle sprains and lower extremity overuse injuries4 .
studies have reported that balance - training
programs that alter the somatosensory input , such as the use of an unstable surface to
induce equalized weight distribution , can be helpful in hemiplegic patients5 .
kim et al . used an ankle board with a slope
in their center to achieve efficient ankle stretching and increased dorsiflexion6 .
they suggested that stretching exercises
using their device resulted in a greater increase in foot pressure compared to that with
stretching exercises using existing inclined boards during ankle dorsiflexion
stretching6 .
therefore , we investigated
the effects of an unstable inclined board on the active and passive ankle rom in patients
with ankle stiffness .
the study included 10 female patients ( mean age , 22.8 5.3 years ; height , 158.9 3.0 cm ;
weight , 61.2 4.7 kg ) with ankle stiffness caused by diseases or medical conditions .
the
patients were selected based on ankle stiffness scores obtained using a symptom checker
tool .
the patients were assigned randomly to two groups ( both n = 5 ) and their height and
weight were measured .
the patients were not cognitively impaired and were capable of
independent walking for 20 m without difficulty .
patients were excluded if they had a
history of or current neurological conditions . before participating ,
the purpose and methods
of the study were explained to the patients , and all provided informed consent , according to
the principles of the declaration of helsinki .
active and passive ankle dorsiflexion were measured using a standard 31.75 4.45 cm clear
plastic goniometer , with one increment , a 360 scale , and a bubble level at each end .
group
1 performed the ankle dorsiflexion stretching exercise using a wooden inclined board , and
group 2 performed ankle dorsiflexion stretching exercises using an air - cushioned inclined
board that provided an unstable surface .
rubber sheets were attached to the inclined boards to prevent falls due to
sliding .
the patients performed ankle dorsiflexion stretching exercises for 5 min , with a
10-min rest between each set , five times a day for 1 week .
the active and passive ankle
dorsiflexion angles were measured bilaterally using the goniometer before and after the
study .
independent and paired t - tests were used to assess within- and between - group differences in
ankle dorsiflexion , with spss for windows ( spss , chicago , il , usa ) .
the amount of active and passive dorsiflexion did not differ between the two groups before
the exercises , with active dorsiflexion angles of 18.2 6.4 and 19.3 4.2 and passive
dorsiflexion angles of 23.4 5.5 and 22.6 5.7 in groups 1 and 2 , respectively ( both p
> 0.05 ) .
the stretching exercises significantly ( p < 0.05 ) increased the active and
passive ankle dorsiflexion angles in both groups compared to those at baseline .
the active
dorsiflexion angle was significantly increased ( p < 0.05 ) in group 2 compared to that in
group 1 after the exercises .
the active dorsiflexion angles before and after the exercises
were 18.2 6.4 and 22.1 5.0 in group 1 and 19.3 4.2 and 28.1 6.9 in group 2 ,
respectively .
the passive dorsiflexion angles did not differ significantly between the
groups after the exercises ( p>0.05 ) and were 23.4 5.5 and 32.2 5.3 in group 1 and
22.6 5.7 and 35.1 6.0 in group 2 before and after exercising , respectively .
the
air - cushioned inclined board provided an unstable surface for performing ankle dorsiflexion
stretching exercises .
the results showed that ankle stretching exercises using the unstable
inclined board significantly increased the active dorsiflexion angle compared to that with
exercises using a wooden inclined board .
we believe that the use of the air - cushioned
inclined board provides somatosensory input and subsequently stimulates active ankle
dorsiflexion by promoting automatic postural control .
sensory proprioception of the ankle
and sensory compression of the plantar are important in the control of sway7 .
an increase in the number of somatosensory
inputs affects the muscles , and the use of an unstable support surface result in many
different somatosensory inputs that limit compensatory action7 .
performing cognitive tasks in a standing posture reduces internal
focus and induces external focus , which promotes automatic postural control8 .
stretching improve the flexibility of
tendons , and changes in the mechanical condition of tendons depend on the type of stretching
performed9 .
, the passive dorsiflexion angles did not differ significantly between the two
groups after ankle stretching exercises .
however , stretching exercises significantly
increased the active and passive ankle dorsiflexion angles in both groups compared to those
at baseline .
these results suggest that passive ankle exercises using a wooden inclined
board only stretch muscle and tissue , while exercises using an unstable inclined board
stretch muscle and tissue , and stimulate activation of the ankle dorsiflexor .
active ankle
dorsiflexion was more effectively improved with ankle stretching exercises using an unstable
inclined board than with exercises using a wooden inclined board .
|
[ purpose ] the present study assessed the effects of using an unstable inclined board on
the active and passive ankle range of motion in patients with ankle stiffness . [ subjects ]
the study included 10 young female patients with ankle stiffness .
[ methods ] the patients
were divided into the following two groups : a group that performed ankle dorsiflexion
stretching exercises using a wooden inclined board and a group that performed stretching
exercises using an air - cushioned inclined board ( unstable inclined board ) .
active and
passive ankle dorsiflexion angles were measured bilaterally using a goniometer .
[ results ]
both inclined boards significantly increased active and passive ankle dorsiflexion .
after
performing ankle stretching exercises , active dorsiflexion significantly increased the
unstable inclined board compared to that using the wooden inclined board .
however , the
passive dorsiflexion angles did not differ significantly between the two groups after
ankle stretching exercises .
[ conclusion ] the use of an unstable inclined board might
stimulate activation of the ankle dorsiflexors in addition to stretching muscle or tissue .
active ankle dorsiflexion was more effectively improved with stretching exercises using an
unstable inclined board than with exercises using a wooden inclined board .
|
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
|
the dermal vascular changes have been evaluated by electron microscopy and shown to be due to change from arterial - type vessels to venules .
the vascular changes may also precede the epidermal alterations , as shown in some studies .
morphometric evaluation of dermal vasculature in psoriasis has shown increased endothelial and luminal volume of vessels compared to control subjects .
studies utilizing native capillaroscopy have demonstrated reduction in microvessel density and length density after pulsed dye laser treatment . however , none of the earlier studies have compared vascular morphology between psoriasis and psoriasiform dermatitis .
this study was aimed at evaluating the morphometric parameters of papillary dermal vessels in psoriasis and to compare these with psoriasiform lesions .
in this study , skin biopsies from 25 cases of psoriasis and 25 of psoriasiform lesions were included .
the diagnosis of psoriasis was confirmed on the basis of clinical features ( pink to red papules with fine silvery scales and positive auspitz sign ) in conjunction with characteristic histologic changes .
the latter included : regular acanthosis with club - shaped rete ridges , suprapapillary epidermal thinning , hypogranulosis , confluent parakeratosis , spongiform pustules of kogoj and/or munro microabscesses .
psoriasiform lesions included in the study were : endogenous eczema : nine cases , hyperkeratotic eczema : seven cases , seborrheic dermatitis : three cases , nummular eczema : 2 cases , and one case each of allergic contact dermatitis , irritant contact dermatitis , lichen simplex chronicus , and pityriasis rosea .
serial sections were stained for cd34 ( biogenex , usa ) using the standard streptavidin - biotin - peroxidase method . using image analysis software ,
the number , area , and length density of microvessel in dermal papillae were calculated from the cd34-stained sections .
the microvessel density was calculated as number of vessels per unit ( mm ) papillary dermal area .
length density of microvessel ( mm / mm or mm ) was calculated by superimposition of cycloid grid on vertical sections of skin biopsy [ figure 1 ] .
microvessel length density is defined as length of microvessels per unit dermal area and was calculated as 2i / l , where i refer to the number of intersections and l is total length of test lines .
ratio of microvessel area to the papillary dermal area was also calculated in both the groups included .
photomicrograph showing immunostaining for cd34 in a case of psoriasis with superimposed cycloid grid for morphometry ( lsab , 200 ) appropriate statistical analysis was performed on the morphometric parameters between the two groups .
on light microscopic evaluation of cd34-stained sections , biopsies from psoriasis showed much greater microvascular staining in the papillary dermis compared to psoriasiform dermatitis [ figure 2 ] .
photomicrograph demonstrating the dermal vessels in psoriasis ( a , lsab , 200 ) and psoriasiform dermatitis ( b , lsab 200 ) morphometric analysis showed that length density of microvessels was significantly higher in psoriasis ( 3.731.45 mm ) compared to psoriasiform lesions ( 2.991.99 mm , p value 0.03 ) .
microvessel density , i.e. , number of vessels per unit dermal area , was also higher in psoriasis ( 71.2840.05 ) than psoriasiform lesions ( 62.732.03 ) , though the difference was not statistically significant .
in contrast to these parameters , the ratio of microvessel area to papillary dermal area was similar in psoriasis ( 0.130.07 ) and psoriasiform lesions ( 0.140.11 ) [ table 1 ] . comparative analysis of dermal microvasculature in psoriasis and psoriasiform dermatitis these three morphometric vascular parameters are depicted in scatter plots [ figures 3a c ] . ( a ) scatter plots of vessel area per unit papillary dermal area ; ( b ) microvessel density ; and ( c ) length density in psoriasis and psoriasiform dermatitis
psoriasis is an autoimmune disease affecting people of all ages , though there is a tendency for onset in early adulthood in patients with genetic transmission . apart from epidermal changes , skin biopsies from patients with psoriasis demonstrate alterations in papillary dermis such as edema and dilated tortuous capillaries .
these dilated capillaries are the source of bleeding points generated by gentle scraping of skin ( auspitz sign ) .
few ultrastructural studies have shown that dermal capillaries in psoriasis have a wider lumen , bridged fenestrations with edematous areas in endothelial cytoplasm .
these changes also correlate with enhanced cutaneous blood flow , including in the perilesional clinically unaffected skin .
it has been proposed that vascular changes in the papillary dermis , including enhanced expression of adhesion molecules , permit leukocyte adhesion and assist in the establishment of inflammatory response .
barton et al . , in their study , demonstrated higher endothelial volume and luminal volume in the lesional psoriatic skin compared to uninvolved skin of patients with psoriasis as well as control subjects .
the authors suggested that increase in these parameters was partly due to the increase in number of vascular profiles . in another study ,
computerized image analysis was performed to demonstrate the great degree of expansion of the vascular plexus in papillary dermis .
the same study also showed significant endothelial proliferation in the papillary dermis of active psoriatic plaques .
this study aimed at evaluating the vascular changes in psoriasis vis - - vis psoriasiform dermatitis .
this comparison has not been performed in the available literature till date , to the best of our knowledge .
we found a significantly higher length density of vessels in psoriasis compared to psoriasiform lesions ( p<0.05 ) .
microvessel density was also higher in psoriasis , though the difference did not reach statistical significance .
these results indicate that though there is some vascular proliferation in response to inflammation in psoriasiform dermatitis , the tortuosity , and elongation of vessels reflected by length density is significant in psoriatic lesions only . with the advancing automation in diagnosis
, these morphometric techniques might assist in differentiation between psoriasis and psoriasiform lesions , especially in early lesions of psoriasis . the vascular proliferation in psoriatic skin
systemic therapy for psoriasis , such as methotrexate , cyclosporine , and tnf antagonists exert their therapeutic effect by immune modulation as well as interference with proangiogenic mediators .
in addition , several molecules such as neovastat ( inhibitor of endothelial cell proliferation ) and inhibitors of vegf receptors are undergoing clinical trials .
hern et al . , performed native capillaroscopy and showed a significant reduction in the microvessel density , image area fraction , length , density and vessel image width after pulsed dye laser treatment of plaque psoriasis .
hence , the superficial microvascular bed in psoriatic skin is a legitimate and easily accessible target for antiangiogenic therapy .
the present study demonstrates significant difference in the papillary dermal vasculature between psoriatic skin and psoriasiform dermatitis . these results , which need to be confirmed in further larger studies ,
|
background : vascular proliferation in the papillary dermis is considered to be an important and probably an early feature of psoriasis .
few morphometric studies have attempted to analyze the vascular changes .
however , no study was found in the available literature comparing vascular changes between psoriasis and psoriasiform dermatitis.materials and methods : skin biopsies from 25 cases each of psoriasis and psoriasiform lesions were immunohistochemically stained for cd34 ( endothelial marker ) .
microvessel density ( mvd ) , microvessel length density and ratio of microvessel area to papillary dermal area were calculated using image analysis software.results:skin biopsies from psoriasis showed higher staining for cd34 on light microscopy . using morphometric techniques ,
microvessel length density was significantly higher in psoriasis compared to psoriasiform lesions ( p value < 0.05 ) .
mvd was also higher in psoriasis , though the difference was not significant .
the ratio of microvessel area to dermal area was almost similar in both the groups.conclusion:our results indicate that vascular tortuousity and dilatation is significant only in psoriatic lesions .
these results may assist in automated diagnosis of skin biopsies .
|
Introduction
Materials and Methods
Results
Discussion
Conclusion
|
toxoplasmosis , a zoonosis caused by obligate intracellular protozoan toxoplasma gondii [ 1 , 2 ] , occurs in about one third of world population . having the cats as definitive hosts , especially the domestic cat
, it shows a wide range of intermediate hosts , including humans . in immunocompetent individuals ,
severe forms are secondary to infection in immunocompromised patients or when acquired during pregnancy , when it may cause congenital disease with severe visual and neurological impairment .
transplacental transmission of t. gondii occurs during the period of acute maternal infection [ 57 ] .
the rate of congenital toxoplasmosis with risk for severe fetal varies from 15 to 68% , depending on gestational age ; the transmission rate is highest in the later stages of pregnancy [ 8 , 9 ] .
the congenital disease is characterized by a broad spectrum of clinical manifestations with neurological , ophthalmological , and systemic involvement .
although most newborns with congenital infection show no signs of infection at birth , up to 85% of these will develop visual impairment in their lives , and 55% will present neurological disorders [ 1012 ] .
the prenatal care , when done properly and from the beginning of the pregnancy , allows early diagnosis of infection , allowing the team more time and resources to treat the fetus .
there are few studies addressing the degree of knowledge on toxoplasmosis of the health professionals . in the few surveys carried out among obstetricians , a deficit in the knowledge about the diagnostic , clinical , and epidemiological aspects of toxoplasmosis
nevertheless , similar surveys were not developed in countries with high prevalence of toxoplasmosis as brazil [ 1820 ] , where a previous study observed little knowledge on the subject from pregnant women , in particular regarding the infection prevention . in a previous work
, the authors observed that the majority ( 81.3% ) of the women sent to a referral service with a diagnosis of toxoplasmosis were coming from public health units and that for only 16% of them treatment was initiated at the origin unit .
this study aims to assess the knowledge about toxoplasmosis by doctors and nurses involved in prenatal care at public health units in brazil .
we conducted a cross - sectional study of 118 physicians and nurses who provide prenatal care at public health units of a midsize brazilian city .
we chose the city of juiz de fora , in minas gerais ( 214551s and 432101w ) , with 500,000 inhabitants and altitude tropical climate .
this city was selected because it is a located in a region of high prevalence of toxoplasma infection and offers the following services for the care of pregnant women : basic health units with the family health program ( fhp ) and three public hospitals .
all physicians and nurses providing care in all basic units of the family health program ( fhp ) , prenatal clinics , and public hospitals were invited to participate .
the study excluded the professionals who did not provide prenatal care last year , who works exclusively in rural areas , and those who did not agree to participate .
data was collected through an anonymous and self - completed questionnaire , including questions related to the professional profile and nine items about toxoplasmosis equally distributed among diagnosis , clinical issues , and prevention ( table 1 ) .
epidata version 3.1 was the software used with double capture and control for data entry .
the analysis was performed in the statistic software statistical package for social sciences ( spss ) version 16.0 . in exploratory analysis of the data
, we described the frequencies of qualitative variables and summary measures , such as mean and standard deviation for continuous variables . in continuous variables
, we checked the data normality ( total number of correct answers in each of the variables : diagnosis , clinical issues , and prevention ) by the kolmogorov - smirnov test . at the 5% level of significance
, the hypothesis of normally distributed data was rejected , which indicated the use of nonparametric tests . to check for differences in the number of correct answers and the different professional performances ( fhp or hospital )
p values < .05 indicated significant differences , as demonstrated by the comparison of medians ( md ) and their respective interquartile ranges ( iqrs ) . in checking the association between qualitative variables ( sex , time from graduation , type of graduation , profession , expertise , previous experience with pregnant women with toxoplasmosis , areas of expertise ) and the total number of correct scored ( up to four correct answers and over four correct answers ) the independence pearson chi - square test was used . for tables with low counts ( less than five ) fisher 's exact test was used .
the cutoff of the total number of correct answers was categorized according to the median of total correct answers .
the study was approved by the ethics in research committee from the institute of clinical research evandro chagas ( instituto de pesquisa clnica : ipec)-fiocruz under the protocol 0029.0.009.000 - 09 .
of the 118 professionals who participated in the study , 112 respondents completed the questionnaire ( 61 physicians and 56 nurses ) , and six of them did not agree to respond the questionnaire .
the population consisted mostly of women ( 80.5% ) , age 39 9.7 , with a degree in public schools ( 88.2% ) and postgraduation stricto and lato sensu .
seventy percent ( 83 ) of professionals worked at primary fhp and 30.0% in public hospitals .
fifteen percent also worked in private hospitals , 13.7% in private clinics , and 12% in educational and/or research institutions .
fifteen percent of the respondents reported having provided care for pregnant women with toxoplasmosis in the past 12 months . in the block of questions related to the diagnosis
, most professionals said that repeated serology for toxoplasmosis in pregnant women is needed for those who are not immune .
regarding prevention , 97.4% of professionals recognized the cat as the animal that eliminates the parasite in the feces , but 51.7% said the dogs also eliminate oocysts .
the largest number of errors was evident in relation to the education of nonimmune pregnant women in regard to not ingesting raw vegetables ( table 1 ) .
the overall median from the hospital was significantly higher than the fhp 's median . comparing the number of correct answers according to the area of the professional expertise
, the professionals who worked in hospitals showed the total number of correct answers on the issues of diagnosis , significantly higher than those who worked in the fhp .
both professionals in hospitals and the fhp had more correct answers to questions about clinical issues and fewer correct answers on prevention ( table 2 ) . in the comparison
according to the professions , the physicians had the highest number of correct answers in the diagnostic and clinical issues . regarding prevention , no significant differences were found between the two professional categories .
the issues of prevention had lower scores both among physicians and nurses ( table 3 ) .
table 4 presents the results of the analysis of the association of the total number of correct answers classified ( up to four or > 4 correct answers ) with several characteristics of the professionals .
greater number of correct answers was observed in professionals with time of graduation up to 10 years and among physicians .
the basic health units ( bhus ) are the main gateway to the health system in brazil , and the professionals who work at the family health strategy have a wide work area .
therefore , it is necessary that health professionals have a general education as the proper conduct of health problems depends crucially on the degree of the professional 's knowledge .
as it is a mother - to - child-(mtc- ) transmitted disease , early diagnosis and treatment can prevent serious and irreversible fetal damage [ 13 , 23 ] .
thus , it is critical that doctors and nurses who provide prenatal care are appropriately trained on the prophylactic , diagnostic , and clinical aspects of the mtc - transmitted diseases . in relation to t.
gondii infection , its complex life cycle , the variable clinical spectrum including mostly oligosymptomatic presentation , and no consensus on guidelines contribute to the ignorance or mistaken notions of professionals with regard to this zoonosis . regarding the contents of the questionnaire ,
the block of questions concerning diagnosis , most professionals have responded correctly that it is necessary to repeat serology for toxoplasmosis in pregnant women who are not immune .
the periodicity of this screening was not asked as the authors opted for a question of minor complexity .
less than half of professionals recognized low avidity as an indicator of recently acquired infection , and an even smaller proportion still knew that igm anti - t .
gondii may remain positive for more than a year . the greatest number of correct answers concentrated on clinical issues , although the responses showed a misunderstanding between the period of greatest transmissibility and the period of increased severity of congenital injuries [ 25 , 26 ] . about prevention ,
although 97.4% of professionals recognize the cat as the animal that eliminates the parasite in feces , professionals attributed this role , which is unique to felidae [ 27 , 28 ] , to other animals as well , especially dogs ( 51.7% ) and pigeons ( 21.6% ) .
the largest number of errors was evident in relation to the orientation of non - immune pregnant women as to not eating raw vegetables .
as oocyst has a major importance in the epidemiology of toxoplasmosis , and taking into account water as an important font of infection and oocyst resistance to chlorine and acetic acid , the authors recommend that eating raw salads and unpeeled fruits should be avoided in nonimmune toxoplasma pregnant women in brazil [ 18 , 2931 ] .
the fact that the overall median number of correct answers and diagnosis from professionals working in hospitals were higher than those of the fhp may in part be explained by the fact that professionals working in hospitals have more training in high - risk prenatal care .
however , a higher number of correct questions of prevention among professionals fhp were expected , since the basic health unit has as one of its main missions educational activities aimed at preventing health problems .
likewise , the median number of correct answers among physicians higher than that of nurses is expected in the diagnosis and clinical issues , as these issues are the object of focus in medical schools than in schools of nursing .
however , the basic knowledge of nurses in issues related to prevention is inconsistent with the role of nurses as health educators .
eleven ( 15.3% ) respondents reported having provided care to pregnant women with toxoplasmosis in the last 12 months , two times the percentage already considered high in a study conducted in the united states , implying that a considerable proportion of doctors and nurses encounter over their lives with at least one case of a pregnant woman with toxoplasmosis . the greatest number of correct answers among the professionals with less training time is consistent with the literature that indicates an inverse correlation between knowledge and years of professional practice , justifying the need for recertification exams in some countries [ 31 , 32 ] .
continued education seems especially useful when targeted to specific groups and disciplines like our target population .
the low ratio observed ( 44.4% ) of correct answers on questions about global toxoplasmosis reinforces the need for action and continuing education programs on this zoonosis .
finally , the authors suggest that similar surveys shall be conducted in other endemic regions and with larger populations . the establishment of a cutoff for the number of correct answers can be a useful tool for a possible intervention , providing support to actions for continuing education about pregnancy and congenital toxoplasmosis for these occupational categories .
|
congenital toxoplasmosis is a potentially severe infection and its prevention is most often based on serological screening in pregnant women .
many cases could be prevented by simple precautions during pregnancy .
aiming to assess the knowledge about toxoplasmosis among professionals working in antenatal care in a high prevalent region , a questionnaire was administered to 118 obstetric nurses and physicians attending at primary care units and hospitals .
the questionnaire was self - completed and included questions on diagnosis , clinical issues , and prevention .
only 44% of total answers were corrected .
lower scores were observed among those with over 10 years of graduation , working in primary care units , and nurses .
errors were mainly observed in questions of prevention and diagnosis . as congenital toxoplasmosis is a mother - to - child ( mtc ) transmitted disease , early diagnosis and treatment can prevent serious and irreversible fetal damage .
thus , doctors and nurses who provide prenatal care must be appropriately trained on prophylactic , diagnostic , and clinical aspects of toxoplasmosis .
the authors suggest that measures should be taken for continuing education regarding toxoplasmosis in pregnancy .
|
1. Introduction
2. Methods
3. Results
4. Discussion
|
it is present , in fact , in compounds with different activities such as inhibitors of p38 map kinase , inhibitors of hiv - ep1 cellular transcription factor , inhibitors of coagulation factor xa , and -chemokine receptor ccr5 antagonists in anti - hiv therapy ; in particular we have focused our work on the development of new cyp51 inhibitors , active both on fungal strains and trypanosoma cruzi .
many literature data evidenced that the pyridine group can efficaciously replace the heme - iron chelating azole moiety present in classical azole cyp51 inhibitors and , therefore , the alkylation of 4-aminopyridine ( 4ap ) represents an important goal in organic synthesis to develop novel classes of antifungal and antiparasitic drugs [ 7 , 8 ] . due to the wide presence of these products ,
the alkylation of 4-aminopyridine ( 4ap ) is therefore an important goal in organic synthesis .
some examples are the efficient condensation of 4ap with alcohols catalyzed by benzaldehyde or copper [ 10 , 11 ] or magnetite , the reaction of 4ap with an acyl chloride , and the following reduction of the amide with lialh4 . the most straightforward method , however , is the direct alkylation of 4ap with alkyl halides , although it suffers from some drawbacks .
the two different nitrogen atoms compete in the alkylation reaction and usually the more nucleophilic pyridine nitrogen atom reacts faster , leading to the corresponding pyridinium salt ( scheme 1 ) [ 14 , 15 ] . in these case , the use of a very strong base is therefore necessary : n - buli was successfully used by singh and coworkers , obtaining n - methyl- and n - ethyl-4-aminopyridines in 7480% yields .
a viable alternative is the enhancement of the nucleophilicity of the amine nitrogen atom ( versus the pyridine one ) , allowing the use of weaker bases .
an example is the activation of 2-aminopyridine as formyl or boc derivative at the amine nitrogen atom , with subsequent deprotonation using sodium hydride , alkylation , and deprotection with trifluoroacetic acid .
the deprotonation of n - boc-2-aminopyridine with nah needs a careful control of the temperature ( 05c ) and is carried out in anhydrous dmf , with a vigorous stirring required to keep the suspension fluid . in this context , we envisaged the possibility to alkylate n - boc-4-aminopyridine ( n - boc-4ap ) using milder reaction conditions , that is , using electrogenerated tetraethylammonium cyanomethanide ( et4nch2cn ) .
this base , the acetonitrile anion , can be easily obtained by cathodic galvanostatic reduction of a solution of acetonitrile containing tetraethylammonium hexafluorophosphate as supporting electrolyte ( scheme 2 ) , without by - products ( the reagent is the electron ) , and it was successfully used by us in a good variety of reactions , such as the selective n - alkylation of bifunctional compounds , the gewald reaction , the synthesis of -lactams , and the synthesis of carbamates . the actual mechanism for the formation of acetonitrile anion is not known , but a hypothesis based on the direct reduction of the tetraethylammonium cation has been reported ( scheme 2 ) .
the high reactivity of this base is ascribable to the large tetraethylammonium counterion , which renders the acetonitrile anion extremely reactive .
moreover , its reaction as a base gives no by - products , as the protonation restores the molecule of solvent .
the reaction of electrogenerated acetonitrile anion with 4ap , followed by an alkyl halide , leads to poor yields in desired compound with the pyridinium salt being the major product .
this prevents the direct use of ch2cn with 4ap . on the other hand , if the amine nitrogen is activated as boc derivative ( n - boc-4ap ) , the deprotonation / alkylation reaction using acetonitrile anion leads to products1 in very high yields ( scheme 3 and table 1 , entries 16 ) .
the classic deprotection with trifluoroacetic acid allows obtaining the desired products2 ( scheme 3 and table 1 , entries 16 ) .
the data in table 1 highlight that the reaction of deprotonation of n - boc-4ap using electrogenerated acetonitrile anion , alkylation with both alkyl and benzyl halides , and deprotection with trifluoroacetic acid is very efficient , with overall yields of 7886% .
however , when the alkylating agent is a bromoacetophenone , the yields in alkylated product are lower and in most cases the deprotection reaction leads to the dealkylation of the starting material ( table 1 , entries 710 ) .
as many biologically active compounds contain the dialkylated 4-aminopyridine moiety , we tried to carry out a second alkylation on products 2a j using acetonitrile anion but , as expected , the high nucleophilicity of the pyridine nitrogen led to the synthesis of the corresponding pyridinium salt .
we thus carried out this second alkylation using strong bases , the most efficient being t - buok in dmso ( scheme 4 ) , although the yields in dialkylated 4ap were not very high .
symmetrically dialkylated 4ap , 4ap was subjected to deprotonation with t - buok in dmso , adding an excess of alkylating agent .
the reaction led to a mixture of mono- and dialkylated 4-aminopyridines , in moderate to acceptable yields .
a selection of synthesized compounds was in vitro tested to evaluate antifungal activity against different strains of c. albicans , c. parapsilosis , and cryptococcus neoformans ; data are reported in table 4 . as can be evidenced the nonsymmetrical dialkylated 4aps 3ac and 3ae showed a moderate antifungal activity towards c. albicans and c. parapsilosis with mic50 values of 32 g / ml and showed an interesting activity against cryptococcus neoformans , with mic50 values of 0.4 and 4 g / ml , respectively .
otherwise , the symmetrical dialkylated 4aps 3cc , 3ee and the boc - protected monoalkylated 4aps 1b , 1e , 1f showed poor antifungal activity with mic50 and mic100 64 g / ml . furthermore , the symmetrical dialkylated 4aps 3cc , 3ee , and 3ff were in vitro tested to evaluate the activity against trypanosoma cruzi , trypanosoma brucei , leishmania infantum , and plasmodium falciparum ; the results are summarized in table 5 .
as can be evidenced , all tested compounds showed a moderate activity versus p. falciparum and an interesting activity towards l. infantum with ic50 values lower than the reference drug miltefosine ; otherwise , they resulted scarcely active against t. cruzi and t. brucei .
in conclusion , we demonstrated the usefulness of electrogenerated acetonitrile anion in the alkylation of n - boc 4-aminopyridines , both from the point of view of the high yields and of the cleanliness of the reaction ( no by - products ) .
the deprotection of n - boc 4-aminopyridines allowed obtaining monoalkylated 4-aminopyridine in very high yields .
the following alkylation , by means of t - buok and alkyl halides , led to nonsymmetrically dialkylated 4-aminopyridine , while symmetrically dialkylated products were obtained directly from 4-aminopyridine by reaction with an excess of t - buok and alkyl halide .
furthermore , it can also be concluded that the monoalkylation of the 4ap leads to inactive products and otherwise interesting activity against fungi and some protozoa can be obtained by dual , symmetrical , or nonsymmetrical dialkylation of the amino group of 4ap ; these active molecules can be considered as lead compound to develop new antifungal and antiprotozoal compounds .
to a solution of di - tert - butyl dicarbonate ( 3 mmol ) in acetonitrile ( 3 cm ) at room temperature 4-aminopyridine ( 3 mmol ) was slowly added .
the solvent was evaporated and the crude 4-[n-(tert - butoxycarbonyl)amino]pyridine ( > 95% ) was used in the electrolyses without further purification .
rf ( 30% ethyl acetate in light petroleum ether ) 0.20 ; h nmr ( 200 mhz , cdcl3 ) 1.53 ( s,9h ) , 6.9 ( bs , 1h ) , 7.32 ( dd , j = 4.8 , 1.6 hz , 2h ) , 8.45 ( dd , j = 4.8 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.2 , 81.7 , 112.3 , 145.6 , 150.3 , 151.9 ; eims , m / z : 194 ( m , 1% ) , 137 ( 2% ) , 121 ( 5% ) , 120 ( 8% ) , 94 ( 50% ) , 78 ( 4% ) , 57 ( 100% ) . constant current electrolyses ( i = 25 ma cm ) were performed under a nitrogen atmosphere , at 20c , using an amel model 552 potentiostat equipped with an amel model 731 integrator .
all the experiments were carried out in a divided glass cell separated through a porous glass plug filled up with a layer of gel ( i.e. , methyl cellulose 0.5% volume dissolved in dmf - et4npf6 1.0 mol dm ) ; pt spirals ( apparent areas 0.8 cm ) were used both as cathode and anode .
mecn - et4npf6 0.1 mol dm was used as solvent - supporting electrolyte system ( catholyte : 20 cm ; anolyte : 5 cm )
after 145 c were passed , the current was switched off and 1 mmol of alkylating agent was added to the catholyte .
the solution was kept under stirring at room temperature for 2 hours ; then the solvent was evaporated under reduced pressure and the residue was purified by flash column chromatography , using a mixture of ethyl acetate / light petroleum ether 2/8 in volume , obtaining the pure products .
flash column chromatography was carried out using merck 60 kieselgel ( 230400 mesh ) under pressure .
gc - ms measurements were carried out on se 54 capillary column using a fisons 8000 gas chromatograph coupled with a fisons md 800 quadrupole mass selective detector .
h and c nmr spectra were recorded at room temperature using a bruker ac 200 spectrometer using cdcl3 as internal standard .
rf ( 80% ethyl acetate in dichloromethane ) 0.60 ; h nmr ( 200 mhz , cdcl3 ) 0.88 ( t , j = 6.5 hz , 3h ) , 1.201.30 ( m , 10h ) , 1.491.86 ( m , 3h ) , 1.50 ( s , 9h ) , 3.69 ( app t , j = 7.6 hz , 2h ) , 7.24 ( dd , j = 6.2 , 1.6 hz , 2h ) , 8.51 ( dd , j = 6.2 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 14.0 , 22.6 , 26.7 , 28.2 , 28.4 , 29.1 , 31.7 , 48.7 , 81.4 , 118.8 , 150.0 , 150.1 , 153.4 .
rf ( 50% ethyl acetate in light petroleum ether ) 0.46 ; h nmr ( 200 mhz , cdcl3 ) 1.48 ( s , 9h ) , 1.862.02 ( m , 2h ) , 2.64 ( t , j = 7.6 hz , 2h ) , 3.74 ( app t , j = 7.6 hz , 2h ) , 7.127.33 ( m , 7h ) , 8.49 ( dd , j = 4.8 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.2 , 30.0 , 33.0 , 48.3 , 81.6 , 118.9 , 126.1 , 128.3 , 128.5 , 141.0 , 149.7 , 150.3 , 153.4 ; eims , m / z : m absent , 212 ( 5% ) , 107 ( 100% ) , 105 ( 5% ) , 91 ( 25% ) , 78 ( 27% ) , 77 ( 12% ) .
rf ( 60% ethyl acetate in light petroleum ether ) 0.58 ; h nmr ( 200 mhz , cdcl3 ) 1.45 ( s , 9h ) , 4.94 ( s , 2h ) , 7.207.37 ( m , 7h ) , 8.46 ( dd , j = 4.8 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.1 , 52.5 , 82.1 , 118.2 , 126.3 , 127.3 , 128.7 , 137.7 , 150.2 , 150.1 , 153.5 ; eims , m / z : m absent , 227 ( 4% ) , 183 ( 14% ) , 91 ( 100% ) , 78 ( 7% ) , 57 ( 51% ) .
tert - butyl ( 2,6-dichlorobenzyl)pyridin-4-ylcarbamate
1d .
rf ( 50% ethyl acetate in light petroleum ether ) 0.60 ; h nmr ( 200 mhz , cdcl3 ) 1.48 ( s , 9h ) , 5.29 ( s , 2h ) , 7.027.21 ( m , 5h ) , 8.41 ( dd , j = 4.8 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.2 , 46.7 , 81.7 , 121.5 , 128.6 , 129.5 , 131.7 , 136.1 , 148.1 , 149.9 , 153.3 ; eims , m / z : 352 ( m , 1% ) , 252 ( 3% ) , 163 ( 6% ) , 161 ( 30% ) , 159 ( 42% ) , 78 ( 76% ) , 51 ( 100% ) .
tert - butyl ( 4-fluorobenzyl)pyridin-4-ylcarbamate
1e .
rf ( 50% ethyl acetate in light petroleum ether ) 0.49 ; h nmr ( 200 mhz , cdcl3 ) 1.45 ( s , 9h ) , 4.89 ( s , 2h ) , 6.977.22 ( m , 6h ) , 8.47 ( app d , j = 6.0 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.1 , 51.8 , 82.2 , 116.6 ( d , j = 21.5 hz ) , 118.4 , 128.1 ( d , j = 8.0 hz ) , 133.4 ( d , j = 3.2 hz ) , 149.9 , 150.3 , 153.4 , 162.2 ( d , j = 245.4 hz ) ; eims , m / z : 302 ( m , 1% ) , 245 ( 4% ) , 201 ( 53% ) , 108 ( 100% ) , 78 ( 42% ) , 57 ( 100% ) .
tert - butyl ( 4-trifluoromethylbenzyl)pyridin-4-ylcarbamate
1f .
rf ( 50% ethyl acetate in light petroleum ether ) 0.41 ; h nmr ( 200 mhz , cdcl3 ) 1.46 ( s , 9h ) , 4.99 ( s , 2h ) , 7.207.36 ( m , 4h ) , 7.61 ( app d , j = 8.4 hz , 2h ) , 8.49 ( app d , j = 6.2 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.1 , 52.2 , 82.5 , 118.1 , 124.0 ( q , j = 271.9 hz ) , 125.7 ( q , j = 3.7 hz ) , 126.6 , 129.7 ( q , j = 32.3 hz ) , 141.9 , 149.8 , 150.4 , 153.3 ; eims , m / z : m absent , 251 ( 9% ) , 158 ( 34% ) , 145 ( 2% ) , 78 ( 25% ) , 69 ( 9% ) , 57 ( 100% ) .
tert - butyl ( 2-oxo-2-phenylethyl)pyridin-4-ylcarbamate
1 g. rf ( 60% ethyl acetate in light petroleum ether ) 0.50 ; h nmr ( 200 mhz , cdcl3 ) 1.46 ( s , 9h ) , 5.09 ( s , 2h ) , 7.26 ( d , j = 6.4 hz , 2h ) , 7.487.68 ( m , 3h ) , 7.99 ( d , j = 8.2 hz , 2h ) , 8.478.55 ( m , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.1 , 55.5 , 82.5 , 118.7 , 127.9 , 128.9 , 133.9 , 134.7 , 150.2 , 153.2 , 193.7 .
tert - butyl ( 2-(4-fluoropheny)-2oxoethyl)pyridin-4-ylcarbamate
1 h. rf ( 50% ethyl acetate in light petroleum ether ) 0.50 ; h nmr ( 200 mhz , cdcl3 ) 1.46 ( s , 9h ) , 5.05 ( s , 2h ) , 7.157.27 ( m , 4h ) , 7.998.06 ( m , 2h ) , 8.52 ( dd , j = 5.0 , 1.4 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.1 , 55.4 , 82.6 , 116.2 ( d , j = 22.0 hz ) , 118.8 , 130.6 ( d , j = 9.4 hz ) , 131.1 ( d , j = 3.2 hz ) , 150.1 , 150.2 , 153.2 , 166.2 ( d , j = 256.1 hz ) , 192.2 .
tert - butyl ( 2-(4-chloropheny)-2oxoethyl)pyridin-4-ylcarbamate
1i .
rf ( 20% ethyl acetate in dichloromethane ) 0.30 ; h nmr ( 200 mhz , cdcl3 ) 1.45 ( s , 9h ) , 5.04 ( s , 2h ) , 7.21 ( dd , j = 4.6 , 1.6 hz , 2h ) , 7.50 ( d , j = 8.8 hz , 2h ) , 7.93 ( d , j = 8.0 hz , 2h ) , 8.51 ( dd , j = 4.6 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.1 , 55.4 , 82.6 , 118.8 , 129.3 , 133.0 , 140.5 , 149.9 , 150.3 , 153.1 , 192.7 .
tert - butyl ( 2-(4-methoxypheny)-2oxoethyl)pyridin-4-ylcarbamate
1j .
rf ( 40% ethyl acetate in dichloromethane ) 0.50 ; h nmr ( 200 mhz , cdcl3 ) 1.46 ( s , 9h ) , 3.90 ( s , 3h ) , 5.04 ( s , 2h ) , 6.99 ( d , j = 9.0 hz , 2h ) , 7.24 ( dd , j = 4.8 , 1.6 hz , 2h ) , 7.97 ( d ,
j = 9.0 hz , 2h ) , 8.50 ( dd , j = 4.8 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.1 , 55.1 , 55.5 , 82.3 , 114.1 , 118.7 , 127.8 , 130.2 , 150.2 , 153.3 , 164.1 , 192.0 . to a solution of 1 ( 1 mmol ) in ch2cl2 ( 5 cm ) , kept at 0c , 1 cm of cf3cooh was added .
the solution was then mixed with aqueous sodium carbonate till ph 8 and extracted with ethyl acetate .
the solvent was removed under reduced pressure and the mixture was purified by flash chromatography , yielding pure compound 2 .
rf ( 20% dichloromethane in ethyl acetate ) 0.16 ; h nmr ( 200 mhz , cdcl3 ) 0.89 ( t , j = 7.2 hz , 3h ) , 1.251.34 ( 6h ) , 1.611.70 ( m , 2h ) , 2.893.12 ( m , 2h ) , 3.153.24 ( m , 2h ) , 5.435.48 ( m , 2h ) , 6.57 ( d , j = 5.2 hz , 2h ) , 8.11 ( d , j = 5.2 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 14.1 , 22.6 , 27.0 , 28.8 , 29.2 , 29.2 , 31.8 , 42.9 , 107.4 , 155.2 .
rf ( ethyl acetate ) 0.46 ; h nmr ( 200 mhz , cd3cn ) 1.912.06 ( m , 2h ) , 2.71 ( t , j = 7.4 hz , 2h ) , 3.173.27 ( m , 2h ) , 4.9 ( bs , 1h ) , 6.626.66 ( m , 2h ) , 7.147.46 ( m , 5h ) , 7.457.97 ( m , 2h ) ; c nmr ( 50 mhz , cd3cn ) 29.8 , 32.5 , 42.0 , 107.3 , 125.9 , 128.3 , 128.4 , 141.0 , 141.5 , 157.8 ; eims , m / z : 212 ( m , 1% ) , 107 ( 100% ) , 91 ( 43% ) , 78 ( 13% ) .
rf ( ethyl acetate ) 0.57 ; h nmr ( 200 mhz , cdcl3 ) 4.46 ( d , j = 6.0 hz , 2h ) , 6.7 ( bs , 2h ) , 7.1 ( bs , 1h ) , 7.217.41 ( m , 5h ) , 8.0 ( bs , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 46.8 , 107.4 , 127.2 , 127.6 , 128.8 , 137.7 , 148.6 , 154.0 ; eims , m / z : 184 ( m , 15% ) , 183 ( 15% ) , 107 ( 5% ) , 91 ( 100% ) , 78 ( 16% ) .
rf ( ethyl acetate ) 0.38 ; h nmr ( 200 mhz , cd3cn ) 4.59 ( s , 2h ) , 6.0 ( bs , 1h ) , 6.68 ( dd , j = 5.2 , 1.4 hz , 2h ) , 7.277.47 ( m , 3h ) , 8.41 ( app d , j = 5.4 hz , 2h ) ; c nmr ( 50 mhz , cd3cn ) 42.2 , 107.4 , 128.7 , 130.4 , 132.8 , 136.0 , 147.3 , 154.6 ; eims , m / z : 256 ( m + 4 , 1% ) , 254 ( m + 2 , 7% ) , 252 ( m , 14% ) , 162 ( 10% ) , 160 ( 67% ) , 158 ( 100% ) , 78 ( 37% ) .
rf ( ethyl acetate ) 0.27 ; h nmr ( 200 mhz , cdcl3 ) 4.38 ( d , j = 5.4 hz , 2h ) , 5.1 ( bs , 1h ) , 6.5 ( bs , 2h ) , 7.027.10 ( m , 2h ) , 7.307.34 ( m , 2h ) , 8.2 ( bs , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 46.3 , 107.8 , 115.8 ( d , j = 21.5 hz ) , 129.0 ( d , j = 8.1 hz ) , 133.1 , 148.5 , 153.8 , 166.8 ( d , j = 205.9 hz ) , ; eims , m / z : 202 ( m , 5% ) , 107 ( 16% ) , 109 ( 100% ) , 78 ( 16% ) .
rf ( ethyl acetate ) 0.25 ; h nmr ( 200 mhz , cdcl3 ) 4.51 ( d , j = 6.0 hz , 2h ) , 6.2 ( bs , 1h ) , 6.6 ( bs , 2h ) , 7.54 ( d , j = 8.0 hz , 2h ) , 7.67 ( d , j = 8.0 hz , 2h ) , 8.1 ( bs , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 45.4 , 107.8 , 124.4 ( q , j = 271.0 hz ) , 125.4 ( q , j = 3.9 hz ) , 128.4 ( q , j = 31.9 hz ) , 127.7 , 143.2 , 146.7 , 155.0 ; eims , m / z : 252 ( m , 80% ) , 183 ( 11% ) , 159 ( 100% ) , 107 ( 52% ) , 78 ( 31% ) .
1-(4-methoxyphenyl)-2-(pyridin-4-ylamine)ethan-1-one
2 g. rf ( 50% ethyl acetate in ethanol ) 0.15 ; h nmr ( 200 mhz , cdcl3 ) 3.90 ( s , 3h ) , 4.57 ( d , j = 3.8 hz , 2h ) , 4.6 ( bs , 1h ) , 6.58 ( d , j = 6.2 hz , 2h ) , 6.98 ( d , j = 9.0 hz , 2h ) , 7.98 ( d , j = 9.0 hz , 2h ) , 8.19 ( d , j = 6.2 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 48.2 , 55.6 , 108.1 , 114.1 , 127.2 , 130.2 , 150.8 , 153.6 , 164.5 , 191.6 . to a solution of 2 ( 1 mmol ) in anhydrous dmso ( 2 cm ) , kept at rt under n2 , 1.5 mmol of t - buok was added .
this mixture was allowed to stir for 20 min at rt ; then 1 mmol of alkyl halide was added and the solution was kept under stirring at rt for 4 h. the solution was then mixed with water and extracted with dichloromethane .
the solvent was removed under reduced pressure and the mixture was purified by flash chromatography , yielding pure compound 3 .
rf ( 80% ethyl acetate in ethanol ) 0.38 ; h nmr ( 200 mhz , cdcl3 ) 0.860.92 ( m , 3h ) , 1.201.40 ( m , 10h ) , 1.631.70 ( m , 2h ) , 3.42 ( app t , j = 7.6 hz , 2h ) , 4.59 ( s , 2h ) , 6.51 ( d , j = 5.2 hz , 2h ) , 7.147.38 ( m , 5h ) , 8.18 ( d , j = 5.2 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 14.1 , 22.6 , 26.9 , 27.0 , 29.2 , 29.4 , 29.7 , 31.8 , 50.7 , 53.4 , 106.9 , 126.2 , 127.3 , 128.8 , 136.8 , 148.9 , 153.6 .
rf ( ethyl acetate ) 0.40 ; h nmr ( 200 mhz , cdcl3 ) 0.860.92 ( m , 3h ) , 1.231.35 ( m , 10h ) , 1.621.72 ( m , 2h ) , 3.42 ( app t , j = 7.8 hz , 2h ) , 4.58 ( s , 2h ) , 6.56 ( app d , j = 6.4 hz , 2h ) , 6.997.16 ( m , 4h ) , 8.17 ( app d , j = 6.4 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 14.1 , 19.2 , 22.6 , 26.9 , 29.2 , 29.3 , 31.7 , 51.3 , 53.3 , 107.4 , 116.1 ( d , j = 21.7 hz ) , 127.9 ( d , j = 8.1 hz ) , 130.9 ( d , j = 3.5 hz ) , 144.9 , 155.3 , 162.3 ( d , j = 246.5 hz ) .
rf ( ethyl acetate : n - hexane : methanol 50 : 33 : 17 ) 0.48 ; h nmr ( 200 mhz , cdcl3 ) 1.912.03 ( m , 2h ) , 2.68 ( t , j = 7.4 hz , 2h ) , 3.43 ( app t , j = 7.8 hz , 2h ) , 4.54 ( s , 2h ) , 6.44 ( app d , j = 5.4 hz , 2h ) , 6.967.36 ( m , 9h ) , 8.15 ( bs , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 28.1 , 33.0 , 49.9 , 52.9 , 106.8 , 115.8 ( d , j = 21.6 hz ) , 126.3 , 127.9 ( d , j = 8.0 hz ) , 128.3 , 128.6 , 132.1 ( d , j = 3.2 hz ) , 140.7 , 147.9 , 153.7 , 162.1 ( d , j = 245.8 hz ) . to a solution of 4ap ( 1 mmol ) in anhydrous dmso ( 2 cm ) , kept at rt under n2 , 2 mmol of t - buok was added .
this mixture was allowed to stir for 20 min at rt ; then 2 mmol of alkyl halide was added and the solution was kept under stirring at rt for 4 h. the solution was then mixed with water and extracted with dichloromethane .
the solvent was removed under reduced pressure and the mixture was purified by flash chromatography , yielding pure compound 3 .
rf ( 80% ethyl acetate in dichloromethane ) 0.20 ; h nmr ( 200 mhz , cd3cn ) 1.841.99 ( m , 4h ) , 2.65 ( t , j = 7.5 hz , 4h ) , 3.29 ( t , j = 7.5 hz , 4h ) , 6.30 ( d , j = 5.6 hz , 2h ) , 7.177.31 ( m , 10h ) , 8.13 ( d , j = 5.6 hz , 2h ) ; c nmr ( 50 mhz , cd3cn ) 28.3 , 33.1 , 49.5 , 106.4 , 126.2 , 128.3 , 128.5 , 141.1 , 140.7 , 152.4 .
rf ( 80% ethyl acetate in ethanol ) 0.32 ; h nmr ( 200 mhz , cdcl3 ) 4.67 ( s , 4h ) , 6.58 ( dd , j = 4.8 , 1.6 hz , 2h ) , 7.197.40 ( m , 10h ) , 8.20 ( dd , j = 4.8 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 53.2 , 107.1 , 126.4 , 127.4 , 128.9 , 136.8 , 150.2 , 153.9 .
di(4-fluorobenzyl)pyridin-4-amine
3ee
. rf ( 80% ethyl acetate in dichloromethane ) 0.40 ; h nmr ( 200 mhz , cdcl3 ) 4.61 ( s , 4h ) , 6.56 ( dd , j = 5.0 , 1.6 hz , 2h ) , 6.997.19 ( m , 8h ) , 8.22 ( dd , j = 5.0 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 52.5 , 107.1 , 115.8 ( d , j = 21.6 hz ) , 128.1 ( d , j = 8.0 hz ) , 132.3 ( d , j = 3.2 hz ) , 150.3 , 153.6 , 162.2 ( d , j = 245.8 hz ) .
rf ( 80% ethyl acetate in dichloromethane ) 0.25 ; h nmr ( 200 mhz , cdcl3 ) 4.73 ( s , 4h ) , 6.58 ( dd , j = 5.0 , 1.6 hz , 2h ) , 7.367.59 ( m , 8h ) , 8.26 ( dd , j = 5.0 , 1.6 hz , 2h ) ; c nmr ( 50 mhz , cdcl3 ) 53.1 , 107.1 , 123.3 ( q , j = 3.7 hz ) , 123.8 ( q , j = 272.3 hz ) , 124.5 ( q , j = 3.8 hz ) , 129.7 , 131.4 ( q , j = 32.4 hz ) , 137.7 , 150.4 , 153.4 .
organisms . for the antifungal evaluation , strains obtained from the american type culture collection ( atcc , rockville , md , usa ) , the german collection of microorganisms ( dsmz , braunschweig , germany ) , and the pharmaceutical microbiology culture collection ( pmc , department of public health and infectious diseases , sapienza university , rome , italy ) were tested .
the strains were candida albicans ( atcc 10231 , atcc 10261 , atcc 24433 , atcc 90028 , 3153 , pmc 1002 , pmc 1011 , and pmc 1030 ) , c. parapsilosis atcc22019 , c. parapsilosis dsm 11224 , c. tropicalis dsm 11953 , c. tropicalis pmc 0908 , c. tropicalis pmc 0910 , c. glabrata pmc 0805 , c. krusei dsm 6128 , and c. krusei pmc 0613 , cryptococcus neoformans ( dsm 11959 , pmc 2102 , pmc 2107 , pmc 2111 , and pmc 2136 ) , dermatophytes ( trichophyton mentagrophytes dsm 4870 , t. mentagrophytes pmc6509 , microsporum gypseum dsm 7303 , and m. gypseum pmc 7331 ) .
all of the strains were stored and grown in accordance with the procedures of the clinical and laboratory standards institute ( clsi ) [ 26 , 27 ] .
the final concentration ranged from 0.125 to 64 g / ml . the compounds were dissolved previously in dmso at concentrations 100 times higher than the highest desired test concentration and successively diluted in test medium in accordance with the procedures of the clsi .
microdilution trays containing 100 l of serial twofold dilutions of compounds in rpmi 1640 medium ( sigma - aldrich , st .
louis , mo , usa ) were inoculated with an organism suspension adjusted to attain a final inoculum concentration of 1.0 101.5 10 cells / ml for yeasts and 0.4 105 10 cfu / ml for dermatophytes .
the panels were incubated at 35c and observed for the presence of growth at 48 h ( candida spp . ) and 72 h ( c. neoformans and dermatophytes ) .
the minimal inhibitory concentration ( mic ) was , for yeasts , the lowest concentration that showed 50% growth inhibition compared with the growth control and , for dermatophytes , the lowest concentration that showed 80% growth inhibition compared with the growth control .
the mic100 was the lowest drug concentration that prevented 100% of growth with respect to the untreated control . according to csi protocols , the fluconazole mic50 and the amphotericin b mic100 were calculated ( 22,23 ) .
the results were expressed as the geometric mean ( g m ) of the mic values .
for the evaluation of antiprotozoal and cytotoxic activity an integrated panel of microbial screens and standard screening methodologies were adopted as previously described on the following organisms : chloroquine - resistant p. falciparum k 1-strain ; l. infantum mhom / ma ( be)/67 amastigote stage ; suramin - sensitive trypanosoma brucei squib-427 strain ; trypanosoma cruzi tulahuen cl2 ( benznidazole - sensitive ) strain ; human fetal lung fibroblast cells ( mrc-5 sv2 ) .
compounds were tested at 5 concentrations ( 64 , 16 , 4 , 1 , and 0.25 g / ml ) to establish a full dose titration and determine the ic50 ( inhibitory concentration 50% ) .
the final in - test concentration of dmso did not exceed 0.5% , which is known not to interfere with the different assays .
|
the use of electrogenerated acetonitrile anion allows the alkylation of n - boc-4-aminopyridine in very high yields , under mild conditions and without by - products .
the high reactivity of this base is due to its large tetraethylammonium counterion , which leaves the acetonitrile anion naked .
the deprotection of the obtained compounds led to high yields in n - alkylated 4-aminopyridines .
nonsymmetrically dialkylated 4-aminopyridines were obtained by subsequent reaction of monoalkylated ones with t - buok and alkyl halides , while symmetrically dialkylated 4-aminopyridines were obtained by direct reaction of 4-aminopyridine with an excess of t - buok and alkyl halides . some mono- and dialkyl-4-aminopyridines
were selected to evaluate antifungal and antiprotozoal activity ; the dialkylated 4-aminopyridines 3ac , 3ae and 3ff showed antifungal towards cryptococcus neoformans ; whereas 3cc , 3ee and 3ff showed antiprotozoal activity towards leishmania infantum and plasmodium falciparum .
|
1. Introduction
2. Results and Discussion
3. Biological Activity
4. Conclusion
5. Materials and Methods
|
germ cell tumors ( gcts ) represent a well - recognized group of heterogeneous neoplasms with diverse clinical , histopathological , diagnostic , and prognostic characteristics .
being the most common solid tumor in the 3rd and 4th decades , gcts assume a particular significance among young adult males .
gonads are the most common primary site of gcts , although a very small proportion is extragonadal in origin .
the most common site of the scarcer extragonadal gcts is the mediastinum , with retroperitoneum being the close runner - up .
immature teratomas , a subtype of gcts , are particularly rare in the mediastinum . in a review of 322 primary mediastinal gcts ,
only 6 immature teratoma cases were identified , which represented 1.8% of all mediastinal gcts and 4% of mediastinal teratomas .
even rarer is a gct with somatic malignant transformation , occurring in around 2% of all male gcts and in 10% to 20% of mediastinal teratomas .
we report herein a case of an aggressive , chemotherapy - resistant immature teratoma harboring foci of sarcoma , melanoma , adenocarcinoma , and squamous cell carcinoma .
a 21-year - old man was referred to our service from a local hospital for further management of a right hemithoracic extra - pleural mass with a provisional diagnosis of immature teratoma .
the patient previously healthy presented to the referring hospital with a 3-week history of productive cough with blood - tinged sputum , right - sided intermittent pleuritic chest pain , and mild shortness of breath .
he also experienced intermittent fever ( 38.5 c ) , nausea , vomiting , anorexia , and significant weight loss .
his past medical , family , travel , contact , and occupational histories are unremarkable . physical examination revealed a thin - built man with normal vital signs .
respiratory examination suggested a reduction of air entry on the right side with no rhonchi , wheezing , or crepitation .
general , cardiac , abdominal , testicular , and lymph - node examinations were unremarkable .
g / l , normal = 135180 ; mean corpuscular volume = 71 fl , normal = 7595 ; mean corpuscular hemoglobin = 23.3 pg , normal = 2430 ) , mild leukocytosis ( total leukocyte count = 12.49 10/l , normal = 3.911.0 ) , thrombocytosis ( platelets = 1061 10/l , normal = 155435 ) , and an elevated lactate dehydrogenase ( ldh ) level ( ldh = 567 u / l increased to 727
notably , -fetoprotein ( afp ) and cancer antigen 199 were elevated ( afp = 9.13 g / l increased to 19.3 g /
l in few weeks , normal < 7 ; cancer antigen 199 = 38.5 u / ml , normal < 27 ) , while carcinoembryonic antigen and -human chorionic gonadotropin levels were within normal limits .
a chest radiograph showed a large , well - defined substernal mass obscuring the right cardiac border .
contrast - enhanced computed tomography ( ct ) at presentation revealed a large ( approximately 4 4 5 cm ) noncalcified , heterogeneous mediastinal mass extending to the right upper lobe and pleura ( fig .
several mediastinal and cardiophrenic lymph nodes were noted , with the largest measuring 1.2 cm in its widest diameter .
no focal lesions in the liver , spleen , pancreas , adrenals , or kidneys were identified .
whole - body combined 18f - fluorodeoxyglucose positron emission tomography / computed tomography ( fdg - pet / ct ) showed an fdg - avid mediastinal mass with a single fdg - avid superior mediastinal lymph node ( fig .
( a , b ) a contrast - enhanced computed tomography scan , using the mediastinal ( a ) and lung ( b ) windows , showing a mediastinal mass that is extending into the adjacent lung . ( a , b ) microscopic examination of needle core biopsy from the mediastinal mass showing predominant immature neuroectodermal elements forming glands and tubules lined by columnar embryonal cells with stratified hyperchromatic nuclei ( a ) and focal areas of a more mature neural tissue ( b ) . a whole - body combined 18f - fluorodeoxyglucose ( fdg ) positron emission tomography / computed tomography scan showing an fdg - avid mediastinal mass with an intense peripheral hypermetabolic activity .
combination chemotherapy with bleomycin ( 30 units iv on days 1 , 8 , and 15 ) , etoposide ( 100 mg / m iv on days 15 ) , and cisplatin ( 20 mg / m iv on days 15 ) was subsequently initiated
. however , notwithstanding the mild improvement initially , the patient showed steady symptomatic deterioration ; after completing two 3-week chemotherapy cycles , he presented to the emergency room with chest pain , cough , and hemoptysis .
although no evidence of pulmonary embolism ( pe ) was found , the study showed an interval progression of the tumor size , reaching around 12 13 12 cm .
the ill - bordered mass appeared to have invaded the lung parenchyma , superior vena cava , and pericardium ( fig .
4a and b ) , while abutting on the carina , right main bronchus , right pulmonary artery , as well as the anterior , inferior , and superior pulmonary veins .
( a , b ) axial ( a ) and coronal ( b ) contrast - enhanced computed tomography scans obtained after receiving 2 chemotherapeutic cycles showing interval progression of size , reaching around 12 13 12 cm in its maximum dimensions .
the case was thoroughly reviewed and discussed in a multidisciplinary team meeting , whose recommendation was to proceed with surgical resection .
the patient underwent clamshell thoracotomy with an r0 resection of the mass along with the right middle and lower lung lobes .
however , he presented 2 months later with extensive liver and bone metastasis , for which he received 1 cycle of palliative radiation .
therefore , the decision was taken to treat him as a case of metastatic melanoma .
one 5-day cycle temozolomide ( 150 mg / m po qday for 5 days ) was administered .
unfortunately , the patient passed away5 and a half months after the initial diagnosis was made .
macroscopic examination of the resected mediastinal mass revealed solid and mucoid - filled cystic components with hemorrhagic cut surface measuring 18 14 13 cm in its maximum dimensions .
microscopically , the tumor consisted of a mixture of various ectodermal , endodermal , and mesodermal elements , of which immature neuroectodermal tissue areas were predominant .
anaplastic , pleomorphic glial tissues with marked nuclear atypia , and brisk mitosis were noted .
these areas stained positive for glial fibrillary acidic protein and represented a glioblastoma component ( fig .
c ) . ( a c ) neural tissue with pleomorphic atypical cells and high nuclear - to - cytoplasmic ratio consistent with glioblastoma ( a , b ) .
immunohistochemical staining of the tissue with glial fibrillary acidic protein ( gfap ) showing positive reaction ( c ) .
other areas of the tumor contained pleomorphic spindle - to - round cells with large hyperchromatic nuclei , prominent nucleoli , and frequent atypical mitosis suggestive of malignant , undifferentiated spindle - cell sarcoma ( fig .
extensive immunohistochemical staining for relevant markers ( smooth muscle actin , desmin , myogenic differentiation 1 , cluster of differentiation [ cd]34 , cd31 , and synaptophysin ) were negative .
atypical , large pleomorphic cells with melanin pigment staining positive for s100 , human melanoma black ( hmb ) 45 , melan a , and microphthalmia - associated transcription factor-1 were present in other areas indicating a melanocytic differentiation consistent with transformation to malignant melanoma ( fig .
areas of endodermal differentiation with glandular structures , some of which showed malignant features consistent with adenocarcinoma , were also noted ( fig .
besides , mature and immature cartilaginous tissues were seen in areas of the tumor ( fig .
regional lymph nodes adjacent to the tumor were positive for metastatic squamous cell carcinoma ( fig .
( a h ) somatic - type malignancies : pleomorphic spindle to round cells and frequent atypical mitosis suggestive of sarcomatous differentiation ( a ) .
malignant spindle cells with melanin pigment ( b d ) which were positive for melan - a ( e ) .
cartilage with numerous lacunae containing small chondrocytes , surrounded by immature mesenchymal cells ( g ) .
a 21-year - old man was referred to our service from a local hospital for further management of a right hemithoracic extra - pleural mass with a provisional diagnosis of immature teratoma .
the patient previously healthy presented to the referring hospital with a 3-week history of productive cough with blood - tinged sputum , right - sided intermittent pleuritic chest pain , and mild shortness of breath .
he also experienced intermittent fever ( 38.5 c ) , nausea , vomiting , anorexia , and significant weight loss .
his past medical , family , travel , contact , and occupational histories are unremarkable . physical examination revealed a thin - built man with normal vital signs .
respiratory examination suggested a reduction of air entry on the right side with no rhonchi , wheezing , or crepitation .
general , cardiac , abdominal , testicular , and lymph - node examinations were unremarkable .
g / l , normal = 135180 ; mean corpuscular volume = 71 fl , normal = 7595 ; mean corpuscular hemoglobin = 23.3 pg , normal = 2430 ) , mild leukocytosis ( total leukocyte count = 12.49 10/l , normal = 3.911.0 ) , thrombocytosis ( platelets = 1061 10/l , normal = 155435 ) , and an elevated lactate dehydrogenase ( ldh ) level ( ldh = 567 u / l increased to 727
notably , -fetoprotein ( afp ) and cancer antigen 199 were elevated ( afp = 9.13 g / l increased to 19.3 g /
l in few weeks , normal < 7 ; cancer antigen 199 = 38.5 u / ml , normal < 27 ) , while carcinoembryonic antigen and -human chorionic gonadotropin levels were within normal limits .
a chest radiograph showed a large , well - defined substernal mass obscuring the right cardiac border .
contrast - enhanced computed tomography ( ct ) at presentation revealed a large ( approximately 4 4 5 cm ) noncalcified , heterogeneous mediastinal mass extending to the right upper lobe and pleura ( fig .
several mediastinal and cardiophrenic lymph nodes were noted , with the largest measuring 1.2 cm in its widest diameter .
no focal lesions in the liver , spleen , pancreas , adrenals , or kidneys were identified .
a ct - guided core needle biopsy was suggestive of immature teratoma ( fig . 2a and b ) . whole - body combined 18f - fluorodeoxyglucose positron emission tomography / computed tomography ( fdg - pet / ct ) showed an fdg - avid mediastinal mass with a single fdg - avid superior mediastinal lymph node ( fig .
( a , b ) a contrast - enhanced computed tomography scan , using the mediastinal ( a ) and lung ( b ) windows , showing a mediastinal mass that is extending into the adjacent lung . ( a , b ) microscopic examination of needle core biopsy from the mediastinal mass showing predominant immature neuroectodermal elements forming glands and tubules lined by columnar embryonal cells with stratified hyperchromatic nuclei ( a ) and focal areas of a more mature neural tissue ( b ) .
a whole - body combined 18f - fluorodeoxyglucose ( fdg ) positron emission tomography / computed tomography scan showing an fdg - avid mediastinal mass with an intense peripheral hypermetabolic activity .
combination chemotherapy with bleomycin ( 30 units iv on days 1 , 8 , and 15 ) , etoposide ( 100 mg / m iv on days 15 ) , and cisplatin ( 20 mg / m iv on days 15 ) was subsequently initiated
. however , notwithstanding the mild improvement initially , the patient showed steady symptomatic deterioration ; after completing two 3-week chemotherapy cycles , he presented to the emergency room with chest pain , cough , and hemoptysis .
although no evidence of pulmonary embolism ( pe ) was found , the study showed an interval progression of the tumor size , reaching around 12 13 12 cm .
the ill - bordered mass appeared to have invaded the lung parenchyma , superior vena cava , and pericardium ( fig . 4a and b ) , while abutting on the carina , right main bronchus , right pulmonary artery , as well as the anterior , inferior , and superior pulmonary veins .
( a , b ) axial ( a ) and coronal ( b ) contrast - enhanced computed tomography scans obtained after receiving 2 chemotherapeutic cycles showing interval progression of size , reaching around 12 13 12 cm in its maximum dimensions .
the case was thoroughly reviewed and discussed in a multidisciplinary team meeting , whose recommendation was to proceed with surgical resection .
the patient underwent clamshell thoracotomy with an r0 resection of the mass along with the right middle and lower lung lobes .
however , he presented 2 months later with extensive liver and bone metastasis , for which he received 1 cycle of palliative radiation .
therefore , the decision was taken to treat him as a case of metastatic melanoma .
one 5-day cycle temozolomide ( 150 mg / m po qday for 5 days ) was administered .
unfortunately , the patient passed away5 and a half months after the initial diagnosis was made .
macroscopic examination of the resected mediastinal mass revealed solid and mucoid - filled cystic components with hemorrhagic cut surface measuring 18 14 13 cm in its maximum dimensions .
microscopically , the tumor consisted of a mixture of various ectodermal , endodermal , and mesodermal elements , of which immature neuroectodermal tissue areas were predominant .
anaplastic , pleomorphic glial tissues with marked nuclear atypia , and brisk mitosis were noted .
these areas stained positive for glial fibrillary acidic protein and represented a glioblastoma component ( fig .
c ) . ( a c ) neural tissue with pleomorphic atypical cells and high nuclear - to - cytoplasmic ratio consistent with glioblastoma ( a , b ) .
immunohistochemical staining of the tissue with glial fibrillary acidic protein ( gfap ) showing positive reaction ( c ) .
other areas of the tumor contained pleomorphic spindle - to - round cells with large hyperchromatic nuclei , prominent nucleoli , and frequent atypical mitosis suggestive of malignant , undifferentiated spindle - cell sarcoma ( fig .
extensive immunohistochemical staining for relevant markers ( smooth muscle actin , desmin , myogenic differentiation 1 , cluster of differentiation [ cd]34 , cd31 , and synaptophysin ) were negative .
atypical , large pleomorphic cells with melanin pigment staining positive for s100 , human melanoma black ( hmb ) 45 , melan a , and microphthalmia - associated transcription factor-1 were present in other areas indicating a melanocytic differentiation consistent with transformation to malignant melanoma ( fig .
e ) . areas of endodermal differentiation with glandular structures , some of which showed malignant features consistent with adenocarcinoma , were also noted ( fig .
besides , mature and immature cartilaginous tissues were seen in areas of the tumor ( fig .
regional lymph nodes adjacent to the tumor were positive for metastatic squamous cell carcinoma ( fig .
( a h ) somatic - type malignancies : pleomorphic spindle to round cells and frequent atypical mitosis suggestive of sarcomatous differentiation ( a ) .
malignant spindle cells with melanin pigment ( b d ) which were positive for melan - a ( e ) .
cartilage with numerous lacunae containing small chondrocytes , surrounded by immature mesenchymal cells ( g ) .
a 40-year , population - based review of finland cancer registry suggested an incidence of 1.8 and 1.0 per 1,000,000 person - years among males and females , respectively .
despite the increasing incidence of gonadal gcts , no temporal change was observed in the incidence of its extragonadal counterpart a finding that is consistent across the studied populations .
several reports suggested the mediastinum to be the most common site of origin of extragonadal gcts in males , while others found the central nervous system to be more common .
teratomas account for 40% to 60% of all mediastinal gcts , with the overwhelming majority being histologically mature .
mediastinal teratomas are more common in males than females , except during the first few years of life .
an uncommon but well - recognized phenomenon that may accompany gcts is the somatic - type malignant transformation , in which a non - gct malignant component is found within the bulk of a cgt or in its metastatic foci .
somatic - type malignancy arising specifically in a teratoma setting remains an exceptionally rare event .
given the remarkable chemo - sensitivity of gcts , chemotherapy is thought to eliminate the bulk of gcts exposing the non - gct , chemo - resistant malignant components when present . possibly due to the pluripotency of gct components
the most common histological subtype of malignant transformation is sarcoma ( predominantly rahbdomyosarcoma ) , followed by adenocarcinoma and primitive neuroectodermal tumors .
squamous cell transformation is rare but may be relatively more common in ovarian teratomas , especially in the mature cystic variants .
an international , multicentric review of 635 patients with extragonadal gcts identified secondary melanoma in 2 cases , which occurred few years later after the primary diagnosis .
few other cases of melanoma either occurring as secondary tumors or in conjugation with the primary mediastinal gct have been reported ( table 1 ) . to our knowledge , this is the first report of combined sarcomatous , carcinomatous , and melanomatous malignant transformation arising in the setting of immature mediastinal teratoma .
clinicopathological characteristics of primary medistinal gcts cases reported in the literature with an associated melanomatous degeneration .
clinically , gcts with somatic - type malignancy tend to be more symptomatic than pure gcts , although symptoms are clinically indistinguishable when present .
symptoms may arise from compression , invasion , or rupture and include chest pain , dyspnea , and cough .
patients may also experience weight loss , fever , malaise , night sweats , nausea , hemoptysis , postobstructive pneumonia , dysphagia , hoarseness , and superior vena cava syndrome .
radiologically , attenuation heterogeneity is a common characteristic of the mass , due to the presence of somatic malignancy ( i.e. , a solid mass ) along with areas of teratomatous , necrotic , or hemorrhagic zones .
invasion to adjacent structures , such as the great vessels , lung , and heart can also be seen .
metastasis to regional lymph nodes , lung , brain , liver , and spleen may be evident at the time of diagnosis or as a recurrence .
since no evidence of testicular involvement was found on physical examination or ultrasonography , testicular biopsy was deemed unnecessary in this case .
the patient 's clinical presentation featured postchemotherapy disease progression that is , tumor growth and symptomatic deterioration .
syndrome and teratoma rupture , respectively , which are infrequently but characteristically seen in mature mediastinal teratomas . however , the rapid tumor progression in this case is likely due to the aggressive nature of the underlying non - gct malignant transformation , especially sarcoma .
in fact , it is likely that the tumor 's poor response to cisplatin - based therapy was due to the presence of sarcomatous component .
elevation of afp or -human chorionic gonadotropin is not uncommon in gcts with malignant transformation .
busmanis and tay suggested the lack of immunohistochemical correlation with the afp serum levels in a pure immature ovarian teratoma with extra - ovarian implants .
the possibility of yolk sac component being present in our case ( given the elevation of afp ) could not be ruled out .
although chemotherapy might have accounted for the eradication of yolk - sac components , the absence of histological evidence led to the designation of this case as an immature teratoma .
given the significant resistance to cisplatin - based chemotherapy , motzer et al suggested using chemotherapeutic agents based on the transformed histology as the strategy of choice for regimen selection .
others advocated initiating gct - standard cisplatin - based regiment and reserving chemotherapeutics of the specific somatic - type transformed malignancy for relapses .
although the rarity of gcts with malignant transformation have not permitted the generation of a strong body of evidence , it is agreed that an aggressive surgical approach with complete resection may be beneficial whenever deemed feasible , since it appears to afford the best survival . as demonstrated by the case
presented herein , the radiologic evidence of postchemotherapeutic interval growth in the context of a known immature teratoma may serve as a useful clue as to the presence of somatic - type transformation , favoring urgent surgical intervention over other treatment modalities ( e.g. , salvage chemotherapy ) .
in fact , it was shown that such a tumor may harbor hematopoietic stem cells within its bulk , providing a possible explanation for the source of the hematologic derangements . with the exception of thrombocytosis
, there were no overly aberrant blood indices in our case that would warrant further investigation .
it is worth mentioning that gcts may predispose to thrombotic events especially during chemotherapy treatment , and a sudden occurrence of hemoptysis raises the suspicion of pe .
ct pulmonary angiogram may be deemed optimal in an emergency setting , it aids in pe diagnosis and , if pe is ruled out , delineation of any structural alterations of the underlying pathology .
favorable factors include younger age , limited disease extent at diagnosis , feasibility of complete resection , the absence of somatic - type malignancy ( especially metastatic sarcoma - type histological transformation ) , and the response to standard chemotherapeutic regimens .
teratomas with malignant transformation have an aggressive course , high recurrence rate , and poor survival when present in the mediastinum . nonetheless , when confined to primary site , such tumors tend to carry mortality risk that is comparable to its teratomatous counterpart with no malignant transformation .
for gcts with somatic - type malignancy , the survival - determining factor may be the histopathological component that is most aggressive .
the patient had an overall survival of around 6 and 2 months from the initial diagnosis and metastasis , respectively .
this seems to mirror the survival of melanoma with metastasis to the liver , brain , or bone , whose median survival was found to be 4.4 months .
the patient also had several of the poor prognostic indicators , including an elevated ldh and leukocytosis .
however , what determines the metastatic potential in gct - associated melanomas remains largely unknown .
although our patient presented with metastatic disease 4 months after the diagnosis , 13 months elapsed before metastatic disease occurred in a previously reported gct - associated melanoma .
somatic - type malignant degeneration is a rare but well - recognized phenomenon that occurs in the settings of gcts . due to its rarity , distinct nature , and remarkable heterogeneity ,
this disease warrants comprehensive reporting of the clinical , radiological , and pathological features . in case of more - than - single transformation
, the clinical outcomes and overall survival may well correlate with that of the most aggressive and poorest prognosis tissue type .
the presence of melanomatous transformation , as illustrated by this case , likely confers aggressive biological behavior .
as the literature is enriched with additional studies , understanding the clinical implications of the distinct malignant transformations will expectedly aid in clarifying the optimal , possibly tailored , management lines .
|
abstractgerm cell tumors ( gcts ) represent a well - recognized group of heterogeneous neoplasms with diverse clinical , histopathological , diagnostic , and prognostic characteristics .
we present a rare case of a locally aggressive , chemotherapy - resistant immature mediastinal teratoma with a peculiar histological finding of a multilineage somatic - type malignant degeneration . a 21-year - old male patient presented with a 3-week history of persistent , blood - tinged productive cough and shortness of breath .
a contrast - enhanced computed tomography ( ct ) scan of the chest showed a heterogeneous mass occupying the right hemithorax and abutting on adjacent structures .
ct - guided biopsy was consistent with immature teratoma .
combination chemotherapy with bleomycin , etoposide , and cisplatin was initiated , albeit without success ; the mass showed interval progression in size , and surgical resection through clamshell incision was performed .
histological assessment of the resected mass confirmed the diagnosis of immature teratoma and revealed an extensive multilineage malignant differentiation into sarcomatous , carcinomatous , and melanomatous components .
the patient underwent an uneventful recovery but presented 2 months later with extensive liver and bone melanomatous metastases . in this report ,
relevant findings from the literature are also highlighted . despite being exceptionally rare ,
such tumors carry poor prognosis . understanding the clinicopathological characteristics and biological behavior of such tumors
may provide an insight into interventions tailored to improve the otherwise dismal disease outlook .
|
Introduction
Case report
Clinical history and findings
Radiology findings
Clinical course
Pathology findings
Discussion
Conclusion
|
parturient with congenital heart block may be asymptomatic but can present with sudden vascular collapse , especially during labour .
few patients with congenital heart block may have sudden cardiac death ( scd ) for which there are no predictors .
we present our experience of spinal anaesthesia in a 29-year - old female with congenital complete heart block for lower segment caesarean section ( lscs ) .
a 29-year - old parturient ( gravida 3 , para 0 , abortion 2 ) was admitted to our hospital with 9 months of amenorrhea .
she had undergone appendicectomy 8 years back under spinal anaesthesia , and the procedure was uneventful .
past obstetric history revealed two abortions and an episode of perioperative seizures during intravenous sedation for medical termination of pregnancy .
the episode was associated with bradycardia non - responsive to atropine , during which she was evaluated as having a congenital complete heart block .
she had been prescribed orciprenaline by a local practitioner and had received orciprenaline 10 mg bd for 2 months preceding the present pregnancy and till 10 days before presenting to the hospital .
it was stopped by our cardiologist as it does not help to increase heart rate in complete heart block .
her general condition was stable ; pulse rate was 46/min and blood pressure ( bp ) was 110/70 mmhg . clinically , cardiorespiratory and central nervous system examinations were normal .
per - abdomen examination showed a foetus in cephalic presentation , uterine height at 38 weeks , foetal heart rate 136 beats / min and regular with clinical and ultrasonographic evidence of reduced liquor .
electrocardiography showed a complete heart block with an atrial rate of 80/min , ventricular rate of 46/min and a narrow qrs complex [ figure 1 ] .
she was accepted for anaesthesia under asa ii and was explained about the anaesthetic technique . patient was kept nil oral for 8 h. tab .
metoclopramide 10 mg the next morning 2 h before the surgery . on the morning of the surgery
, the patient was taken for cardiac catheterization in supine position with a wedge under the right buttock and temporary pacemaker insertion ( ventricular , ventricle inhibition
the position of the lead in the right ventricle was confirmed by fluoroscopy , with a lead shield cover over the abdomen of the patient .
a pacemaker can also be inserted using electrocardiographic and echocardiographic guidance to avoid foetal exposure to ionizing radiation , but our cardiologist could not get enough window to do the same . immediately after the procedure ,
a spinal anaesthesia was given in the l3-l4 interspace with a total of 1.5 ml , which is a combination of a 1.0 ml hyperbaric 0.5% bupivacaine with 0.5 ml fentanyl ( 25 g ) .
intra - operatively , the first episode of hypotension after the spinal anaesthesia was treated by increasing the pacing rate to 70 beats / min and the second episode was treated by 3 mg of intravenous ( iv ) ephedrine .
a healthy male baby weighing 2.5 kg was born with an apgar score of 8/10 in the 1 minute and 9/10 in the 5 minute .
post - operatively , the pacemaker rate was changed to 60 beats / min and the patient was shifted to the post - operative ward for continuous monitoring .
post - operative pain relief was achieved with iv tramadol 50 mg and diclofenac 75 mg iv alternatively 8 hourly .
the temporary pacemaker was removed after 24 h and the patient was haemodynamically stable after the removal .
her post - natal period was uneventful and she was discharged on the 5 day with an advice for the placement of permanent pacemaker as early as possible .
heart block may be congenital or acquired . congenital heart block may occur alone or in association with other cardiac abnormalities .
conversely , in isolated complete heart block , 8590% of all births live beyond the neonatal period , even up to late adulthood .
if congenital complete heart block occurs alone , then it is relatively benign , as the block to conduction is at the level of the av node .
the ventricular pacemaker is proximal to the bifurcation of the bundle of his , and therefore the qrs complexes are narrow , and the ventricular conduction system intact .
the rate is relatively high and can vary from 40 to 80 beats / min , and may increase with exercise , atropine or sympathomimetics .
acquired heart block in children or early adulthood is mostly secondary to cardiac surgery involving closure of perimembranous or infundibular ventricular septal defect ( vsd ) or muscle bundle resection near the conduction tissues , but can occur as an isolated condition also . in the chronic type ,
the atrio ventricular ( av ) junction or bundle branches are usually involved , the qrs complexes are wide and the heart rate is lower and is not increased by exercise or atropine .
prophylactic placement of a pacemaker is not indicated in an asymptomatic pregnant patient with complete heart block as it does not cause unusual problems.[35 ] if the patient is symptomatic during her first and second trimesters , then the placement of a permanent pacemaker is indicated .
we used temporary pacing in this patient because the patient 's heart rate was resistant to exercise and atropine , and the same can not adapt to her changing bp .
increase in the heart rate during labour is essential to increase in the cardiac output and to maintain the haemodynamics , which is not possible in the patient .
hence , for a safe delivery , temporary pacemaker insertion was essential , which ideally we did before operative delivery .
there are quite a few anaesthetic problems in patients with complete heart block undergoing incidental surgeries .
the anaesthetic technique that least alters the cardiac stability should be wisely planned and executed for the procedure .
general anaesthesia carries a potential risk to these patients because both the inhalational and the intravenous agents alter the haemodynamics to such an extent to put them in peril.[68 ] if general anaesthesia is planned , drugs with minimal effects in depressing the heart rate have to be preferred , such as ketamine for induction , pancuronium for relaxation and isoflurane for maintenance .
combined spinal epidural is another option , but we did not opt for it due to cost constraints and brevity of surgery .
modi et al . successfully managed such a case with the epidural anaesthetic technique . in our patient ,
we opted for intrathecal opioids , especially fentanyl , which gives adequate anaesthesia with minimal effects on the cardiovascular system .
we added 25 g of fentanyl with almost half the usual dose of hyperbaric 0.5% bupivacaine .
we did use 3 mg of vasopressor ephedrine , but such a single low dose to maintain adequate haemodynamic stability was found satisfactory to us .
we did insert a temporary pacemaker to compensate for any possible haemodynamic eventuality that can occur during anaesthesia , and the immediate post - partum period .
the pacemaker rate was set at 50/min in order to preserve her native rhythm till her blood pressure is maintained in the intra - operative period .
long - term pacing suppresses the native rhythm , and our intention was to avoid it .
also , the fluoroscopy time for positioning the lead was minimized to less than 10 s. the patient was discharged with an advice for permanent pacemaker implantation ( ppi ) . as per the recent guidelines , ppi is indicated for all congenital complete heart blocks ( class 2a / b ) as trials have shown that there is a subgroup of patients who may have scd for which there are no predictors available .
to conclude , we successfully managed a case of congenital complete heart block for operative delivery with temporary pacemaker in situ with intrathecal low - dose bupivacaine
|
congenital complete heart block could be absolutely asymptomatic . increased awareness of suspecting an atrioventricular heart block in patients with slow heart rate and electrocardiograph examination
will ensure recognition of this problem .
the possibility of sudden cardiac death in these patients should not be forgotten .
the goal in the peri - operative anaesthetic management is to preserve the heart rate and maintain haemodynamic stability .
herein , we present a case of congenital complete heart block posted for elective caesarean section for an obstetric indication .
we would like to highlight the advantage of bupivacaine fentanyl combination in maintaining haemodynamic stability and peri - operative heart rate control with temporary pacemaker .
|
INTRODUCTION
CASE REPORT
DISCUSSION
|
the study protocol was approved by the tianjin hospital ethics committee , and informed consent of all patients was obtained .
the inclusion criteria were 1 ) the injury time of hip fractures was < 24 h before admission and fracture diagnosis by x - ray examinations in our emergency department , 2 ) patient participated in physical examinations each year and blood glucose was normal ( < 6.1 mmol / l ) with ordinary values of glycosylated hemoglobin ( hba1c ) , 3 ) a pair of lower limb venous vascular ultrasound examinations confirmed no deep venous thromboembolism ( dvt ) , 4 ) osteoporosis was confirmed by bone density examination , and 5 ) orthopedic surgery was not completed within 1 week after admission to the hospital or no operation was performed ( conservative treatment ) .
the exclusion criteria were 1 ) the time of hip fracture injury was > 24 h before admission ; 2 ) diagnosed type 1 or type 2 diabetes ; 3 ) need of emergency operation within 1 week after hip fracture ; 4 ) the occurrence of dvt , pulmonary embolism , or thyroid disease ; and 5 ) renal / liver failure .
blood and urine routine , liver and kidney function , calcium , phosphorus , and serum cardiac enzyme concentrations , insulin level , and serum lipids were tested after admission .
electrocardiography ( ecg ) , cardiorespiratory function test , ultrasonic cardiography , chest x - ray , lower limb venous vascular ultrasound , and bone mineral density measurements of the bilateral hip were conducted .
closed reduction , bone traction treatments , and prevention of dvt were administered to all patients with hip fractures on the day of admission ( 10 ) and they continued to take their cardiac medications .
all patients who met the inclusion criteria were recorded with a standardized form throughout the study course .
the fasting blood glucose ( fbg ) was examined at 6:00 a.m. every morning from the 1st to the 7th day after admission , and ecgs were measured at admission and from the 1st to the 7th day after admission at 10:00 a.m. every morning . if clinical symptoms indicated and/or electrocardiographic changes suggested a myocardial infarction , additional blood samples were drawn from the antecubital vein in order to examine creatine kinase ( ck ) , ck - mb isoenzyme , and troponin - i values .
ami diagnosis criteria met at least two of following : 1 ) the ck - mb isoenzyme and/or troponin - i concentration elevated above the hospital laboratory 's myocardial infarction threshold ; 2 ) persistent st - t segment changes , new q waves , or left bundle branch block occurred ; 3 ) serious precordial chest pain lasting for 30 min or more .
stress hyperglycemia diagnosis criteria were as follows : 1 ) patients did not have a previous diabetes history , 2 ) admission or in - hospital fasting plasma glucose levels of 7 mmol / l or more in two or more tests ( 13 ) , and 3 ) normal hba1c values .
, chicago , il ) software was applied by a professional statistician to complete the analyses .
the period of follow - up interviews was 3 months after discharge , and mortality was recorded by the investigators via telephone calls and/or outpatient clinic visits .
consecutive hip fracture patients ( n = 1,257 ) were selected for the analysis during a 5-year period from february 2007 to february 2012 .
the study protocol was approved by the tianjin hospital ethics committee , and informed consent of all patients was obtained .
the inclusion criteria were 1 ) the injury time of hip fractures was < 24 h before admission and fracture diagnosis by x - ray examinations in our emergency department , 2 ) patient participated in physical examinations each year and blood glucose was normal ( < 6.1 mmol / l ) with ordinary values of glycosylated hemoglobin ( hba1c ) , 3 ) a pair of lower limb venous vascular ultrasound examinations confirmed no deep venous thromboembolism ( dvt ) , 4 ) osteoporosis was confirmed by bone density examination , and 5 ) orthopedic surgery was not completed within 1 week after admission to the hospital or no operation was performed ( conservative treatment ) .
the exclusion criteria were 1 ) the time of hip fracture injury was > 24 h before admission ; 2 ) diagnosed type 1 or type 2 diabetes ; 3 ) need of emergency operation within 1 week after hip fracture ; 4 ) the occurrence of dvt , pulmonary embolism , or thyroid disease ; and 5 ) renal / liver failure .
blood and urine routine , liver and kidney function , calcium , phosphorus , and serum cardiac enzyme concentrations , insulin level , and serum lipids were tested after admission .
electrocardiography ( ecg ) , cardiorespiratory function test , ultrasonic cardiography , chest x - ray , lower limb venous vascular ultrasound , and bone mineral density measurements of the bilateral hip were conducted .
closed reduction , bone traction treatments , and prevention of dvt were administered to all patients with hip fractures on the day of admission ( 10 ) and they continued to take their cardiac medications .
all patients who met the inclusion criteria were recorded with a standardized form throughout the study course .
the fasting blood glucose ( fbg ) was examined at 6:00 a.m. every morning from the 1st to the 7th day after admission , and ecgs were measured at admission and from the 1st to the 7th day after admission at 10:00 a.m. every morning . if clinical symptoms indicated and/or electrocardiographic changes suggested a myocardial infarction , additional blood samples were drawn from the antecubital vein in order to examine creatine kinase ( ck ) , ck - mb isoenzyme , and troponin - i values .
ami diagnosis criteria met at least two of following : 1 ) the ck - mb isoenzyme and/or troponin - i concentration elevated above the hospital laboratory 's myocardial infarction threshold ; 2 ) persistent st - t segment changes , new q waves , or left bundle branch block occurred ; 3 ) serious precordial chest pain lasting for 30 min or more .
stress hyperglycemia diagnosis criteria were as follows : 1 ) patients did not have a previous diabetes history , 2 ) admission or in - hospital fasting plasma glucose levels of 7 mmol / l or more in two or more tests ( 13 ) , and 3 ) normal hba1c values .
chicago , il ) software was applied by a professional statistician to complete the analyses .
the period of follow - up interviews was 3 months after discharge , and mortality was recorded by the investigators via telephone calls and/or outpatient clinic visits .
patients ( n = 1,257 ) who met the inclusion criteria were enrolled in this clinical observation . among the patients , the average age was 75.20 7.71 years and 64.52% were women .
the most prevalent comorbidity was hypertension ( 57.68% ) , followed by coronary artery disease ( cad ) or family history of cad ( 49.90% ) , dyslipidemia ( elevation of ldl cholesterol [ ldl - c ] , 49.27% ; triglyceride , 39.14% ; and low levels of hdl cholesterol [ hdl - c ] , 21.40% ) , obesity ( 37.63% ) , and smoking ( 28.16% ) .
the frequency of stress hyperglycemia was 47.89% ( 602/1,257 ) and that of ami was 9.31% ( 117/1,257 ) .
figure 1 shows the correlation between time of fracture and fbg . in the stress hyperglycemia group ,
repeated anovas showed that at each time point in the stress hyperglycemia group , the blood glucose values were significantly different ( p < 0.05 ) , whereas in the non - hyperglycemia patients , they did not change ( p = 0.10 ) ( fig .
the incidence of ami in the stress hyperglycemia group was significantly higher ( 12.46% [ 75/602 ] ) than in the non - hyperglycemia group ( 6.41% [ 42/655 ] , p < 0.05 ) .
the number of amis was correlated with the occurrence of hyperglycemia and was significantly higher 3 days after injury in the hyperglycemia group ( p < 0.05 ) , whereas the temporal distribution of ami in the non - hyperglycemia group was not different between 3 and > 3 days after injury ( table 2 ) . in all patients with ami
, nstemi events occurred more often than stemi ( 62.39% [ 73/117 ] vs. 37.61% [ 44/117 ] ) , but stemi significantly prevailed in the stress hyperglycemia group ( p < 0.05 ) . in table 3 , risk factors for ami and their significance are listed . a multiple logistic regression analysis using a forward stepwise ( conditional ) method revealed that stress hyperglycemia is an independent risk factor for the development of ami ( relative risk [ rr ] 2.130 [ 95% ci 1.4313.172 ] ) .
patients with stress hyperglycemia after hip fracture were 2.130-fold more likely to develop an ami than the non - hyperglycemia individuals .
characteristics of myocardial infarction in the two groups at the end of 3 months follow - up , there were no differences in mortality in patients with versus without stress - induced hyperglycemia ( 6.98% [ 42/602 ] vs. 4.43% [ 29/655 ] , p = 0.051 ) .
in our study , we found that the frequency of stress hyperglycemia was 47.89% and the incidence of ami was 9.31% in patients after hip fractures .
the incidence of ami in the stress hyperglycemia group was 12.46% , whereas the overall ami incidence in patients without hip fractures in china is 2.6% ( 14 ) , indicating that the incidence of ami in patients after hip fractures appears higher than overall ami incidence in china .
others have reported that the increased incidence of ami after hip fracture was related to osteoporosis ( 4,14 ) .
osteoporosis and cardiovascular diseases share common risk factors like poor general health status , lifestyle , nutrition , hormone secretion , vitamin d deficiency , systemic inflammation - related ( e.g. , c - reactive protein , interleukin-6 , or tumor necrosis factor ) diseases , and medications through different mechanisms both in bone and arteries ( 1525 ) .
patients with osteoporotic fractures are likely to have coexisting blood vessel diseases , but in our study , we found that particularly stress hyperglycemia plays a vital role , because the incidence of ami in the stress hyperglycemia group was 2.130 higher than in the non - hyperglycemia group . in patients with hip fractures ,
stress hyperglycemia was the sole significant independent risk factor for the development of ami ( 95% ci 1.4313.172 ) .
acute fractures induce stress hormone secretion of glucocorticoid , glucagon , adrenaline , thyroxin , somatotropin , and others , which is called the stress response .
during the stress response , activation of serial hormones induces insulin resistance , resulting in hyperglycemia and the associated risk factors present then in patients with acute traumata ( 26 ) .
it is well known that stress hyperglycemia is associated with oxidative stress , inflammatory responses , damaging of coronary microcirculation , and markedly worsened signal transduction pathways of endogenous cardioprotective responses .
hyperglycemia can induce adp - induced platelet aggregation and increase plasma catecholamine , which is associated with vulnerable plaque evolution , the promotion of microcircular dysfunctions , and thrombogenesis ( 2737 ) .
our present work indicates that for patients without a history of diabetes , stress - induced hyperglycemia plays a key role in the risk of developing ami .
our present analysis revealed that most ( 62.67% [ 47/75 ] ) amis occurred within the first 3 days after hip fractures , which is in accordance with the literature ( 2 ) . in the stress hyperglycemia group ,
blood glucose peaked at 23 days after hip fracture and then declined gradually , indicating a coincidence of ami with the peak time of fbg .
the cause of a more frequent stemi occurrence in stress - induced hyperglycemia might be that raised glucose levels contribute to platelet activation and thereby enhanced platelet - mediated thrombogenesis ( 38 ) , which develops into completely occlusive thrombi .
in addition , our study showed stress hyperglycemia ranges of 6.19.7 mmol / l after hip fractures for the first time , and we recommend that stress - induced hyperglycemias after hip fracture should be identified early .
a limitation of our study was that we did not investigate an effect of inflammatory factors and/or stress hormones on fbg levels , which lead to increased ami risk .
in addition , we did not compare the results of this study with patients suffering from diabetes , and we did not find differences between the stress hyperglycemia and no hyperglycemia groups regarding comorbid disorders and/or drug administrations , e.g. , statin against hypertension .
therefore we can not completely rule out that other factors also contributed to the incidence of myocardial infarctions .
moreover , patients were not accurately diagnosed for diabetes in the longer term , and because blood glucose levels largely fluctuate , we did not specifically treat the hyperglycemias . up until now , there has been no clear agreement on whether it is necessary to control glucose levels in these patients .
in addition , we only show the significance of hyperglycemia on the incidence of ami during admission ; we did not show clinical outcome data at follow - up .
however , this study underlines the importance of understanding the indication for adopting appropriate methods to identify stress - induced hyperglycemia in correlation with ami after hip fracture .
we recommend that in patients , even without previous diabetes , fbgs and ecgs should be monitored for at least the first 7 days after hip fractures .
this might be helpful for the endocrinologist , cardiologist , and orthopedic surgeon to timely detect amis .
we conclude that stress - induced hyperglycemia increased the risk of ami in patients with hip fractures .
|
objectiveto investigate the risk of acute myocardial infarction ( ami ) following stress hyperglycemia after hip fracture.research design and methodsfrom february 2007 to february 2012 , we carried out a prospective observational analysis of 1,257 consecutive patients with no history of diabetes who suffered hip fractures .
fasting blood glucose ( fbg ) and glycosylated hemoglobin tests as well as electrocardiography , ultrasonic cardiography , and chest x - ray examinations were performed after admission .
all selected hip fracture patients were divided into stress hyperglycemia and non - hyperglycemia groups according to their fbg , and the incidence of ami was monitored.resultsamong the patients enrolled , the frequency of stress hyperglycemia was 47.89% ( 602/1,257 ) and that of ami was 9.31% ( 117/1,257 ) , and the occurrence of ami in the stress hyperglycemia group was higher than in the non - hyperglycemia group ( 12.46 vs. 6.41% , p < 0.05 ) . in the stress
hyperglycemia patients , fbg reached maximum levels at 23 days after hip fractures and then decreased gradually . the ami incidence ( 62.67% [ 47/75 ] ) of
the stress hyperglycemia group was highest in the initial 3 days after hip fracture , significantly coinciding with the fbg peak time ( p < 0.05 ) . in all patients with ami ,
non
st - segment elevation myocardial infarction occurred more often than st - segment elevation myocardial infarction ( 62.39% [ 73/117 ] vs. 37.61% [ 44/117]).conclusionsstress - induced hyperglycemia after hip fracture increased the risk of ami .
|
RESEARCH DESIGN AND METHODS
Patient selection
Procedures
Statistical analyses
Clinical follow-up
RESULTS
CONCLUSIONS
|
technological advances in three - dimensional ( 3d ) imaging such as cone - beam computed tomography ( cbct ) appear to offer significant advantages in both quality and quantity of data representing true anatomy
,
.
the increased use of computed tomography in dentistry has also spurred the improvement of existing software designed primarily for dental use , as well as the development of new software
.
however , it is necessary to select protocols and to assess the reliability and accuracy of landmark identification in 3d images
.
development of 3d imaging into a practical and usable cephalometric method was hindered for many years by technologic limitations , high costs , and high radiation doses
.
advances in cbct technology make its use feasible for specific indications , such as impacted teeth , craniofacial anomalies , airway and temporomandibular joint disorders . in these cases ,
cbct images could be used for routine cephalometric purposes beyond the diagnostic purposes for which they were primarily taken .
this has been extensively done for cephalometric measurements
, but to a much lesser extent for maxillofacial computed tomography imaging
. whether cbct should be used routinely in all orthodontic patients
the voxel is the smallest unit of ct images and its size has an influence on image spatial resolution
. by decreasing the voxel size
the image spatial resolution increases as there is a decrease in partial volume averaging
. on the other hand , by decreasing the voxel size , the scan time increases , as does the probability of patient movement
.
this is especially of concern in the case of younger individuals , who constitute the overwhelming majority of orthodontic patients .
this issue of increased radiation dose to children , and specifically for orthodontic purposes , has caused widespread concern and has been the source of much discussion even in the lay literature
. in order to verify the clinical reliability of measurements on multiplanar sections and 3d reconstructions derived from cbct , it is also necessary to evaluate the measurements using anatomic landmarks without the use of metallic markers .
the most recent studies involving cbct scans have shown reproducibility and accuracy of cephalometric measurements performed on lateral cephalograms reformatted from cbct images
or in 3d - cbct images
. however ,
most of the studies used radiopaque markers
and these might have an influence on the accuracy and reproducibility of the measurements .
the presence of metallic markers eliminates errors from landmark identification because metallic landmarks can be easily identified and located with a high degree of accuracy and precision .
the aim of this study was to verify the influence of voxel size on the accuracy and reproducibility of linear measurements of the mandible performed without metallic markers on 3d cbct images , comparing multiplanar sections and 3d rendering images .
approval for this study was received from the ethics committee at the university of so paulo , bauru , brazil .
ten dry adult human mandibles , selected from the collection of dry skulls of the department of anatomy from the bauru school of dentistry were scanned . to fit the inclusion criteria , the mandibles had to be well preserved with no large metallic restorations on the teeth , in order to avoid scattering and artifacts .
the sample size of each group was calculated based on an alpha significance level of 0.05 and beta of 0.2 to achieve 80% of power
. to provide soft - tissue equivalent attenuation ,
the mandibles were positioned with the mandibular plane parallel and the midsagittal plane perpendicular to the floor , in accordance with the manufacturer s instructions for performing the scans in the clinical setting .
the cbct images were acquired using the i - cat classic 3d dental imaging system ( i - cat classic , imaging sciences international , hatfield , pa , usa ) .
each mandible was scanned twice : once with a 0.20 voxel resolution ( 36.12 mas , 40 sec , fov of 8 cm , 120 kv ) and once with a 0.40 voxel resolution ( 18.45 mas , 20 sec , fov of 8 cm , 120 kv ) .
cbct data were exported in dicom ( digital imaging and communication in medicine ) multi - file format and imported into dolphin11.5 software ( dolphin imaging & management solutions , patterson technology , chatsworth , ca , usa ) . a computer a505-s6975 ( satellite , toshiba , tokyo , japan ) with a dedicated 512-mb video card on a 17-in crystalbrite lcd flat - panel color screen with a maximum resolution of 1440x900 pixels
measurements were made separately on the multiplanar sections ( 2d ) and on 3d rendering images display window of dolphin 3d .
the multiplanar window displays the sagittal , coronal and axial slices , as well as the 3d rendered image .
it was possible to visualize the 3 projections and the 3d rendered image together or choose one of the four images in full screen .
linear measurements of 3d coordinates were obtained using several craniometric anatomic landmarks ( figure 1 ) . for multiplanar groups ,
each landmark ( figure 2 ) was identified and marked on the three orthogonal slices simultaneously ( axial , sagittal and coronal ) .
the corresponding linear measurements ( figure 3 ) were determined electronically with the measurement tool on the sectional images . for performing the linear measurements which landmarks were not located in the same sectional image , as c mf , the first mark of a line was performed in the sectional image showing the first landmark and the second mark was performed on a different section showing the second landmark . as the volume rendering was reoriented , the positions of the landmarks were verified in the axial , sagittal and coronal slices and , if necessary , relocated .
figure 1frontal ( left ) and sagittal ( right ) views of mandible showing the points
figure 2points .
reference in the anatomical view and in the cone - beam computed tomography ( cbct ) images
figure 3definitions of mandibular linear measurements
for 3d rendering images , the landmarks were identified directly on the surface for 3d images .
the measurements were performed twice by 2 blinded independent examiners ( tmff and ja ) .
the physical measurements of the dry mandibles were obtained with a high - precision digital caliper ( mitutoyo , mitutoyo sul americana , suzano , sp , brazil ) , identical to those used in similar studies
. in this initial study , we selected landmarks that are relatively easy to identify
linear measurements were identified on two occasions , one month apart , by one operator ( mlp ) .
the mean of the two measurements of each distance was calculated and used as the gold standard to evaluate the accuracy of the cbct 3d images .
the measurements were grouped into five groups : physical measurements ( g1 ) ; measurements performed on multiplanar slices with 0.20 mm voxel size ( g2 ) ; measurements performed on multiplanar slices with 0.40 mm voxel size ( g3 ) ; measurements performed on 3d images with voxel size of 0.20 mm ( g4 ) and measurements performed on 3d images with voxel size of 0.40 mm ( g5 ) .
intraclass correlation and bland - altman test was used to evaluate the intra- and interobserver reliability .
analysis of variance for repeated measures ( anova ) and dunnett s test were used to compare the groups on cbct images and the physical measurements .
all tests were performed with statistica software 6.0 ( statistica for windows ; statsoft , tulsa , ok , usa ) and spss version 10 ( spss , spss inc .
measurements were made separately on the multiplanar sections ( 2d ) and on 3d rendering images display window of dolphin 3d .
the multiplanar window displays the sagittal , coronal and axial slices , as well as the 3d rendered image .
it was possible to visualize the 3 projections and the 3d rendered image together or choose one of the four images in full screen .
linear measurements of 3d coordinates were obtained using several craniometric anatomic landmarks ( figure 1 ) . for multiplanar groups ,
each landmark ( figure 2 ) was identified and marked on the three orthogonal slices simultaneously ( axial , sagittal and coronal ) .
the corresponding linear measurements ( figure 3 ) were determined electronically with the measurement tool on the sectional images . for performing
the linear measurements which landmarks were not located in the same sectional image , as c mf , the first mark of a line was performed in the sectional image showing the first landmark and the second mark was performed on a different section showing the second landmark .
as the volume rendering was reoriented , the positions of the landmarks were verified in the axial , sagittal and coronal slices and , if necessary , relocated .
figure 1frontal ( left ) and sagittal ( right ) views of mandible showing the points
figure 2points .
reference in the anatomical view and in the cone - beam computed tomography ( cbct ) images
figure 3definitions of mandibular linear measurements
for 3d rendering images , the landmarks were identified directly on the surface for 3d images .
the measurements were performed twice by 2 blinded independent examiners ( tmff and ja ) .
the physical measurements of the dry mandibles were obtained with a high - precision digital caliper ( mitutoyo , mitutoyo sul americana , suzano , sp , brazil ) , identical to those used in similar studies
. in this initial study , we selected landmarks that are relatively easy to identify .
linear measurements were identified on two occasions , one month apart , by one operator ( mlp ) .
the mean of the two measurements of each distance was calculated and used as the gold standard to evaluate the accuracy of the cbct 3d images .
the measurements were grouped into five groups : physical measurements ( g1 ) ; measurements performed on multiplanar slices with 0.20 mm voxel size ( g2 ) ; measurements performed on multiplanar slices with 0.40 mm voxel size ( g3 ) ; measurements performed on 3d images with voxel size of 0.20 mm ( g4 ) and measurements performed on 3d images with voxel size of 0.40 mm ( g5 ) .
intraclass correlation and bland - altman test was used to evaluate the intra- and interobserver reliability .
analysis of variance for repeated measures ( anova ) and dunnett s test were used to compare the groups on cbct images and the physical measurements .
all tests were performed with statistica software 6.0 ( statistica for windows ; statsoft , tulsa , ok , usa ) and spss version 10 ( spss , spss inc . , chicago , il , usa ) .
the measurements for investigator 1 showed excellent reliability with intraclass correlation coefficients ( icc ) ranging from 0.93 to 1 ( icc average g2:0.97 ; g3:0.97 ; g4:0.98 and g5:0.98 ) .
additionally , the mean absolute differences between the first and second measurements were less than 0.40 mm ( mean average difference g2:0.11 mm and g3:0.02 mm ) for multiplanar images and less than 0.64 mm ( mean average difference g4:0.14 mm and g5:0.03 mm ) for 3d model images . for investigator 2 , the results also showed excellent reliability , with icc coefficients ranging from 0.88 to 0.99 ( icc average - g2:0.96 ; g3:0.95 ; g4:0.97 and g5:0.97 ) .
table 1intraobserver and interobserver variation for each group and analysis methods [ intraclass correlation coefficient ( icc ) and bland - altman ] multiplanar images3d model imagesvariable g2g3g4g5 0.2 voxel ( n=10)0.4 voxel ( n=10)0.2 voxel ( n=10)0.4 voxel ( n=10)intraobserver obser veradsd95% ciiccadsd95% ciiccadsd95% ciiccadsd95% ciiccc c
10.010.36 - 0.69 ; 0.711 - 0.010.15 - 0.31 ; 0.2910.120.67 - 1.20 ; 1.440.990.180.77 - 1.34 ; 1.700.98 2 - 0.410.75 - 1.89 ; 1.070.98 - 0.680.75 - 2.16 ; 0.800.970.170.87 - 1.54 ; 1.880.980.460.73 - 0.98 ; 1.900.98mf mf
1 - 0.130.46 - 1.03 ; 0.770.99 - 0.190.49 - 1.14 ; 0.760.980.170.72 - 1.25 ; 1.590.96 - 0.210.49 - 1.17 ; 0.750.98 2 - 0.301.18 - 2.16 ; 2.010.93 - 0.080.68 - 1.41 ; 1.250.97 - 0.210.82 - 1.82 ; 1.400.96 - 0.031.33 - 2.63 ; 2.570.92c mf
1 - 0.041.09 - 2.18 ; 2.100.970.310.67 - 0.99 ; 1.610.980.640.78 - 0.90 ; 2.180.980.260.81 - 1.32 ; 1.840.98 2 - 0.581.21 - 2.95 ; 1.790.95 - 0.920.97 - 2.81 ; 0.970.960.480.92 - 1.33 ; 2.290.970.420.75 - 1.04 ; 1.880.98c mf
10.190.84 - 1.46 ; 1.840.990.240.90 - 1.53 ; 2.010.970.390.67 - 0.93 ; 1.710.980.100.70 - 1.28 ; 1.480.99 2 - 0.660.81 - 2.24 ; 0.920.97 - 0.811.27 - 3.30 ; 1.680.940.100.73 - 1.34 ; 1.540.990.630.85 - 1.03 ; 2.290.97c mf
10.401.22 - 2.0 ; 2.800.950.051.40 - 2.68 ; 2.780.930.041.00 - 1.93 ; 2.010.960.261.06 - 1.83 ; 2.350.95 2 - 1.050.90 - 2.82 ; 0.720.91 - 0.291.01 - 2.27 ; 1.690.95 - 0.090.61 - 1.28 ; 1.100.990.410.68 - 0.92 ; 1.740.97c mf
10.121.40 - 2.62 ; 2.860.94 - 0.041.13 - 2.27 ; 2.190.950.090.90 - 1.67 ; 1.850.970.090.73 - 1.35 ; 1.530.98 2 - 0.650.88 - 2.36 ; 1.060.96 - 0.690.72 - 2.10 ; 0.720.96 - 0.020.99 - 1.96 ; 1.920.970.061.01 - 1.93 ; 2.050.97mco lco ( right)1 - 0.030.39 - 0.80 ; 0.740.98 - 0.070.40 - 0.84 ; 0.700.980.140.28 - 0.42 ; 0.700.99 - 0.030.45 - 0.91 ; 0.850.98 20.260.47 - 0.66 ; 1.180.970.180.71 - 1.22 ; 1.580.950.770.54 - 0.29 ; 1.830.900.680.55 - 0.40 ; 1.760.92mco lco ( left)10.390.44 - 0.47 ; 1.250.94 - 0.380.53 - 1.42 ; 0.660.930.010.35 - 0.67 ; 0.690.98 - 0.050.53 - 1.10 ; 1.000.95 2 - 0.050.53 - 1.08 ; 0.990.950.300.81 - 1.29 ; 1.890.88 - 0.080.43 - 0.93 ; 0.770.980.140.33 - 0.50 ; 0.780.98lco lco
10.300.38 - 0.34 ; 0.940.99 - 0.220.50 - 1.22 ; 0.780.99 - 0.030.31 - 0.65 ; 0.591 - 0.150.70 - 1.52 ; 1.220.99 2 - 0.300.70 - 1.09 ; 1.690.980.150.67 - 1.15 ; 1.450.990.770.53 - 0.28 ; 1.820.970.780.53 - 0.27 ; 1.830.97mco mco
1 - 0.070.35 - 0.76 ; 0.620.990.080.54 - 0.98 ; 1.140.98 - 0.220.35 - 0.91 ; 0.470.99 - 0.120.50 - 1.11 ; 0.870.99 2 - 0.330.51 - 1.34 ; 0.680.98 - 0.620.35 - 1.31 ; 0.070.97 - 0.080.35 - 0.78 ; 0.620.99 - 0.051.29 - 0.62 ; 0.520.99total10.11 0.970.02 0.970.14 0.980.03 0.98 2 - 0.41 0.96 - 0.35 0.950.18 0.970.35 0.97interobserverc c
1 x 2 - 0.540.98 - 2.46 ; 1.380.960.000.49 - 0.95 ; 0.960.99 - 0.110.88 - 1.84 ; 1.630.98 - 0.150.56 - 1.25 ; 0.950.99mf mf
1 x 21.000.61 - 0.20 ; 2.190.910.940.54 - 0.12 ; 1.990.923.591.490.66 ; 6.510.263.251.450.38 ; 6.090.45c mf
1 x 2 - 1.201.18 - 3.51 ; 1.150.93 - 1.491.09 - 3.63 ; 0.640.90 - 2.420.80 - 3.99 ; -0.840.84 - 2.560.66 - 3.86 ; -1.260.82c mf
1 x 20.631.05 - 1.45 ; 2.700.970.060.80 - 1.51 ; 1.640.980.071.20 - 2.30 ; 2.430.96 - 0.340.57 - 1.46 ; 0.790.99c mf
1 x 2 - 1.091.34 - 3.72 ; 1.530.89 - 1.070.94 - 2.91 ; 0.790.92 - 2.421.38 - 5.13 ; 0.300.70 - 2.471.06 - 4.56 ; -0.370.69c mf
1 x 20.400.60 - 0.78 ; 1.590.98 - 0.790.81 - 0.79 ; 2.380.950.070.68 - 1.28 ; 1.410.980.100.58 - 1.05 ; 1.240.99mco lco ( right)1 x 2 - 1.770.54 - 2.83 ; -0.710.69 - 1.840.50 - 2.83 ; -0.860.67 - 0.180.84 - 1.83 ; 1.460.92 - 0.490.46 - 1.40 ; 0.410.95mco lco ( left)1 x 2 - 1.430.54 - 2.49 ; -0.360.63 - 1.390.63 - 2.63 ; -0.140.63 - 1.130.84 - 2.79 ; 0.520.72 - 0.730.56 - 1.85 ; 0.380.86lco lco
1 x 2 - 1.420.55 - 2.50 ; -0.340.92 - 1.450.45 - 2.34 ; -0.550.93 - 0.091.16 - 2.35 ; 2.190.96 - 0.150.93 - 1.96 ; 1.670.97mco
mco
1 x 21.990.461.08 ; 2.890.762.120.431.27 ; 2.960.761.340.610.14 ; 2.530.861.170.480.21 ; 2.130.90total -0.34 0.86 - 0.49 0.87 - 0.13 0.82 - 0.24 0.86ad : average difference ; sd = standard deviation ; ci : confidence interval ; icc : intraclass correlation coefficient
ad : average difference ; sd = standard deviation ; ci : confidence interval ; icc : intraclass correlation coefficient cbct multiplanar reconstruction measurements showed good to high interobserver reliability with icc ranging from 0.63 to 0.99 ( mean average g2=0.86 and g3=0.87 ) with the absolute differences between the first and second measurements ranging for 0 to 2.12 mm ( mean average difference g2:-0.34 mm and g3:-0.49 mm , table 1 ) . for 3d reconstruction measurements , independently of the voxel size ( g4 or g5 ) , most of the variables presented good to high interobserver reliability , with the exception of inter - mental foramen width ( mf mf ) that had fair reliability on 0.2 mm voxel images ( g4=0.26 ) and moderate reliability for 0.4 voxel size ( g5=0.45 ) .
the mean values and standard deviations of the physical measurements and of the cbct measurements for all groups are presented in table 2 .
two variables ( mf mf and c mf ) at groups g2 and g3 were significantly smaller than physical measurements ( g1 ) . for 3d rendering images ( g4 and g5 ) , only two variables out of 10 were not significantly smaller than the physical measurements .
no significant difference was found between the voxel size both for 3d rendering and multiplanar sections .
table 2mean ( mm ) and standard deviation ( sd ) of linear measurements for dry mandibles compared to cone - beam computed tomography ( cbct ) images with different voxel sizes [ analysis of variance ( anova ) for repeated measures and dunnett s tests ] physical measurementmultiplanar images3d model images variableg1g2g3g4g5p 0.2 voxel0.4 voxel0.2 voxel0.4 voxel ( n=10)(n=10)(n=10)(n=10)(n=10 ) meansdmeansdmdmeansdmdmeansdmdmeansdmd c c
101.033.98101.154.150.11100.974.01 - 0,06101.124.060,09101.184.260.140.697mf mf
49.17
3.0347.91
2.98 - 1.2647.91
2.81 - 1,2647.16
2.64 - 2,0147.76
3.01 - 1.420.000*c mf
76.43
4.7176.50
4.420.0776.24
4.17 - 0,1875.59
4.60 - 0,8475.51
4.49 - 0.920.000*c mf
104.41
4.63103.82
4.79 - 0.58103.56
4.26 - 0,85102.76
4.38 - 1,64103.10
4.47 - 1.310.000*c mf
75.16
3.9975.66
3.640.5075.69
3.580,5374.48
3.46 - 0,6874.25
3.30 - 0.910.000*c mf
102.65
3.92102.44
4.02 - 0.21102.33
3.76 - 0,32101.29
3.90 - 1,36101.38
3.81 - 1.270.000*mco lco
21.42
2.4321.68
2.280.2621.69
2.280,2619.61
2.08 - 1,8120.07
2.19 - 1.350.000*mco lco
21.37
2.0521.28
1.67 - 0.0921.02
1.68 - 0,3419.31
1.82 - 2,0619.14
1.74 - 2.230.000*lco lco
124.25
4.05124.00
4.03 - 0.25123.78
4.10 - 0,47122.73
4.21 - 1,53122.74
4.04 - 1.510.000*mco mco
83.873.0984.072.860.2084.132.970,2684.072.810,283.762.98 - 0.110.251sd , standard deviation ; md ,
mean difference * statistically significantdifferent letters represent statistically significant differences ( dunnett s test )
sd , standard deviation ; md , mean difference statistically significant different letters represent statistically significant differences ( dunnett s test )
the availability of this technology is undoubtedly expanding the use and application of 3d imaging in the field of orthodontics
.
however , while cbct provides many advantages , patient radiation dose is still higher than conventional cephalometric and panoramic digital imaging modalities
.
this study was performed to evaluate the reliability and accuracy of linear measurements between common landmarks of the mandible taken from cbct multiplanar sections and 3d volumetric renderings with different voxel sizes .
in addition , larger voxel sizes are consistent with the alara principle , keeping the dose as low as reasonably achievable for the intended purpose of the scan
.
most of the previous studies
performed to validate cbct for 3d cephalometrics measured linear distances between anatomical landmarks in dry skulls in combination with radiopaque markers .
cephalometric analyses are subject to the influence of the examiner , landmark identification and type of image analyzed ( 2d sections or 3d image ) .
the major question of this study was if the difficulty in identifying the landmarks in 3d images can influence the reliability and accuracy of the measurements , and for this reason , metallic markers were not used .
this method is close to the real clinical procedures and is more clinically relevant than making measurements between well - defined metallic landmarks .
there was excellent intraobserver reliability for both examiners with the mean icc for all groups above 0.95 and mean average differences less than 0.41 mm , independently of the protocol analyzed ( table 1 ) .
these differences are not clinically significant for orthodontic / surgical diagnosis and treatment planning , and acceptable for skeletal measurements
.
additionally , good to high interobserver precision was identified for all groups , with exception of measurement mf
the main explanations for the good reliability found in this study are the use of landmarks which are easily identifiable , the calibration between observers and the high spatial definition of cbct images .
the differences between multiplanar sections and 3d volume rendering images were probably because landmarks on multiplanar images were identified on the orthogonal slices simultaneously instead of being identified directly on the surface of 3d reformatted images .
our study also evaluated the accuracy of measurements performed in cbct images ( table 2 ) .
two variables at g2 and g3 and 8 variables at g4 and g5 were significantly smaller than physical measurements ( g1 ) .
( 2009 ) , who showed a tendency for the cbct measurements to slightly underestimate the gold standard .
the consistency of the differences we found suggests a systematic error in the 3d model measurement method .
one possible explanation is that this error might have been introduced by the measurement software , mainly building line .
although few software systems currently have a 3d cephalometric module , most of them have not been tested or validated
.
the results of the validation measurements for a specific machine or software can not be extrapolated to the entire cbct machine and software
.
most of them are designed differently and software updates are constant and frequent . in addition , the difference ranged from 0.06 to 2.23 mm . in agreement with previous reports
, the average difference in our study is below clinical significance . according to grauer , et al .
( 2009 ) , generating measurements in 3d volumetric images rather than simultaneously in sets of 2d multiplanar images introduces error because of the difficulty involved in locating landmarks in 3d space and the inaccuracies of the user entered threshold used for the construction of 3d virtual surface models . rendered
for this reason , greater caution is suggested for linear measurements performed on 3d models
.
the spatial resolution of the image has an influence in the accuracy of the measurements
.
most studies on cbct with a large field of view used 0.4 mm voxel size
, whereas others with a small field of view used a 0.2 mm or smaller voxel sizes
.
none of the measurements showed significant difference between the voxel sizes , independently of the method of measurement ( table 2 ) .
( 2010 ) , who have suggested that 0.4 mm voxel resolution is adequate for performing measurements of craniofacial structures .
while small voxel sizes increase the image resolution , they also may increase the image noise
.
the benefits of a shorter scanning time , with its reduced likelihood of patient movement and especially the lower exposure dose compensates for the poorer resolution . besides
, large measurements are less influenced by small differences in spatial resolution
. on the other hand , the diagnostic ability of cbct images for evaluating small structures appears to be influenced by voxel size .
the two most common voxel sizes used in orthodontics 0.3 and 0.4 mm provide lower spatial resolution than smaller voxel sizes .
orthodontic scans with resolutions of 0.3 and 0.4 voxels are ideal for general treatment planning but should be used with caution if the goal is to assess small variations in bone thickness
.
( 2009 ) investigated the usefulness of cbct for identifying artificial external root resorption in images with voxel sizes of 0.4 , 0.3 , and 0.2 mm .
they concluded that , even though the results were the same for the different voxel sizes , diagnosis was easier at smaller voxel sizes of 0.3 and 0.2 mm .
although the benefits of a shorter scanning time satisfy the as low as reasonably achievable principle , the risks of misdiagnosis and treatment complications must also be weighed .
consequently , a scanning protocol with a 0.40 mm voxel size might not be suitable for every patient .
voxel size may vary according to the size of the structures to be analyzed as well as to the level of detail desired . in this study
, we measured distances that are in excess of the resolutions 0.2 and 0.4 mm of the scan , so that voxel size would not very likely influence the measurements .
unlike in conventional cephalometrics , where all the landmarks are identified in one image , namely , the lateral cephalogram , with cbct each landmark must be identified in three different images ( axial , coronal and sagittal ) , making the process of performing the measurements in cbct images more time - consuming for the orthodontist .
further , cbct demands a higher radiation dose than traditional cephalometric images . for these reasons ,
its use should be limited to specific indications , for example , patients with impacted teeth , or those with facial asymmetries or craniofacial anomalies where cbct is better able to quantify the differences between the right and the left side of craniofacial structures .
evidence - based will tell whether future advances , especially in terms of dose reduction , will make cbct appropriate for routine use in all orthodontic patients .
linear measurements obtained on multiplanar 2d cbct images with 0.2 and 0.4 voxel sizes using i - cat scanner and dolphin software are reliable and accurate for clinical diagnosis and treatment planning .
caution should be taken in linear measurements on 3d rendering images , because the measurements were reliable , but not accurate .
reducing the voxel size from 0.4 to 0.2 mm does not influence the accuracy and reliability of measurements of large craniofacial structures on cbct-3d images .
|
objective the purpose of this study was to determine the accuracy and reliability of two methods of measurements of linear distances ( multiplanar 2d and tridimensional reconstruction 3d ) obtained from cone - beam computed tomography ( cbct ) with different voxel sizes.material and methods ten dry human mandibles were scanned at voxel sizes of 0.2 and 0.4 mm .
craniometric anatomical landmarks were identified twice by two independent operators on the multiplanar reconstructed and on volume rendering images that were generated by the software dolphin. subsequently , physical measurements were performed using a digital caliper .
analysis of variance ( anova ) , intraclass correlation coefficient ( icc ) and bland - altman were used for evaluating accuracy and reliability ( p<0.05).results excellent intraobserver reliability and good to high precision interobserver reliability values were found for linear measurements from cbct 3d and multiplanar images .
measurements performed on multiplanar reconstructed images were more accurate than measurements in volume rendering compared with the gold standard .
no statistically significant difference was found between voxel protocols , independently of the measurement method.conclusions linear measurements on multiplanar images of 0.2 and 0.4 voxel are reliable and accurate when compared with direct caliper measurements .
caution should be taken in the volume rendering measurements , because the measurements were reliable , but not accurate for all variables .
an increased voxel resolution did not result in greater accuracy of mandible measurements and would potentially provide increased patient radiation exposure .
|
INTRODUCTION
MATERIAL AND METHODS
Image measurements
Statistical analyses
RESULTS
DISCUSSION
CONCLUSIONS
|
positive peritoneal washings are considered to be an important factor in management of patients with gynecological malignancies .
patients with positive peritoneal washings have been found to have worse prognosis than patients without malignant cells in cytology .
positive peritoneal washing in endometrial cancer is associated with an increased risk of recurrence and poor survival .
factors that can lead to false negative peritoneal cytology include inadequate volume of peritoneal fluid examined , obscuration of the tumor cells by florid mesothelial reaction , blood , excessive dilution of malignant cells , or small foci of peritoneal metastasis such that the tumor cells are not easily identified .
conventional smear may display cell clumping in one corner of the smear , obscuring of cells by mucus , haemorrhage , inflammatory cells , and debris along with multilayering of cells which will hamper cytological interpretation of the smear .
liquid - based cytology ( lbc ) uses the principle of cell filtration ( thinprep ) or cell enrichment ( surepath ) .
consequently , the smears are homogenously stained , circular smears of 22 mm diameter in thinprep and 13 mm diameter in surepath , which contain 5070000 well - preserved cells per smear .
the cells are uniformly distributed throughout the circular smear , which is devoid of background debris and obscuring by red blood cells ( rbcs ) or inflammatory cells . because of uniform staining and absence of background debris , the nuclear morphology is clear on lbc smear .
thus , lbc preparations can overcome the shortcomings of conventional smears to a large extent and improve the diagnostic accuracy .
a similar study in the english literature has explored the efficacy of thinprep lbc technique .
the aim of the present study was to evaluate gynecological cancers causing positive peritoneal cytology and to evaluate diagnostic accuracy of lbc using bd surepath lbc technique in peritoneal cytology .
an audit of peritoneal fluid / washing / lavage ( january 2012 to july 2013 ) was performed with identification of corresponding gynecologic specimens .
the samples were centrifuged and the smears were prepared from the sediment using a conventional technique ; the samples were then processed as per the manufacturer 's instructions for surepath lbc technique .
however , suspicious cases at the time of initial reporting were reported in the light of corresponding histopathologic diagnosis to categorize all cytology samples as positive or
( it is the standard practice in our department that smears with equivocal features on cytology are reported after examining the corresponding histopathological sections ) .
all the positive peritoneal wash samples were reviewed by two cytopathologists ( rt , ng ) to analyze cytological features in various gynecological malignancies and to study the difference in cytomorphology in conventional versus surepath lbc technique .
peritoneal fluid / washings comprised a total of 393 samples out of 12971 samples of exfoliative cytology ( excluding cervical pap samples ) received in the department during the study period .
age range for ovarian epithelial neoplasms ( 1573 ; mean 46.6 years ) , germ cell tumors ( 1456 ; mean 27 years ) , sex - cord stromal tumors ( 1970 ; mean 48 years ) , and uterine malignancies ( 2685 ; mean 57 years ) was noted .
a total of 83/393 ( 21.1% ) were positive and 310/393 ( 78.9% ) were negative for malignancy on peritoneal cytology .
out of the 393 samples of peritoneal washes , corresponding histology was available in 352 cases only .
rest of the cases either did not undergo surgery or were lost to follow - up .
out of 83 positive peritoneal washings , 69 samples had ovarian malignancies and 5 had uterine causes .
no record of histopathology of the remaining 9 cases with positive peritoneal cytology was available .
out of a total of 84 histopathologically diagnosed ovarian serous carcinoma cases , 55 ( 65.5% ) cases had positive peritoneal cytology [ figure 1a and b ] .
only 3 cases had a few clusters in conventional smears and the diagnosis was based mainly on lbc samples .
tumor cells were seen in peritoneal cytology in 1/8 ( 12.5% ) cases of borderline serous tumor ( atypical proliferative serous tumor ) with a few tiny papillaroid clusters of tumor cells . this case
four out of 9 cases ( 44.4% ) of mucinous adenocarcinoma were positive [ figure 1c and d ] with clusters as well as scattered tumor cells with mild nuclear atypia embedded in abundant mucinous material .
chicken - wire type of capillary network associated with mucin was also appreciated in 3 out of 4 cases .
lbc helped to identify malignant cells in 2 cases , which were darkly stained and embedded in thick mucin in conventional smears .
pseudomyxoma peritonei was seen in two cases of ovarian mucinous cystadenoma with mucinous neoplasm involving the appendix .
the tumor was present in ovary as well as the appendix ; hence , ovarian involvement was not secondary . thick mucinous material along with benign mesothelial cells were seen in these cases .
mucinous material was seen metachromatically staining in conventional smears ; however , the same was also represented as thick greenish mucin pools in lbc smears .
three out of 4 ( 75% ) cases of dysgerminoma , squamous cell carcinoma in teratoma ( n = 1 ) [ figure 2a and b ] , 2/4 ( 50% ) yolk sac tumor , and 1/13 ( 7.7% ) granulosa cell tumor had positive peritoneal cytology .
cytology samples revealed dispersed population of intermediate - sized tumor cells with high n / c ratio , coarse chromatin , prominent nucleoli , and scanty vacuolated cytoplasm in cases of dysgerminoma .
these were better seen in conventional mgg smears as compared to lbc samples due to even smaller size and intense staining in lbc smear .
the case of squamous cell carcinoma showed tumor cells having squamoid differentiation and marked variation in size and shapes of the tumor cells .
cases of yolk sac tumor showed obviously malignant cell clusters admixed with florid mesothelial cell proliferation .
the main clue to diagnosis of a germ cell tumor in fluid cytology was round - to - oval tumor cells with very prominent nucleoli and vacuolated cytoplasm .
the case of granulosa cell tumor revealed classic morphology with dispersed population of round - to - angulated tumor cells and a few tumor cells arranged in acinar - like arrangement around pink staining bodies .
the cases of dysgerminoma , yolk sac tumor , and granulosa cell tumor were initially reported as positive for malignancy on cytology .
positive peritoneal washing in ovarian serous carcinoma ( a ) cps ( mgg 40 ) ; ( b ) corresponding lbc ( pap stain 40 ) ; positive peritoneal washing in ovarian mucinous carcinoma ( c ) cps ( mgg 100 ) ;
( d ) corresponding lbc ( pap stain 100 ) positive peritoneal washing in squamous cell carcinoma arising in ovarian teratoma ( a ) cps ( mgg 100 ) ; ( b ) corresponding lbc ( pap stain 100 ) ; positive peritoneal washing in ovarian serous carcinoma ( c ) cps showing only mesothelial cells ( mgg 40 ) ; ( d ) corresponding lbc showing a cluster of malignant cells ( pap stain 100 ) uterine causes included 2/45 ( 4.4% ) cases of endometrioid adenocarcinoma , ( 25% ) cases of clear cell carcinoma , ( 50% ) cases of carcinosarcoma , and 1/4 ( 25% ) cases of cervix carcinoma [ tables 1 and 2 ] .
on review , 14 cases were identified in which peritoneal washings were reported as negative , both on conventional smear as well as the lbc smear , whereas histopathological examination of the omentum revealed tumor deposits .
further review of the smears ( both conventional and lbc smears ) of these cases failed to reveal any tumor cells .
distribution of ovarian neoplasms distribution of uterine neoplasms there were 2 cases of sertoli leydig cell tumor , 3 cases of fibroma thecoma , 15 cases of mature cystic teratoma of ovary , and 5 cases of leiomyosarcoma , where the peritoneal cytology was negative .
the histopathological diagnoses of other cases with negative peritoneal washings included leiomyoma , adenomyosis , follicular , endometriotic and simple cyst of ovaries , serous cystadenomas , mucinous cystadenoma , cervicitis , salpingitis , etc .
distribution of cases with negative peritoneal cytology all the positive cases ( n = 83 ) were reviewed by two cytopathologists mainly concentrating on cytologic features , which were different in two techniques ( conventional and lbc sure path ) .
lbc smears were easier to study , small circular smears of 13 mm diameter , with uniformly distributed , concentrated cells .
all the lbc smears were devoid of hemorrhagic background and debris , which was a confounding feature in conventional smears of 257 out of 393 cases . in our department , conventionally ,
after that , conventional smear stained with papanicolaou stain is examined , followed by lbc smear .
thus , it was observed that conventional smears of 4 cases had no cells , however , lbc smears showed presence of tumor cell clusters .
these cases on follow - up histopathology were diagnosed as ovarian high - grade serous carcinoma with omental metastases . there were 6 cases whose conventional smears had low cellularity ( less than three tumor cell clusters in the entire smear ) , however , lbc smears displayed uniformly present tumor cells [ figure 2c and d ] , indicating a better diagnostic accuracy of the lbc technique .
peritoneal wash cytology helps in the management of gynecological malignancies by identifying microscopic intraperitoneal metastatic deposits and helps in the surgical staging of these tumors . during
second - look laparotomy with negative peritoneal cytology , the patient may be considered disease free and chemotherapy may be discontinued accordingly .
serous ovarian carcinoma was found to be the most common gynecological malignancy resulting in positive peritoneal cytology in the present study .
we also observed positive peritoneal cytology in 3 cases of dysgerminoma , 2 cases of yolk sac tumor , and 1 case of granulosa cell tumor .
one case showed squamous cell carcinoma arising in teratoma , which led to shedding of malignant squamoid cells in the peritoneal wash fluid .
jacques et al . in 1991 studied multiple peritoneal cytologies collected during laparotomy for gynecologic malignancies and showed 23/61 cases of positive peritoneal cytology due to ovarian causes .
positive peritoneal cytology is associated with high recurrence rate and decreased survival in endometrial cancer .
( 1981 ) found that stage i endometrial carcinoma cases with negative peritoneal cytology did not recur , whereas tumor recurrence at shorter interval was observed in cases with positive peritoneal cytology .
however , according to yazigi et al . ( 1983 ) , positive peritoneal cytology did not influence prognosis in stage i endometrial carcinoma .
tubal transportation , exfoliation from extrauterine tumor deposits , contamination from manipulation during endometrial sampling or intracavitary therapy or other procedures , and lymphatic spread have been proposed as factors contributing to the spread of malignant cells in the peritoneal cavity .
one out of two cases of carcinosarcoma of the uterus also showed positivity for malignant cells in peritoneal lavage in the present study .
tumor cells are commonly observed from the carcinomatous component of carcinosarcoma because this component is less cohesive and is easily shed into washing samples .
selvaggi and jacques ( 1991 ) reported negative peritoneal cytology in all 33 cases of cervical carcinoma in their study .
false positive peritoneal washings , where peritoneal washings are reported as positive in absence of any known tumor in the patient has been described previously .
we did not observe any such case with false positive peritoneal cytology , similar to ziselman et al .
this observation may be explained by the fact that all the suspicious cases were reported along with corresponding gynecologic specimens .
there were 14 false negative cases ( negative peritoneal washings but had omental tumor deposits on histology ) in the present study .
. this could be because of dilution of sample with blood and less number of malignant cells in fluid .
four cases in the present study had only blood in conventional smears , and malignant cells could be identified on lbc smears only . in another 6 cases ,
conventional smears had less than three clusters of malignant cells , whereas lbc smears helped to concentrate and better delineate the tumor cells .
the negative peritoneal cytology may be explained by poor distribution or infrequent exfoliation of tumor cells .
this shortcoming is overcome in lbc technique where preservative solution lyses the rbcs and cell enrichment process results in uniform homogenous concentration of cells within the circular smear .
the excessive amount of fluid may also dilute the tumor cells , making their identification difficult in conventional preparations .
these factors may result in false negative peritoneal cytology which can be detrimental to the patient 's clinical outcome .
lbc preparation offers advantage in hemorrhagic and paucicellular samples improving the diagnostic accuracy of peritoneal washings . residual material from lbc samples can also be utilized for ancillary techniques such as immunocytochemistry ( icc ) .
icc can directly be done on lbc smears . however , in our study , we did not perform immunocytochemistry on lbc sample for diagnosis .
lbc smears also reduce the time of observer to hunt for tumor cells because cells are uniformly distributed and concentrated in the circular smear .
these factors result in improved productivity by reducing the inadequate rate and increasing the diagnostic accuracy of peritoneal cytology .
in conclusion , papillary serous carcinoma is the most common ovarian malignancy leading to positive peritoneal cytology and presenting at advanced stages .
endometrioid adenocarcinoma rarely leads to positive peritoneal cytology , especially when myometrial invasion is more than 50% and/or with parametrial involvement .
lbc technique leads to concentration of tumor cells causing reduction in false negative cases , especially in hemorrhagic and paucicellular cases , thereby improving the diagnostic accuracy of peritoneal washings .
the correct identification of malignant cells in peritoneal wash fluids is essential for the proper management of gynecological cancer , especially early stage carcinomas .
|
context : peritoneal washing is performed for staging of gynecologic tumors to detect subclinical intraperitoneal metastases.aim:the aim of the present study was to assess the impact of surepath liquid - based cytology ( lbc ) in peritoneal washing in various gynecological malignancies.settings and design : an audit of peritoneal - fluid / washing ( january 2012 to july 2013 ) was performed with corresponding gynecologic specimens .
all peritoneal washings were processed using both conventional and lbc technique .
suspicious cases on cytology were reported along with gynecologic specimens.results:there were a total of 393 peritoneal fluids .
eighty - three ( 21.1% ) were positive for malignancy , and the corresponding histology was available in 352 ( 89.6% ) cases .
sixty - nine positive samples had ovarian malignancies and 5 had uterine causes .
there were 9 cases of peritoneal washings in which no histopathology was available .
the most common cause of positive peritoneal cytology was ovarian serous carcinoma in 55/84 ( 65.5% ) cases .
other causes included mucinous cystadenocarcinoma , dysgerminoma , squamous cell carcinoma in teratoma , yolk sac tumor , and granulosa cell tumor .
uterine causes included 2/45 ( 4.4% ) cases of endometrioid adenocarcinoma , ( 25% ) cases of clear cell carcinoma , ( 50% ) cases of carcinosarcoma , and ( 25% ) cervix carcinoma . on review of positive cases
( n = 83 ) , 10 cases were identified , which had nil ( n = 4 ) to low cellularity ( <3 tumor clusters / smear ; n = 6 ) on conventional smears , and were confirmed malignant on lbc.conclusions:the most common ovarian malignancy causing positive peritoneal cytology is papillary serous carcinoma .
endometrioid adenocarcinoma rarely leads to positive peritoneal cytology .
lbc technique leads to concentration of tumor cells causing reduction in false negative cases , especially in hemorrhagic and low - cellular cases .
|
Introduction
Patients and Methods
Results
Discussion
Conclusion
Financial support and sponsorship
Conflicts of interest
|
endometrial cancer ( ec ) is one of the most common gynecologic malignancies , occupying the sixth place in malignant tumor throughout the world with an increasing trend .
the initial treatment of ec is comprehensive staging surgery , which consists of total hysterectomy , bilateral salpingo - oophorectomy , pelvic and para - aortic lymphadenectomy and peritoneal cytology .
laparotomy is the traditional approach , but laparoscopic surgery is widely proposed recent years since it was first reported using for the treatment of ec in 1992 by childers and surwit . in lots of retrospective and prospective studies ,
laparoscopy has been showed with many advantages compared to laparotomy , such as smaller incision , less pain , and faster recovery .
it seems that laparoscopic surgery for ec also has similar recurrence rate and survival outcome with laparotomy procedure .
however , most of these studies have focused on patients with early stage or low - risk disease and few specifically on patients with advanced stage or high risk .
what 's more , data of long - term survival outcomes about laparoscopic surgery remain limited .
willis et al have published an article about laparoscopic hysterectomy in a high - risk series of patients with ec , showed that laparoscopy provide all the benefits inherent in minimal access surgery with no compromise in terms of outcomes , but there was no laparotomy data as control .
therefore , to compare the long - term safety and efficacy between laparoscopic surgery and laparotomy in patients with high - risk ec , we conducted a retrospective analysis based on our decade of clinical data using propensity score matching ( psm ) in this research .
a total of about 400 medical case notes of patients with histologically confirmed ec who has been received surgical treatment in beijing chao - yang hospital affiliated capital medical university from 2000 to april 2014 were reviewed .
we selected subjects according to the following criteria : high - risk ( grade 2 , histologic nonendometrioid , myometrial invasion 1/2 , positive pelvic or para - aortic nodes or stages ii
iv ) patients who had undergone comprehensive surgical staging in the form of peritoneal cytology , total hysterectomy , bilateral salpingo - oophorectomy , pelvic lymphadenectomy and para - aortic lymphadenectomy or sampling , + / cytoreductive surgery by the same experienced surgical team via laparotomy ( total abdominal hysterectomy , tah ) or laparoscopy ( total laparoscopic hysterectomy , tlh ) .
para - aortic lymphadenectomy was performed when para - aortic lymph nodes sampled positive and nonendometrioid carcinomas .
patients who were enrolled ( excepted staged i ag2 with histologic endometrioid ) received postoperative adjuvant therapy including 3 to 6 cycles of chemotherapy ( paclitaxel , 175 mg / m + carboplatin , area under the concentration
time curve [ auc ] 5 or adriamycin , 45 mg / m + cisplatin , 50 mg / m + paclitaxel , 160 mg / m ) and vaginal cuff brachytherapy or pelvic irradiation ( 45 gy over 5 weeks ) .
patients had antibiotic prophylaxis ( cephalosporins or quinolones ) half an hour before operation and venous thrombosis prophylaxis after operation by using molecular weight enoxaparin ( 0.4 ml every day for 45 days ) and lower extremity sequential compression device or graduated compression stocking .
patients associated with severe cardiopulmonary disease ( myocardial infarction , heart failure , or cerebral infarction ) , other malignancies or a bulky uterus 12 weeks of gestation were excluded . because our data source was case notes and the missing data were few , we deleted the individuals with missing data .
the collected data of patients included age , body mass index ( bmi ) , the scope of operation , operating time , blood loss , intraoperative and postoperative complications , first flatus time , postoperative hospital stay , surgical stage , histologic type and grade , number of lymph nodes dissection , and clinical outcome .
the staging of patients was done according to the international federation of gynecology and obstetrics ( figo ) 2009 staging classification .
intraoperative complications included injuries to bladder , ureter , intestine or large vessels , bleeding requiring blood transfusion , conversion from laparoscopy to laparotomy and transferred to intensive care unit ( icu ) for any unintended reasons .
postoperative complications included fever ( > 38c ) after 24 hours postoperatively , wound infection or dehiscence , deep - vein thrombosis ( dvt ) , and pulmonary embolism ( pe ) within 30 days after operation .
the overall survival ( os ) was defined as the period from the date of surgery to death or the date of last contact for living people .
follow - up is completed by telephone , e - mail , or outpatient service .
all procedures were approved by the ethics committee for human experiments of the capital medical university . because parts of the baseline characteristics were statistical different between patients received laparoscopic surgery and laparotomy
eight covariates entered in the propensity model , including age , bmi , the scope of operation , myometrial invasion , pathologic type and grade , lymph node metastasis , and surgical stage .
propensity scores were calculated using a nonparsimonious multivariable logistic regression model to estimate the conditional probability of a patient receiving a surgery approach .
then a 1:1 match between the laparoscopy and laparotomy groups was performed by using the nearest available neighbor matching . after matching ,
data of patient baseline characteristics , operation outcome and incidence of intraoperative and postoperative complication were compared using t test , chi - square test , or fisher exact test .
survival data were estimated using kaplan meier curves and compared between groups by log - rank statistics . the 5- and 10-year survival rate and os were reported .
all of the statistical analysis was completed by spss 19.0 ( spss version 19.0 ; spss , inc . , chicago , il ) . statistical significance was defined as p < 0.05 .
three hundred eighty - two case notes were reviewed including 255 patients with high - risk ec . among these 225 patients ,
31 individuals with severe cardiopulmonary disease , other malignancies or uterus 12 weeks of gestation were excluded ; 4 individuals with missing surgical or pathological data were deleted .
finally , a total of 220 patients with high - risk ec met the study criteria , 107 were treated by laparotomy and 113 by laparoscopy .
table 1 provides details of patient characteristics across the laparotomy and laparoscopy group pre- and postpropensity score matching . before matching
, significant differences were not found in age , myometrial invasion , pathological type , and grade and lymph node metastasis between 2 groups but in bmi , scope of operation , and figo stage which could be the confounding factors . after matching , 81 pairs of patients were matched successfully . in the matched cohorts
baseline patient characteristics pre- and postpropensity score matching the mean operative time was similar between 2 groups , 253 minutes in laparoscopy group and 258 minutes in laparotomy group .
the mean blood loss were significant less in laparoscopy group ( 107 ml ) than laparotomy group ( 414 ml ) ( p < 0.01 ) .
the laparoscopy cohort also has significant shorter first flatus time ( 2.0 days vs. 2.4 days , p < 0.01 ) and postoperative hospital stay ( 14.7 days vs. 17.7 days , p = 0.02 ) than laparotomy which meant that patients received laparoscopic surgery had more rapid recovery than that received laparotomy .
the mean numbers of pelvic lymph nodes dissected by laparoscopy were 16.6 compared 21.9 by laparotomy with significant difference ( p < 0.01 ) .
however , the mean numbers of para - aortic lymph nodes were comparable between 2 groups ( p = 0.11 ) ( table 2 ) .
comparison of operation outcome intraoperative complications were significant different between the 2 groups ( 6.2% for laparoscopy and 25.9% for laparotomy , p < 0.01 ) .
the patients received transfusion during surgery in laparotomy group was statistically more than that in laparoscopy ( p < 0.01 ) . for the incidence of transferred to icu on account of poor cardiopulmonary resuscitation , it was also statistically higher in patients treated by laparotomy than by laparoscopy . moreover , no injury to bladder , ureter , or large vessels occurred in both 2 groups , only 1 patient in laparotomy group had the intestine injury because of heavy pelvic adhesion that had been received intestinal repair .
there was no significant difference in postoperative complications between the 2 groups ( 19.8% for laparoscopy and 29.6% for laparotomy , p = 0.14 ) .
dvt occurred more often in laparotomy patients than laparoscopy patients , 14.8% versus 3.7% ( p = 0.01 ) .
six patients developed pe in laparotomy group compared 1 in laparoscopy with no statistically difference ( table 3 ) .
comparison of intraoperative and postoperative complications clinical outcome of 2 groups was shown in table 4 .
the median follow - up was 45 months ( rang 5176 ) . during the follow - up time
, there were 16 ( 9.9% ) recurrences which were similar between the 2 treatment cohorts , 7 ( 8.6% ) were observed in laparotomy group at peritoneal , liver , lung , vaginal , and abdominal incision versus 9 ( 11.1% ) in laparoscopy group at peritoneal , liver , lung , and vaginal .
twenty patients died , 11 in the laparotomy group and 9 in the laparoscopy group .
the kaplan meier estimate for survival rate in laparotomy and laparoscopy at 5 years was 89.2% , 85.3% and at 10 years was 75.8% , 85.3% , respectively .
no statistically significant difference was found between the 2 groups when os was compared ( p = 0.97 ) ( figure 1 ) .
it showed that no significant difference between laparotomy and laparoscopy cohorts ( 75.8% vs. 85.3% , p = 0.97 ) .
three hundred eighty - two case notes were reviewed including 255 patients with high - risk ec . among these 225 patients ,
31 individuals with severe cardiopulmonary disease , other malignancies or uterus 12 weeks of gestation were excluded ; 4 individuals with missing surgical or pathological data were deleted .
finally , a total of 220 patients with high - risk ec met the study criteria , 107 were treated by laparotomy and 113 by laparoscopy .
table 1 provides details of patient characteristics across the laparotomy and laparoscopy group pre- and postpropensity score matching . before matching
, significant differences were not found in age , myometrial invasion , pathological type , and grade and lymph node metastasis between 2 groups but in bmi , scope of operation , and figo stage which could be the confounding factors . after matching , 81 pairs of patients were matched successfully . in the matched cohorts
the mean operative time was similar between 2 groups , 253 minutes in laparoscopy group and 258 minutes in laparotomy group .
the mean blood loss were significant less in laparoscopy group ( 107 ml ) than laparotomy group ( 414 ml ) ( p < 0.01 ) .
the laparoscopy cohort also has significant shorter first flatus time ( 2.0 days vs. 2.4 days , p < 0.01 ) and postoperative hospital stay ( 14.7 days vs. 17.7 days , p = 0.02 ) than laparotomy which meant that patients received laparoscopic surgery had more rapid recovery than that received laparotomy .
the mean numbers of pelvic lymph nodes dissected by laparoscopy were 16.6 compared 21.9 by laparotomy with significant difference ( p < 0.01 ) .
however , the mean numbers of para - aortic lymph nodes were comparable between 2 groups ( p = 0.11 ) ( table 2 ) .
intraoperative complications were significant different between the 2 groups ( 6.2% for laparoscopy and 25.9% for laparotomy , p < 0.01 ) . none of the laparoscopic cases was converted into open procedures .
the patients received transfusion during surgery in laparotomy group was statistically more than that in laparoscopy ( p < 0.01 ) . for the incidence of transferred to icu on account of poor cardiopulmonary resuscitation
, it was also statistically higher in patients treated by laparotomy than by laparoscopy . moreover , no injury to bladder , ureter , or large vessels occurred in both 2 groups , only 1 patient in laparotomy group had the intestine injury because of heavy pelvic adhesion that had been received intestinal repair .
there was no significant difference in postoperative complications between the 2 groups ( 19.8% for laparoscopy and 29.6% for laparotomy , p = 0.14 ) .
dvt occurred more often in laparotomy patients than laparoscopy patients , 14.8% versus 3.7% ( p = 0.01 ) .
six patients developed pe in laparotomy group compared 1 in laparoscopy with no statistically difference ( table 3 ) .
the median follow - up was 45 months ( rang 5176 ) . during the follow - up time
, there were 16 ( 9.9% ) recurrences which were similar between the 2 treatment cohorts , 7 ( 8.6% ) were observed in laparotomy group at peritoneal , liver , lung , vaginal , and abdominal incision versus 9 ( 11.1% ) in laparoscopy group at peritoneal , liver , lung , and vaginal .
twenty patients died , 11 in the laparotomy group and 9 in the laparoscopy group .
the kaplan meier estimate for survival rate in laparotomy and laparoscopy at 5 years was 89.2% , 85.3% and at 10 years was 75.8% , 85.3% , respectively .
no statistically significant difference was found between the 2 groups when os was compared ( p = 0.97 ) ( figure 1 ) .
it showed that no significant difference between laparotomy and laparoscopy cohorts ( 75.8% vs. 85.3% , p = 0.97 ) .
for accurately estimating the effect of intervention , randomized controlled trial ( rct ) is considered the optimal approach but often limited by ethic and other problems .
psm can eliminate the imbalance between groups and reduce the effects of confounding to achieve a random effect in observational studies . in the present study ,
the patient baseline characteristics of laparotomy and laparoscopy group were adequately balanced after matching , and then , the surgery outcomes achieved comparability .
this retrospective study demonstrated that laparoscopic technique is effective and even safer compared with laparotomy for patients with high - risk ec . in 2009 , gynecologic oncology group ( gog ) compared laparoscopy and laparotomy for comprehensive surgical staging of ec in a randomized multicenter trial which involved 2616 patients and reported that laparoscopic surgery resulted in fewer complications and shorter hospital stay but longer operation time . in another large randomized trial published in 2010 , laparoscopic surgery was associated with a shorter hospital stay and less amount of blood loss , but no beneficial effect was observed in terms of major complications and operation time .
however , our data showed evidence of less blood loss , lower proportion of major complications and shorter hospital stay with laparoscopy versus laparotomy and a similar operation time .
operation time is much related to the experience of the surgical team and learning curve .
as the improvement of surgical technique , it is a trend that laparoscopic operation time becomes shorter .
moreover , our study indicated that the first flatus time was shorter in laparoscopy group than in laparotomy .
patients treated with laparoscopic surgery had smaller incision and less pain compared to those treated with laparotomy .
laparoscopy can also bring out an earlier resumption of bowel function and shorter hospital stays .
we found that the postoperative hospital stays in our data were longer than that reported in most of the previously studies and just similar to kong 's study in korea .
kong reported a mean hospitalization of 16.4 days for laparoscopy and 23.3 days for laparotomy .
but in gog study , the median hospital stays was 3 days for laparoscopy and 4 days for laparotomy .
patients in china are accustomed to a longer hospitalization , which is mainly decided by the social factors . in chinese traditional opinion , patients are willing to discharge from hospital only when they have complete recovery ( after removing drainage tube and urinary catheter or taking out stitches ) , what 's more , they need not worry about the medical expense because of the coverage of insurance .
so , some patients even have been in hospital until the end of the first chemotherapy . in the present study ,
intraoperative complications were significantly lower in the laparoscopy group and no injuries to bladder , ureter , or large vessels occurred .
the incidence of major complications can be highly reduced when the procedure was performed by experience surgeon .
however , it seems that laparoscopy had no benefit in the incidence of postoperative complications .
but we found dvt occurred significantly often in patients treated by laparotomy than those treated by laparoscopy . in gog study , there was no significant difference in incidence of venous thromboembolism ( vte ) and pe between different approaches .
in contrast , fader 's study showed that the laparoscopy cohort had significantly fewer episodes of vte .
lots of studies indicate that the combination of underlying malignancy and pelvic surgery is the risk for developing a vte . in a japanese large research about risk factors for vte , the age ( 50 or 55 years ) , a diagnosis of cancer , operating time ( 4 hours ) , blood loss ( 1000 ml ) , and blood transfusion ( 2000 ml ) were reported significantly related to postoperative vte .
it may account for the difference of dvt incidence between the 2 groups that laparotomy had longer operating time and more blood loss in our study . what 's more , trendelenburg position during laparoscopic surgery may play a role in preventing vte .
most of the previous studies demonstrate that the pelvic or para - aotic lymph nodes count removed by laparoscopy approach were similar to those dissected by laparotomy .
laparoscopy has been proven to be even more efficient in lymphadenectomy because of its clearer vision .
however , obermair and kalogiannidis reported lower number of lymph node dissections in the laparoscopy group . in the present study , the mean number of dissected para - aotic lymph nodes was comparable between the 2 groups , but the pelvic lymph nodes were significant fewer in laparoscopy cohort than laparotomy .
two large rcts reported by benedett and kitchener showed no evidence that lymphadenectomy could decrease the risk of recurrence or mortality rate in patients with early ec .
conversely , another retrospective research gave a result that lymphadenectomy improved the survival of patients with high - risk ec and stage i b to stage iv .
moreover , an article published in lancet in 2010 reported that para - aotic had a significantly benefit in terms of os for patients with ec at mediate or high risk .
it seems that the difference of pelvic lymph nodes count between the laparoscopy and laparotomy groups had not influenced the os in our study .
lots of previous studies have proven that laparoscopy surgery was similar to laparotomy in short - term survival . a meta - analysis reported in 2012
based on 8 rcts showed that there was no significant difference in disease - free survival , cancer - related survival , or os .
cho et al reported a similar 5-year os and progression - free survival between laparoscopy and laparotomy groups based on 10-year experience . in 2012 , the gog published the survival data of their large rct in 2009 that the 5-year os was almost identical in both laparoscopy and laparotomy cohorts at 89.8% . between 2000 and 2010
, we conducted a randomized trial and reported that the 5-year os rates were 91% of laparotomy and 96% of laparoscopy with no significantly difference . in another retrospective study focus on high - grade ec in 2012 , progression - free and os were not significantly different between the surgical cohorts at 44-month median follow - up . in the present study , parts of patients were follow - up more than 10 years . with a median follow - up time of 45 months ,
the 5- and 10-year survival rate for the laparotomy were 89.2% and 75.8% compared with 85.3% and 85.3% for the laparoscopy .
there was no significant difference in os between 2 cohorts and also the recurrence rate .
although the patients enrolled were not assigned to different approaches randomly which was a limitation of our study , we performed a psm and successfully to control a certain confounding factors yet .
when randomized trial was limited by the objective conditions , psm might be a feasible alternative to reduce selection bias . in conclusion
, it seems that laparoscopic surgery is as effective as laparotomy in the long term and can be safely carried out in patients with high - risk ec for surgery treatment .
furthermore , other long - term follow - up data on high - risk ec of randomized trials is still needed to confirm these results .
|
abstractthe aim of this study was to compare the long - term safety and efficacy of laparoscopic surgery and laparotomy for high - risk endometrial cancer ( ec).a retrospective analysis based on our decade of clinical data of patients with high - risk ec who were comprehensively surgically staged by laparotomy or laparoscopy was performed .
the surgical outcomes were compared between different approaches using propensity score matching ( psm).eighty - one pairs of patients from the initial 220 enrolled ones were matched by psm .
the mean operative time is similar between laparotomy and laparoscopy groups ( 258 minutes vs. 253 minutes ) .
the laparoscopy cohort has less blood loss ( 107 ml vs.414 ml , p < 0.01 ) , shorter hospital stay ( 14.7 days vs. 17.7 days , p = 0.02 ) and significant fewer intraoperative complications ( 6.2% vs. 25.9% , p < 0.01 ) .
the pelvic lymph nodes dissected by laparoscopy ( 16.4 ) were significant less than that dissected by laparotomy ( 21.9 ) .
the 5- and 10-year survival rate for laparotomy were 89.2% and 75.8% compared with 85.3% and 85.3% for the laparoscopy .
there was no significant difference in overall survival ( p = 0.97).laparoscopy is as effective as laparotomy in the long term and can be safely carried out in patients with high - risk ec for surgery treatment .
|
INTRODUCTION
MATERIALS AND METHODS
RESULTS
Baseline Characteristics
Operation Outcome
Intraoperative and Postoperative Complication
Clinical Outcome
DISCUSSION
|
the patient was a 41-week - gestational - age infant girl with a birth weight of 3300 g. after an uneventful pregnancy , the patient was delivered spontaneously and healthy until this episode . at 35 days of age , the patient was admitted to our hospital with a one - day history of anorexia , vomiting and recurrent diarrhea . on admission , conscious level was glasgow coma scale e2 v2 m2 , and blood pressure was 104/flat . physical examination revealed a distended and tender abdomen .
laboratory values were significant for a c- reactive protein ( crp ) of 1.7 mg / dl ( normal range was less than 0.3 mg / dl ) .
the patient was suspected intestinal obstruction due to enteritis , antibiotics were started ( abpc / mcipc and amk ) .
4 hours after admission , she was pale with a temperature of 39.2 c and 220 per minutes pulse .
blood pressure was low , and the fevers , vomiting , and diarrhea persisted and worsened despite antibiotics .
she failed to respond to medical managements and died 27 hours after admission due to multiple organ failures .
the cyst was unilocular , contained watery fluid , did not communicate with the adjacent intestinal lumen ( fig .
microscopic finding of the ileocecal duplication cyst had an undefined mucosal lining though it resembled pyrolic glands or duodenal glands .
duplications of the alimentary tract can be located anywhere between the mouth and the anus.4 they are most commonly found in the ileum ( 30% ) , ileocecal valve ( 30% ) , duodenum ( 10% ) , stomach ( 8% ) , jejunum ( 8% ) , colon ( 7% ) and rectum ( 5% ) with the half of the all lesions may contain ectopic gastric mucosa.5,6 most alimentary tract duplications are cystic ( 80%90% ) , with the remainder being tubular.7 they have three common features , hollow structure that has a wall of smooth muscle often shared with the contiguous intestinal tract , lined by mucous membrane similar to some part of the alimentary canal , and usually attached to some part of the alimentary tube.8 the clinical symptoms are highly variable from minor digestive problems to intestinal obstruction , gastrointestinal bleeding , or perforation.9 most patients present before the age of 2 years with obstructive symptoms , pain , or an abdominal mass .
the presence of gastric or pancreatic mucosa can lead to peptic ulceration , perforation , and hemorrhage .
early diagnosis and treatment reduces the risk of dangerous complications , such as bowel perforation , bleeding , obstruction , and malignant alteration .
resection of duplication alone is the treatment of choice , and in uncomplicated cases laparoscopic resection is advised .
surgically treated intestinal duplications have good prognosis , however , such duplications can have a fatal consequence if not properly treated .
possible complications like bowel perforation , bleeding , obstruction , and malignant alterations are the reason why all duplications should be surgically treated at the time of diagnosis.10 alimentary tract duplications can be easily mis - diagnosed as other disorders.5 pyloric and duodenal duplications can mimic hypertrophic pyloric stenosis or choledochal cyst.11,12 ovarian cyst should be considered in female patients , and adolescent patients can sometimes have a presumptive diagnosis of crohn s disease.5 yousefzadeh et al13 showed that duplication of the colon and rectum clinically presented as abdominal pain , vomiting and chronic constipation , a significant number of patients were thought to have hirschsprung s disease . in our fatal case ,
there are two reasons for this diagnostic difficulty : first , fevers , vomiting , and recurrent diarrhea persisted and worsened despite antibiotics .
the patient was suspected intestinal obstruction due to enteritis , however , actually the ileocecal duplication cyst led to intestinal obstruction and necrotizing ileum ; second , the patient died in an instant due to multiple organ failures .
if this patient was considered for exploratory laparotomy , she might be able to be alive .
however , the patient was getting worse very quickly due to multiple organ failures and showed intractable psvt .
exploratory laparotomy was impossible . it is demonstrated a high association of other anomalies in these patients including skeletal and urologic abnormalities,13,14 however , in this case , there were no other congenital anomalies .
sudden unexpected death of infants associated with duplication cysts is extremely rare with only 4 cases reported in medical literature to date ( table 1 ) .
puligandla ps et al5 and byard rw15 reported the patients presented with group b streptococcal sepsis and clostridium perfringens sepsis , respectively .
kibayashi et al16 reported the patient died at home after being diagnosed as having gastroenteritis .
in present case , though blood , stool and cyst cultures were negative , the patient had high fever and crp level elevated .
infection is suspected to be a risk factor for sudden and unexpected death in infants with duplication cysts . in all cases ,
this potential diagnosis should be borne in mind for a patient who complains of abdominal symptoms with an unknown cause , and duplication cyst should be recognized as a fatal cause in infant .
|
duplications of the alimentary tract are very rare . a one - month - old female presented with symptoms of anorexia , vomiting and continuous watery diarrhea .
the plain abdominal radiograph showed thickened intestinal wall and signs of small bowel obstruction .
the fevers , vomiting , and continuous wartery diarrhea persisted despite antibiotics , and worsened .
the patient failed to respond to medical managements , 27 hours after admission , the patient died due to multiple organ failures .
the autopsy was performed , small bowel obstruction due to an ileocecal duplication cyst ( 3 3 cm ) was recognized .
the ileocecal duplication cyst was attached to the ileum which was changed edematous and necrotic .
this potential diagnosis should be borne in mind for a patient who complains of abdominal symptoms with an unknown cause , and duplication cyst should be recognized as a fatal cause in infant .
|
Report of Case
Discussion
|
a stroke is defined by the who as rapidly developing clinical signs of a focal ( or global ) disturbance of cerebral function , with symptoms lasting 24 h or longer or leading to death , with no apparent cause other than of vascular origin . taking into consideration the cause , the mechanism and the character of morphological lesions ,
there are two types of stroke : ischemic and haemorrhagic [ 2 , 3 ] .
eighty percent of strokes are of ischemic origin [ 2 , 4 ] , and their most common cause is thrombosis of the atherosclerotic internal carotid artery ( ica ) and/or cerebral arteries ( ca)20 % .
a stroke , including an ischemic one , is of particular interest to the forensic pathologist aiming on establishing the cause of death and its mechanism , rather than for giving an opinion in cases of suspected medical malpractice .
neck and/or head trauma may very rarely constitute a cause of thrombosis of the ica and ca without any pre - existing pathology [ 58 ] .
it most commonly results from direct , penetrating or non - penetrating ( blunt ) neck trauma in the region of large cervical vessels ; less frequently it is attributed to indirect head trauma . in such cases
the autopsy result and available clinical information has to allow the forensic pathologist to prove the existence of neck and/or head trauma and establish its causal connection with ica thrombosis and exclude other , known , non - traumatic causes of the latter .
we present two cases of ica thrombosis with concomitant middle cerebral artery ( mca ) thrombosis secondary to the thrombus present in the ica lumen , in which the postmortem examination , the medical records regarding hospitalization and records of investigation indicated trauma as the cause of thrombosis .
a 57-year old male carpenter sustained an injury in an occupational accident in the carpenter s warehouse .
he was struck very hard in the face , in the region of his left cheek by an irregular - shaped wood fragment which looked like a big splinter ( measuring about 26 cm in length , and with the greatest cross - sectional dimensions of 3.5 1.5 cm ) that broke off from the board being processed on the carpenter s machine ( fig . 1 ) .
first aid was provided in the emergency department in the local hospital , where the presence of a large , wooden fragment that thrust deep into the left cheek and penetrated through the parapharyngeal space into the neck was diagnosed . on admission
he was then transferred to the otolaryngological ward , where a physical examination , including an evaluation of his neurological status revealed : maintained consciousness , narrow and symmetric pupils , circulatory and respiratory sufficiency and bp of 180/80 mmhg .
the ct examination revealed the presence of a foreign body penetrating the left maxillary sinus with a comminuted fracture of the anterior and inferior wall below the external surface of the base of the skull , and a left occipital condyle fracture with concomitant breaking and impression into the posterior cranial fossa and soft tissues of the neck .
no cerebral lesions , including traumatic , or features of raised intracranial pressure were observed ( fig . 2 ) .
the ct angiography allowed the diagnosis of an occlusion of the ica by a thrombus localised at the level of the bifurcation of the common carotid artery ( cca ) and at the level of c1 , adjacent to the foreign body .
the laboratory findings were as follows : d - dimer 20921.25 g / l ( n < 500 ) ; pt and aptt within normal limits .
once the diagnostic procedures were finished , a surgical intervention was performed on the day of admission .
the preparation of the internal jugular vein revealed the medial translocation and compression of the carotid vessels . the foreign body was removed .
post - interventional ct revealed the thrombotic occlusion of the left ica and mca and a hypodense area supplied by the mca and the concomitant left hemisphere oedema of the brain .
the patient died on the 6th post - operative day exhibiting symptoms of intracranial hypertension and a brain oedema.fig .
2case 1 : localisation of the foreign body ( ct ) case 1 : wooden splinter removed during the surgical procedure case 1 : localisation of the foreign body ( ct )
the findings of the medico - legal autopsy were as follows : a thrombus occluding the left ica and the proximal segment of the left mca , a large infarction area in the temporal lobe and adjacent parts of the frontal and occipital lobes of the left hemisphere of the brain , a large brain oedema with features of subfalcine herniation and transtentorian herniation , a comminuted fracture of the left maxilla and an occipital bone fracture in the region of the occipital condyle with the fragment impression .
the histopathological examination revealed the occlusion of the left ica by the thrombus ( h + e ( fig .
3 ) , masson , gomori , verhoeff and ptah ) , focal intimal haemorrhages and features of a mechanical injury the rupture of the artery wall in the form of an irregular fissure in the tunica media ( fig . 4 ) .
the samples of the macroscopically changed left cerebral hemisphere revealed ischemic necrosis with polymorphonuclear leukocytes reaction , oedema and hyperaemia , while secondary , focal , non - reactive perivascular haemorrhages were found in the brainstem .
other organs showed no significant histopathological lesions , apart from morphological indices of circulatory disturbances.fig .
3case 1 : the internal carotid artery wall with the adjacent thrombus ( h + e)fig .
4case 1 : damaged tunica media , damaged and dissected intima , as well as fragments of the thrombus within the internal carotid artery ( a h + e and b verhoeff ) case 1 : the internal carotid artery wall with the adjacent thrombus ( h + e ) case 1 : damaged tunica media , damaged and dissected intima , as well as fragments of the thrombus within the internal carotid artery ( a h + e and b verhoeff )
a 32-year old female patient was brought to the hospital after being battered what resulted in police intervention . on admission
the patient was conscious , complaining about a left upper extremity contracture that started 2 weeks prior to admission .
she was generally healthy , with no chronic diseases , taking no medication , apart from being a smoker .
for the previous month she had been repeatedly battered by her partner ( about twice a week ) with a fist and open hand to the face , neck and back , as well as pulled by the hair .
the last battery that took place 1 day prior to admission resulted in a short - term loss of consciousness . on admission
the following body injuries were described in her medical record : resorbing bruises on the face , including a wound of the lower lip mucosa , and the upper extremities , . on admission
the patient was sleepy with left - sided hemiparesis with symptoms concerning mainly the upper extremity , blood pressure was 110/80 mmhg , a fundus examination revealed no pathological lesions .
d - dimer 1 200 ng / ml ( n < 500 ) , aptt slightly shortened 25.69 s ( n : 2836 ) , aptt ratio and pt within normal limits .
ct revealed the presence of a hypodense area in the right hemisphere region supplied by the mca , whereas mri scan confirmed the presence of an ischemic brain damage .
thrombotic occlusion of the ica and mca was visible in mri ( fig . 5 ) and angioct ( fig .
, the patient died on the 12th day after admission presenting symptoms of raised intracranial pressure with a concomitant brainstem injury secondary to a brain oedema.fig .
5case 2 : occlusion of the right internal carotid and middle cerebral artery marked with an arrow ( mri)fig .
6case 2 : occlusion of the right internal carotid artery marked with an arrow ( angioct ) case 2 : occlusion of the right internal carotid and middle cerebral artery marked with an arrow ( mri ) case 2 : occlusion of the right internal carotid artery marked with an arrow ( angioct ) the findings of the medico - legal autopsy were as follows : a thrombus occluding the right ica and the mca , thrombosis of the internal jugular veins , as well as the superior sagittal sinus and transverse sinus , an extensive infarction area in the right hemisphere region supplied by the mca ; a brain oedema with features of right - sided subfalcine herniation and transtentorian herniation , carotid and basal ca with no atherosclerotic lesions .
a histopathological examination revealed an occlusion of the right ica by the thrombus ( h + e , masson , gomori , verhoeff and ptah)a muscular type artery with normal wall structure , the lumen filled with thrombus fragments , mechanical injury of the arterial wall
rupture of the intima and tunica media ( fig . 7 ) . internal jugular veins normal wall structure , occluded by thrombi .
there were dominating circulatory disturbances in the brain in the form of very significant capillary - venous hyperaemia resulting from an impaired cerebral venous return secondary to jugular vein and dural sinuses thrombosis.fig .
7case 2 : damaged tunica media and intima of the internal carotid artery with intact adventitia ( a h + e and b verhoeff ) case 2 : damaged tunica media and intima of the internal carotid artery with intact adventitia ( a h + e and b verhoeff )
observed in both cases , focal ischemic brain injury was secondary to the thrombosis of the ica and mca .
cerebral vascular lesions may be causally associated with the sustained head and/or neck trauma with concomitant ica thrombosis and ischemic brain damage [ 5 , 713 ] .
they can , however , accompany head and/or neck trauma , yet be independent of trauma and have other morbid origins [ 2 , 4 ] . in both cases we considered the most common , known , possible non - traumatic causes for ica thrombosis , including atherosclerosis , which is the most common cause for ica and ca thrombosis .
the endothelium and the connective tissue cap injury expose the blood coagulation activating factors , which induce thrombus formation .
the endothelium injury itself with exposure of the basal membrane may predispose to thrombus formation as well .
the presence of stable atherosclerotic plaque may be complicated by arterial thrombosis , and this refers to cerebral vessels as well .
however , vessel stenosis has to be significant , i.e. the blood flow transforms from being laminar into post - stenotic turbulent [ 1417 ] both the in vivo neuroradiologic diagnostic imaging modalities , including ct angiography , and the postmortem examination with a histopathological assessment did not reveal the atherosclerosis of ica or mca in any of the discussed cases . other factors favouring carotid arteries thrombosis include : hypertension , diabetes , hypercholesterolaemia , obesity and smoking [ 2 , 4 , 18 ] .
such factors accelerate the course of atherosclerosis , of which a common complication is thrombosis .
the analysis of both patients medical records , including the laboratory findings obtained during hospitalisation allowed diabetes and lipid metabolism disorders to be ruled out .
the woman did not manifest features of hypertension , whereas unstable aterial pressure values , i.e. labile hypertension , was observed in the male patient .
no lesions in the small - sized cerebral vessels that could have been responsible for the ischemic stroke were found during the postmortem examination , including the neuropathologic examination , within the ischemic , as well as in the non - ischemic areas .
no arteritis , amyloid angiopathy , vascular malformations nor lesions observed in the course of hypertensive encephalopathy were found .
the presence of a thrombus inside the ica and/or ca , and secondary focal ischemic brain damage may result from embolism [ 2 , 4 , 18 , 19 ] .
the most common source of such embolic material are thrombi localised in the left side of the heart ( auricle of the left atrium in patients with atrial fibrillation ) , parietal thrombi in the left ventricle whose formation is associated with myocardial infarction or more frequently aneurysm of the heart in the area of an extensive post - infarction scar or left ventricle dilatation , e.g. in the course of dilated cardiomyopathy or endocarditis , especially of bacterial origin
the embolism in the ica and ca may then develop in patients with atrial septal defect and deep vein thrombosis .
the postmortem examination did not , however , reveal any of these conditions in the discussed patients .
a significantly less common cause of stroke are haematological disorders , which are responsible for about 1 % of all strokes and occur slightly more frequently in young subjects .
the laboratory findings performed during hospitalisation and autopsies allowed haematological disorders to be ruled out as factors predisposing to thrombosis .
the elevation of d - dimer level was the sole abnormal parameter observed on the coagulation profile and was associated with present ica and mca thrombosis in both cases .
oral contraceptives ( oc ) should be considered as a possible cause for thrombosis in the female patient .
oc are described in neurology as one of the ethological factors of ica and ca thrombosis . in the presented case the anamnesis collected on the patient s admission to
the hospital indicated that she did not take any medication , most probably including oc .
however , should she have taken oc , they did not significantly influence the development of ica thrombosis .
arterial thrombosis in such cases is very rare in women below 35 years of age [ 21 , 22 ] .
the annual incidence of ischemic stroke in women in this age group is 13 cases/100 000 women .
the following risk factors are considered to increase the risk of developing thrombosis in subjects using oc : hypertension , diabetes , hyperlipidemia and smoking [ 2025 ] .
oc predispose more strongly to the development of venous , rather then arterial thrombosis , which results from the haemodynamic properties of blood flow in these two types of vessels .
other risk factors for venous thrombosis are slowed blood flow and thrombophilia , whereas vessel wall injury ( endothelium ) , including the atherosclerotic plaque are characteristic risk factors for arterial thrombosis . despite doubts concerning her taking
oc , the patient s age ( 32 years old ) and lack of other , above - mentioned risk factors and also atherosclerosis allow oc to be ruled out as a possible cause of ica and mca thrombosis .
the above - mentioned exclusions and the circumstances preceding the disease established in the course of the investigation indicate the traumatic background of the ica thrombosis in both cases .
the ct and ct angiography performed in the first patient revealed the presence of a foreign body adjacent to the left ica on the level of c1 , whereas medial translocation and compression of carotid vessels caused by the foreign body were found during the surgical procedure .
the histopathological examination revealed features of mechanical arterial injury , namely the rupture of the tunica media with focal haemorrhages into the intima and tunica media .
the above - mentioned conditions indicate direct trauma as the cause of thrombosis . apart from the arterial occlusion by the thrombus found in the histopathological sections obtained from the second patient , the mechanical injury to the arterial wall in the form of the intima and tunica media rupture were also noted .
such findings and the information obtained during investigation indicate indirect trauma as the cause of arterial thrombosis .
post - traumatic ica thrombosis may result from a penetrating ( e.g. knife , bullet or other foreign body
splinter , as in case 1 ) or non - penetrating ( blunt ) trauma .
there are two types of the latter , namely direct ones concerning the neck in the region of the carotid artery , and indirect concerning distant regions , e.g. head .
penetrating neck trauma caused by the above - mentioned tools , leads to arterial wall injury in the form of contusion or vessel wall dissection with the formation of intramural haematoma , outer arterial layer damage ( adventitia ) and the formation of pseudoaneurysm or segmental vessel spasm .
suppurative inflammation may also develop in the periarterial space , should the patient survive long enough after the trauma .
the above - mentioned primary vessel wall injuries , associated with an injury limited to at least endothelium and intima , predispose to the development of thrombosis with subsequent vessel occlusion and ischemic stroke .
the ica segment localised a few centimetres over the cca bifurcation is the vessel segment most commonly injured in the course of non - penetrating ( blunt ) ica traumas , both direct and indirect .
they result in the rupture of the intima and/or tunica media , with the possible formation of intramural haematoma , i.e. a condition called dissecting aneurysm .
the thrombotic occlusion of the vessel lumen occurs a few hours or even days or weeks after the trauma [ 5 , 27 , 28 ] .
six to ten percent of patients manifest such symptoms within the first hour after trauma , another 50 % after 10 h and in 5773 % of cases the asymptomatic period lasts more than 24 h. only 1735 % of such patients develop ischemic symptoms after such a period .
the above - mentioned facts indicate that in some cases of traumatic ica thrombosis there is a variably long asymptomatic period between the trauma and the manifestation of neurological symptoms of ischemic brain damage .
some authors compare it to intervallum lucidum occurring in patients with epi- and/or subdural haematoma .
it results from thrombus propagation within the damaged vessel and may hamper the process of establishing the causal connection between the trauma and the ica thrombosis , especially , as according to some authors , the process itself may last up to several years [ 8 , 9 , 27 ] , which in our opinion is hardly probable . according to unterharnscheidt traumatic ica thrombosis may result from : ( 1 ) direct neck trauma ( e.g. boxers ) ; ( 2 ) direct head trauma ( including insignificant ) with force transfer to the neck region ; ( 3 ) oral cavity trauma ; ( 4 ) fractures of the neuro- and splanchnocranium bones ; ( 5 ) whiplash injuries ; ( 6 ) strangling or other manual procedures in the carotid artery region ( e.g. massage ) and ( 7 ) compression by seatbelt in the course of road traffic accidents .
non - penetrating ica injuries result from blunt head and/or neck trauma . a head trauma with a violent and powerful head rotation and hyperextension of the cervical spine results in some topographic alterations , including the ica course .
it may lead to ica compression by the transverse processes of c3 or c1c2 vertebral bodies and predisposes to thrombosis due to the secondary rupture of intima and tunica media by an intact adventitia .
ica thrombosis may result from neck trauma , as well . by certain head positions , in which the artery is covered only with skin and fascia ,
it would be pressed against the c1 and c2 or transverse processes of c3c5 with secondary damage to intima , and even tunica media . in both of the discussed cases the thrombosis could have been caused by a segmental artery spasm on the injury site , which is impossible to establish during a postmortem examination . in 1974 crissey and bernstein identified four basic mechanisms of ica injuries , and thus four types of damage .
type i damage results from a direct blow to the neck and most commonly concerns older patients , frequently having advanced ica atherosclerosis .
a similar mechanism occurs by a sudden powerful hyperflexion of the cervical spine with a compression of the ica between the mandible and spine .
type ii ica damage is caused by the hyperextension of the cervical spine with a head and neck rotation in the opposite direction .
such a mechanism results in injury to the ica segment that is overstretched over the lateral masses of the first two cervical vertebrae .
the discussed mechanisms of ica injuries , apart from a direct blow to its region , are most commonly observed in motorcyclists participating in road traffic accidents .
type iii ica damage is most commonly observed in children and accompanies the intraoral trauma caused by a foreign body e.g. by falling on a hand - held or more frequently mouth - held object , e.g. pencil .
the establishment of the causal connection between neck and/or head trauma and ica thrombosis and its cerebral consequences requires the exclusion of non - traumatic factors in the first line disease - associated , being the most common cause of thrombosis .
this is very important as such trauma is most frequently a result of a criminal offence and brings the perpetrator to criminal and civil liability .
the establishment of a causal connection may be problematic or even impossible in victims who apart from traumatic injuries suffer from diseases or factors predisposing to thrombosis .
|
the following manuscript presents two cases of ischemic stroke secondary to traumatic internal carotid artery thrombosis with concomitant middle cerebral artery thrombosis occurring very rarely in the medico - legal practice . penetrating neck trauma due to an occupational accident and multiple head and neck trauma secondary to battery
were described .
the autopsy and histopathological examination as well as the analysis of available medical records , including radiological examinations , and records of investigation indicated the sustained trauma to be the cause of the thrombosis .
|
Introduction
Case 1
Case 2
Discussion
Conclusions
|
alzheimer disease ( ad ) is a rapidly expanding health crisis affecting over 26 million people , with the prevalence expected to rise dramatically ( brookmeyer et al . , 2007 ) .
research examining ad biomarkers suggests a rise in underlying pathology a decade or more before the onset of dementia , and continuing throughout the early stages of the disease ( bateman et al . , 2012 ; benzinger et al . , 2013 ; jack et al . ,
there is need to translate such biomarkers from a laboratory setting into a clinical environment to assist with disease diagnosis and prognosis .
the hallmarks of ad pathology are the formation of amyloid beta ( a ) plaques and the aggregation of tau into neurofibrillary tangles ( nfts ) ( braak and braak , 1995 ; hardy and higgins , 1992 ) .
early elevations in such pathology are subsequently followed by hypometabolism , structural atrophy , and cognitive impairment ( bateman et al . , 2012 ; benzinger et al . , 2013 ; jack et al . , 2010 ) .
atrophy of cortical and subcortical gray matter has long been noted in ad ( e.g. fox et al . , 1996 ; gordon et al . , 2013 ; scheltens et al . , 1992 ) .
less attention has been paid to white matter damage and declines tied to ad disease progression .
early work with computed tomography ( ct ) images noted increased incidence of white matter leukoaraiosis in individuals with ad ( blennow et al . , 1991 ; rezek et al . ,
similar results were found with the introduction of magnetic resonance imaging ( mri ) ( barber et al . , 1999 ; fazekas et al . , 1987 ) .
in this initial work the most common way to characterize white matter damage was to use semi - quantitative scales ( fazekas et al .
, 1987 ; kapeller et al . , 2003 ; scheltens et al . , 1995 )
to grade the severity of white matter hyperintensities ( wmhs ) on t2-weighted or fluid - attenuated inversion recovery ( flair ) scans . at a pathological level ,
higher ratings on these scales are associated with both cognitive decline ( debette et al .
. , 2008 ) and cortical atrophy ( capizzano , 2004 ; schmidt et al . , 2005 ) .
in general there is a rising interest on the clinical importance of wmh across diseases ( debette and markus , 2010 ) .
the relationship between wmh and amyloid is complex and has not been fully evaluated , although there are suggestions that both contribute to cognitive impairment ( provenzano et al . , 2013 ) .
white matter damage may be both a downstream result of elevated a levels , as well as a marker of comorbid pathology ( e.g. cardiovascular disease ) .
a leads to oxidative damage and the formation of free radicals ( hensley et al .
1996 ) , and the administration of a damages oligodendrocytes in vitro ( roth et al . , 2005 ) and in vivo ( jantaratnotai et al . , 2003 ) .
conversely damage to myelin releases iron molecules that promote a oligomerization ( bartzokis et al .
an initial rise in a would damage white matter , which in turn would elevate a levels , subsequently leading to more white matter damage in a continuing cyclical process .
alternatively , white matter lesions from a secondary process ( e.g. head injury ) may release iron , and initiate or accelerate the pathological influences of a on white matter .
such results can been seen in the literature as circulating levels of a are associated with wmh ( gurol et al . , 2006 ) , and baseline levels of white matter lesions predict an accelerated accumulation of amyloid over time ( grimmer et al . , 2012 ) .
using semi - quantitative scales , white matter lesions have often been seen in individuals with compromised cardiovascular systems ( breteler et al . , 1994 ;
consistent with these results , there has been a suggestion that ad may have a larger vascular component than often recognized ( bartzokis , 2011 ; de la torre , 2010 ; launer , 2002 ) . indeed , in epidemiological studies , cardiovascular risk factors such as diabetes or stroke lead to increased risk of ad ( luchsinger et al . ,
damage to the cardiovascular system , such as a thickening and sclerosis of arteries , may lead to an impaired drainage of molecules such as a ( huang et al .
, it is of interest to know whether the incidence of wmhs provides any diagnostic value above and beyond levels of a pathology in the brain .
white matter damage in the brain can be assessed using visual ratings of wmh , quantification of wmh volumes , and using diffusion tensor imaging ( dti ) .
while there is a clear utility to quantifying damage using dti and wmh volumetric measurement , visual rating scales are an easily obtained radiological measure available across both research and clinical settings . here
we examine the relationships between a deposition , white matter damage , and dementia in a population of cognitively normal , very mildly demented , and mildly demented individuals .
based upon prior work in the literature , we hypothesize that more severe semi - quantitative ratings of white matter damage will be related to an impaired cognitive status .
middle aged and older adults were drawn from studies on aging and dementia conducted through the knight alzheimer 's disease research center ( adrc ) at washington university in st .
louis . based upon the clinical dementia rating ( cdr ) scale ( morris , 1993 ) participants were classified as cognitively normal ( cdr = 0 , n = 397 , female = 256 ) , very mildly demented ( cdr = 0.5 , n = 51 , female = 20 ) , or mildly demented ( cdr = 1 , n = 11 , female = 1 ) . individuals with dementia
using these diagnoses individuals whose dementia was thought to be from a non - alzheimer cause ( e.g. lewy bodies , vascular dementia , depression ) were excluded from all analyses .
the population ranged in age from 42 to 90 , with a mean age of 68.5 years ( table 1 ) .
all participants underwent a structural imaging session as well as positron emission tomography ( pet ) to estimate amyloid deposition using [ c ] pittsburgh compound b ( pib ) ( klunk et al . , 2004 ) .
all procedures where approved by washington university 's institutional review board and were conducted in accordance with the declaration of helsinki .
high - resolution t2-weighted images were acquired on a siemens trio 3 t scanner ( siemens medical systems , iselin , nj ) .
scan parameters were : repetition time ( tr ) of 3200 ms , echo time ( te ) of 455 , flip angle ( fa ) = 120 , with a 256 256 field of view , and a 1 mm isotropic resolution . a trained neurologist ( s.n . ) , blind to clinical diagnosis , examined the t2-weighted images .
the presence and severities of wmh were rated using criteria outlined by fazekas et al .
briefly , periventricular hyperintensities ( pvwmhs ) were rated as follows : 0 absence of wmh ; 1 caps or pencil - thin linings ; 2 halos ; and 3 irregular pvh extending into deep white matter . ratings of wmh in the deep white matter ( dwmh ) were rated as follows : 0 absence of wmh , 1 solitary foci ; 2 the beginning aggregation of foci ; and 3 large confluent areas of wmh .
examples are given in fig . 1 and distributions of scores across the three clinical groups
binding potentials were calculated for multiple regions of interest ( rois ) derived from freesurfer using a cerebellar reference for regions - of - interest . the raw time
activity curve for each region was adjusted by a csf dilution factor in a given voxel to yield partial volume corrected data .
an average across both left and right lateral orbitofrontal , interior parietal , precuneus , rostral middle frontal , superior frontal , superior temporal , and middle temporal rois yielded the mean cortical binding potential ( mcbp ) .
all analyses used mcbp as a continuous variable . as supplementary analyses , individuals were also codified as pib positive or negative using a previously published value from our center of unadjusted mcbp of 0.18 ( vlassenko et al . , 2011 ) .
a second supplementary analysis defined the cutoff on partial - volume corrected mcbp data derived from a roc analysis comparing 212 cognitively normal individuals to 59 cdr = 0.5 with an ad diagnosis . using this approach the partial - volume adjusted mcbp cutoff was determined to be .23 , which was the point that maximized the youden index ( sensitivity + specificity 1 ) .
, individuals were also codified as pib positive or negative using a previously published value from our center of unadjusted mcbp of 0.18 ( vlassenko et al . , 2011 ) .
a second supplementary analysis defined the cutoff on partial - volume corrected mcbp data derived from a roc analysis comparing 212 cognitively normal individuals to 59 cdr = 0.5 with an ad diagnosis . using this approach the partial - volume adjusted mcbp cutoff was determined to be .23 , which was the point that maximized the youden index ( sensitivity + specificity 1 ) .
1 . we assessed the presence ( 1 ) or absence ( 0 ) of a history of hypertension , heart attack , atrial fibrillation , angioplasty , bypass surgery , congestive heart failure , stroke , transient ischemic attack , and diabetes .
height information and weight information were used to calculate bmi , and coded for the presence or absence of obesity ( bmi > 30 ) .
an aggregate vascular risk factor was created by summing the scores of all variables ( possible range of 010 ) ( actual range 06 , mean 1.14 , median 0 ) . across the entire sample
there was a modest bivariate correlation between a history of hypertension and pvwmh ( r = .21 , p <
.0001 ) and dwmh ( r = .19 , p < .0001 ) scores .
similarly the summary vascular risk score significantly correlated with both pvwmh ( r = .17 , p < .001 ) and dwmh ( r = .16 , p < .001 ) .
unsurprisingly , a history of hypertension and the vascular risk were highly correlated ( r = .65 , p <
initial stepwise logistic regressions examined the effects of age , gender , mcbp and a measure of white matter damage ( either dwmh or pvwmh ) .
models additionally allowed for an interaction between white matter damage and mcbp to enter the model .
models tested whether each predictor discriminated between cognitively normal individuals and demented individuals ( combined cdr = 0.5 and 1 ) . for the gender variable women were coded as 0 and males as 1 .
for each predictor the exponentiation of the beta coefficient ( exp(b ) ) indicates the odds ratio , or the change in relative log odds of being in the tested group ( e.g. cdr > 0 ) relative to the reference group ( e.g. cdr = 0 ) with a one unit change in the predictor .
if the odds ratio for a given predictor ( e.g. age ) is > 1 , then an increase in the predictor indicates a greater likelihood to be in the test group ( e.g. cdr > 0 ) . if the odds ratio is < 1 , then the outcome is more likely to be in the reference group ( e.g. cdr = 0 ) . for all analyses
an increase in the exp(b ) indicated greater cognitive impairment . an additional way to examine the data
was presented by calculating the area under the curve ( auc ) values from receiver operating characteristics ( roc ) curves separating cognitively normal individuals from demented individuals .
the first model looked at the auc only using the initial covariates of age and gender .
a second set of models examined the auc using the covariates and then one predictor of interest ( i.e. mcbp , pvwmh , or dwmh ) .
a final set of models examined the auc when using covariates , mcbp , and either of the wmh measures .
significant changes in the auc were assessed using delong 's test for correlated roc curves ( delong et al . , 1988 ) using a package implemented in r ( robin et al . , 2011 ) .
these analyses test whether each predictor of interest ( e.g. wmh , mcbp , age ) significantly differs between each level of the dependent variable ( cdr 0 , 0.5 , and 1 ) .
the structure of these models was set to be identical to that revealed by the stepwise logistic regression comparing cognitively normal to all demented individuals .
middle aged and older adults were drawn from studies on aging and dementia conducted through the knight alzheimer 's disease research center ( adrc ) at washington university in st .
louis . based upon the clinical dementia rating ( cdr ) scale ( morris , 1993 ) participants were classified as cognitively normal ( cdr = 0 , n = 397 , female = 256 ) , very mildly demented ( cdr = 0.5 , n = 51 , female = 20 ) , or mildly demented ( cdr = 1 , n = 11 , female = 1 ) . individuals with dementia
using these diagnoses individuals whose dementia was thought to be from a non - alzheimer cause ( e.g. lewy bodies , vascular dementia , depression ) were excluded from all analyses .
the population ranged in age from 42 to 90 , with a mean age of 68.5 years ( table 1 ) .
all participants underwent a structural imaging session as well as positron emission tomography ( pet ) to estimate amyloid deposition using [ c ] pittsburgh compound b ( pib ) ( klunk et al . , 2004 ) .
all procedures where approved by washington university 's institutional review board and were conducted in accordance with the declaration of helsinki .
high - resolution t2-weighted images were acquired on a siemens trio 3 t scanner ( siemens medical systems , iselin , nj ) .
scan parameters were : repetition time ( tr ) of 3200 ms , echo time ( te ) of 455 , flip angle ( fa ) = 120 , with a 256 256 field of view , and a 1 mm isotropic resolution .
a trained neurologist ( s.n . ) , blind to clinical diagnosis , examined the t2-weighted images .
the presence and severities of wmh were rated using criteria outlined by fazekas et al .
briefly , periventricular hyperintensities ( pvwmhs ) were rated as follows : 0 absence of wmh ; 1 caps or pencil - thin linings ; 2 halos ; and 3 irregular pvh extending into deep white matter . ratings of wmh in the deep white matter ( dwmh ) were rated as follows : 0 absence of wmh , 1 solitary foci ; 2 the beginning aggregation of foci ; and 3 large confluent areas of wmh .
examples are given in fig . 1 and distributions of scores across the three clinical groups
the intraclass correlation was .91 for periventricular ratings and .98 for deep white matter ratings .
binding potentials were calculated for multiple regions of interest ( rois ) derived from freesurfer using a cerebellar reference for regions - of - interest . the raw time
activity curve for each region was adjusted by a csf dilution factor in a given voxel to yield partial volume corrected data .
an average across both left and right lateral orbitofrontal , interior parietal , precuneus , rostral middle frontal , superior frontal , superior temporal , and middle temporal rois yielded the mean cortical binding potential ( mcbp ) . all analyses used mcbp as a continuous variable . as supplementary analyses ,
individuals were also codified as pib positive or negative using a previously published value from our center of unadjusted mcbp of 0.18 ( vlassenko et al . , 2011 ) .
a second supplementary analysis defined the cutoff on partial - volume corrected mcbp data derived from a roc analysis comparing 212 cognitively normal individuals to 59 cdr = 0.5 with an ad diagnosis . using this approach the partial - volume adjusted mcbp cutoff was determined to be .23 , which was the point that maximized the youden index ( sensitivity + specificity 1 ) .
, individuals were also codified as pib positive or negative using a previously published value from our center of unadjusted mcbp of 0.18 ( vlassenko et al . , 2011 ) .
a second supplementary analysis defined the cutoff on partial - volume corrected mcbp data derived from a roc analysis comparing 212 cognitively normal individuals to 59 cdr = 0.5 with an ad diagnosis . using this approach
the partial - volume adjusted mcbp cutoff was determined to be .23 , which was the point that maximized the youden index ( sensitivity + specificity 1 ) .
we assessed the presence ( 1 ) or absence ( 0 ) of a history of hypertension , heart attack , atrial fibrillation , angioplasty , bypass surgery , congestive heart failure , stroke , transient ischemic attack , and diabetes .
height information and weight information were used to calculate bmi , and coded for the presence or absence of obesity ( bmi > 30 ) .
an aggregate vascular risk factor was created by summing the scores of all variables ( possible range of 010 ) ( actual range 06 , mean 1.14 , median 0 ) . across the entire sample
there was a modest bivariate correlation between a history of hypertension and pvwmh ( r = .21 , p < .0001 ) and dwmh ( r = .19 , p < .0001 ) scores .
similarly the summary vascular risk score significantly correlated with both pvwmh ( r = .17 , p < .001 ) and dwmh ( r = .16 , p < .001 ) .
unsurprisingly , a history of hypertension and the vascular risk were highly correlated ( r = .65 , p < .00001 ) .
initial stepwise logistic regressions examined the effects of age , gender , mcbp and a measure of white matter damage ( either dwmh or pvwmh ) . models additionally allowed for an interaction between white matter damage and mcbp to enter the model . models tested whether each predictor discriminated between cognitively normal individuals and demented individuals ( combined cdr = 0.5 and 1 ) . for the gender variable women were coded as 0 and males as 1 . for each predictor the exponentiation of the beta coefficient ( exp(b ) ) indicates the odds ratio , or the change in relative log odds of being in the tested group ( e.g. cdr > 0 ) relative to the reference group ( e.g. cdr = 0 ) with a one unit change in the predictor .
if the odds ratio for a given predictor ( e.g. age ) is > 1 , then an increase in the predictor indicates a greater likelihood to be in the test group ( e.g. cdr > 0 ) . if the odds ratio is < 1 , then the outcome is more likely to be in the reference group ( e.g. cdr = 0 ) .
for all analyses an increase in the exp(b ) indicated greater cognitive impairment . an additional way to examine the data
was presented by calculating the area under the curve ( auc ) values from receiver operating characteristics ( roc ) curves separating cognitively normal individuals from demented individuals .
the first model looked at the auc only using the initial covariates of age and gender .
a second set of models examined the auc using the covariates and then one predictor of interest ( i.e. mcbp , pvwmh , or dwmh ) .
a final set of models examined the auc when using covariates , mcbp , and either of the wmh measures .
significant changes in the auc were assessed using delong 's test for correlated roc curves ( delong et al . , 1988 ) using a package implemented in r ( robin et al . , 2011 ) .
these analyses test whether each predictor of interest ( e.g. wmh , mcbp , age ) significantly differs between each level of the dependent variable ( cdr 0 , 0.5 , and 1 ) .
the structure of these models was set to be identical to that revealed by the stepwise logistic regression comparing cognitively normal to all demented individuals .
there were more males in the cdr 0.5 ( = 12.2 , p < .0005 ) and cdr 1 ( = 14.1 , p < .0005 ) groups relative to cdr 0 .
compared to cdr 0 individuals , cdr 0.5 individuals were older ( t = 7.0 , p < .000001 ) , had lower mmse ( 13.2 , p < .000001 ) , greater mcbp ( t = 8.3 , p <
.00001 ) , and a greater proportion of pib+ individuals ( = 45.2 , p < .000001 ) . similarly relative to cdr 0
individuals those with a cdr = 1 were older ( t = 3.81 , p < .001 ) , had lower mmse ( 18.9 , p <
.00001 ) , and a greater proportion of pib+ individuals ( = 22.7 , p < .000005 ) .
cdr 1 individuals had lower mmse than cdr 0.5 individuals but did not significantly differ in any other way .
as age and gender differed across groups , they were considered as covariates when examining the effects of wmh and amyloid on cognition .
the full results from the initial logistic regressions are presented in table 2 . for all significant effects ,
an increase in the odds ratio for the measure of interest ( e.g. age ) indicated a greater likelihood of being cognitively impaired .
when examining mcbp alongside pvwmh there was a significant effect of age ( exp(b ) 1.08 , p < 0.001 ) , gender ( exp(b ) 3.50 , p < 0.001 ) , mcbp ( exp(b ) 3.68 , p < 0.0001 ) , and pvwhm ( exp(b ) 2.03 , p < 0.01 ) . when examining mcbp alongside dwmh there was a significant effect of age ( exp(b ) 1.10 , p < 0.001 ) , gender ( exp(b ) 3.60 , p < 0.001 ) , mcbp ( exp(b ) 3.63 , p < 0.0001 ) , and dwmh ( exp(b ) 1.52 , p < 0.05 ) .
the interaction terms between mcbp and wmh were non - significant and not entered into either model .
2 , which depicts the distribution of the fazekas scores for periventricular white matter hyperintensities ( pvwmhs ) and deep white matter hyperintensities ( dwmhs ) across the three groups . treating amyloid deposition as a dichotomous pib+/ variable did not substantively change the models ( supplementary tables 1 and 2 ) .
it is possible to gain a bit more understanding of the size of these effects by calculating the roc auc . when predicting dementia status solely from the covariates ( age and gender ) the auc is .831 . when adding mcbp to the model the auc significantly increased to .875 ( z = 2.41 ,
there were non - significant increases in the auc when adding only the pvwmh ( auc.849 , z = 1.34 , p = .18 ) or dwmh ( auc = .836 , z = .5242 , p = .60 ) to the covariate only model . including both mcbp and pvwmh led to an auc of .886 ( z = 2.63 , p < .01 ) and auc of .876 when using mcbp dwmh ( z = 2.26 , p = <
while significant relative to the covariate only models , the increase in auc in the models combining mcbp and wmh was not significantly different from models considering mcbp alone . combined with the results from the logistic regressions , this suggests only modest effects of wmh above amyloid deposition .
the full results from the multinomial logistic regressions are presented in tables 3 and 4 .
these results are quite similar to the logistic regressions above when comparing cognitively normal individuals to either group of demented individuals . when contrasting those with very mild dementia ( cdr 0.5 ) to those with mild dementia ( cdr 1 ) there were no significant effects , although gender , mcbp , and dwmh demonstrated modest trends .
as wmh are thought to be vascular in nature ( breteler et al . , 1994 ) , we additionally examined the initial models predicting dementia status with mcbp and measures of wmh while including a history of hypertension , or an aggregate vascular risk factor in the model .
a history of hypertension was not significant in either model ( pvwmh or dwmh , see table 5 ) and only slightly lowered the contributions of wmh to the model , but such effects were still significant .
the aggregate vascular risk factor was significant in both models , but again the effects on the wmh scores were minor ( table 6 ) .
there were more males in the cdr 0.5 ( = 12.2 , p < .0005 ) and cdr 1 ( = 14.1 , p < .0005 ) groups relative to cdr 0 .
compared to cdr 0 individuals , cdr 0.5 individuals were older ( t = 7.0 , p < .000001 ) , had lower mmse ( 13.2 , p < .000001 ) , greater mcbp ( t = 8.3 , p <
.00001 ) , and a greater proportion of pib+ individuals ( = 45.2 , p < .000001 ) . similarly relative to cdr 0
individuals those with a cdr = 1 were older ( t = 3.81 , p < .001 ) , had lower mmse ( 18.9 , p <
.00001 ) , and a greater proportion of pib+ individuals ( = 22.7 , p < .000005 ) .
cdr 1 individuals had lower mmse than cdr 0.5 individuals but did not significantly differ in any other way .
as age and gender differed across groups , they were considered as covariates when examining the effects of wmh and amyloid on cognition .
the full results from the initial logistic regressions are presented in table 2 . for all significant effects ,
an increase in the odds ratio for the measure of interest ( e.g. age ) indicated a greater likelihood of being cognitively impaired .
when examining mcbp alongside pvwmh there was a significant effect of age ( exp(b ) 1.08 , p < 0.001 ) , gender ( exp(b ) 3.50 , p < 0.001 ) , mcbp ( exp(b ) 3.68 , p < 0.0001 ) , and pvwhm ( exp(b ) 2.03 , p < 0.01 ) .
when examining mcbp alongside dwmh there was a significant effect of age ( exp(b ) 1.10 , p < 0.001 ) , gender ( exp(b ) 3.60 , p < 0.001 ) , mcbp ( exp(b ) 3.63 , p < 0.0001 ) , and dwmh ( exp(b ) 1.52 , p < 0.05 ) .
the interaction terms between mcbp and wmh were non - significant and not entered into either model .
2 , which depicts the distribution of the fazekas scores for periventricular white matter hyperintensities ( pvwmhs ) and deep white matter hyperintensities ( dwmhs ) across the three groups . treating amyloid deposition as a dichotomous pib+/ variable did not substantively change the models ( supplementary tables 1 and 2 ) .
it is possible to gain a bit more understanding of the size of these effects by calculating the roc auc . when predicting dementia status solely from the covariates ( age and gender ) the auc is .831 . when adding mcbp to the model the auc significantly increased to .875 ( z = 2.41 , p <
there were non - significant increases in the auc when adding only the pvwmh ( auc.849 , z = 1.34 , p = .18 ) or dwmh ( auc = .836 , z = .5242 , p = .60 ) to the covariate only model . including both mcbp and pvwmh led to an auc of .886 ( z = 2.63 , p < .01 ) and auc of .876 when using mcbp dwmh ( z = 2.26 , p = < .05 ) . while significant relative to the covariate only models , the increase in auc in the models combining mcbp and wmh was not significantly different from models considering mcbp alone
combined with the results from the logistic regressions , this suggests only modest effects of wmh above amyloid deposition .
the full results from the multinomial logistic regressions are presented in tables 3 and 4 .
these results are quite similar to the logistic regressions above when comparing cognitively normal individuals to either group of demented individuals . when contrasting those with very mild dementia ( cdr 0.5 ) to those with mild dementia ( cdr 1 ) there were no significant effects , although gender , mcbp , and dwmh demonstrated modest trends .
as wmh are thought to be vascular in nature ( breteler et al . , 1994 ) , we additionally examined the initial models predicting dementia status with mcbp and measures of wmh while including a history of hypertension , or an aggregate vascular risk factor in the model .
a history of hypertension was not significant in either model ( pvwmh or dwmh , see table 5 ) and only slightly lowered the contributions of wmh to the model , but such effects were still significant .
the aggregate vascular risk factor was significant in both models , but again the effects on the wmh scores were minor ( table 6 ) .
in the current analyses we examined whether measures of amyloid deposition and white matter damage predict cognitive impairment .
we found that both amyloid burden , estimated by pib mcbp , as well as measures of wmh independently discriminate between cognitively normal individuals and those with very mild or mild dementia .
this is unsurprising as the correlation between ratings of dwmh and pvwmh lesions was .58 ( p
the fazekas visual rating of white matter disease was associated with a greater risk of ad .
this can clearly been seen in how the distribution of the fazekas scores shifts across groups in fig . 2 .
individuals who are cognitively normal have highly right skewed distributions , with the vast majority of individuals having no observable white matter damage . as dementia severity increases ,
the scores become more and more left skewed as higher proportions of the population accrue white matter damage . although the graphs in fig .
2 suggest differences between cdr = 0.5 and 1 individuals the effects were not statistically significant .
this is most likely due to the modest sample size of mildly demented subjects ( cdr = 1 , n = 11 ) .
alternatively these markers may possess nonlinear trajectories that do not significantly differ across graded levels of dementia severity . in our cohort
all of the individuals with a cdr = 1 had a primary clinical diagnosis of dementia of the alzheimer 's type at baseline .
of the 51 individuals with a cdr rating of 0.5 , 21 had a dat diagnosis at baseline and 14 received a diagnosis at a later clinical point .
along with the high levels of amyloid deposition , this would suggest that the majority of the impaired cohort are representative of an ad trajectory .
there were no significant differences in age , gender composition , or severity of white matter damage between those with and without an ad diagnosis .
this suggests that the white matter effects are not being driven by a small subsample of individuals who are demented but are not on an ad trajectory .
many healthy older adults show elevated levels of amyloid without significant cognitive impairment ( arriagada et al . , 1992 ) .
additionally there are numerous risk factors for a clinical diagnosis of ad including a family history of ad ( breitner et al . , 1988 ) , the apoe 4 genotype ( corder et al . , 1993 ) ,
head trauma ( mortimer et al . , 1985 ) , and diabetes ( luchsinger et al . , 2001 ) .
the additive effects in our study of whm and pib deposition are consistent with a broader view of ad that suggests that multiple factors can influence substantial cognitive decline and dementia .
for example after head trauma there is a deposition of a in the cortex , and this deposition is increased in individuals with the apoe 4 genotype ( mayeux et al . , 1995 ) .
the results of our analyses are consistent with a large body of prior work linking white matter damage with impaired cognition ( bozzali et al . , 2002 ;
, 2010 ; frisoni et al . , 2007 ; head et al . , 2004 ; prins et al . , 2004 ) .
an increased prevalence of white matter damage is thought to be mainly vascular in nature , as hypertension , stroke , and diabetes all lead to increases in white matter lesions ( debette and markus , 2010 ; pico et al .
there were modest correlations ( rs < .2 ) between measures of vascular health and wmh .
further an aggregate measure of vascular risk was itself predictive of dementia status above the effects of amyloid deposition and wmh .
however measures of wmh were still predictive of dementia status after including vascular risk as a covariate .
this may be due to the fact that our measures of vascular health are imperfect as they are derived from self - report and do not account for medication usage . although the examinations presented here indicated unique statistical effects of age , mcbp , and wmh this does not necessarily mean that the biological processes these values represent are truly orthogonal processes .
one of the greatest challenges facing ongoing studies of ad is how to tease apart the contributions of comorbid pathologies .
large cohort studies are just now reaching the point to investigate modifiers ( e.g. cardiovascular health , stroke , diabetes , head trauma ) of longitudinal ad biomarker trajectories .
individuals suffering from autosomal dominant ad become demented at a much earlier age than those with sporadic ad . due to the young age of onset of clinical symptoms ,
this population may provide greater insight into the relationship between white matter damage and ad removed from age - related comorbidities such as cardiovascular health .
the current work demonstrates a significant relationship between both levels of amyloid deposition and white matter damage on cognition .
the sample contained only a modest number of mildly demented individuals ( cdr = 1 ) , limiting our ability to detect differences with progressive increases in dementia severity .
as commonly done in the literature , our visual ratings were made on t2-weighted sequences , although more precise measures may have been obtained using a more optimal sequence ( e.g. flair ) .
additionally , although visual ratings of wmh such as the fazekas rating scale have a clear utility , they do not provide the fine - grained detail as would be obtained from a quantification of wmh volume . finally , although the fazekas scale separates lesions into periventricular and deep white matter scores ,
three - dimensional renderings of typical lesions suggest that such lesions are part of the same process rather than representing disparate effects .
the work presented here demonstrated the detrimental influences of advancing age , amyloid deposition in the brain , and white matter damage .
these factors independently discriminated healthy controls from very mildly and mildly demented individuals . from a clinical standpoint , this suggests that easily available radiological measures of white matter health could be an asset in disease diagnosis in addition to psychometric testing and ad biomarkers such as amyloid imaging .
1distributions of unadjusted mean cortical binding potentials in cognitively normal ( left ) and demented ( right ) participants .
distributions of unadjusted mean cortical binding potentials in cognitively normal ( left ) and demented ( right ) participants .
|
background and purposeelevated levels of amyloid deposition as well as white matter damage are thought to be risk factors for alzheimer disease ( ad ) .
here we examined whether qualitative ratings of white matter damage predicted cognitive impairment beyond measures of amyloid.materials and methodsthe study examined 397 cognitively normal , 51 very mildly demented , and 11 mildly demented individuals aged 4290 ( mean 68.5 ) .
participants obtained a t2-weighted scan as well as a positron emission tomography scan using 11[c ] pittsburgh compound b. periventricular white matter hyperintensities ( pvwmhs ) and deep white matter hyperintensities ( dwmhs ) were measured on each t2 scan using the fazekas rating scale .
the effects of amyloid deposition and white matter damage were assessed using logistic regressions.resultslevels of amyloid deposition ( ps < 0.01 ) , as well as ratings of pvwmh ( p < 0.01 ) and dwmh ( p < 0.05 ) discriminated between cognitively normal and demented individuals.conclusionsthe amount of amyloid deposition and white matter damage independently predicts cognitive impairment .
this suggests a diagnostic utility of qualitative white matter scales in addition to measuring amyloid levels .
|
Introduction
Materials and methods
Study population
T
Clinical ratings
PiB imaging
Measures of vascular health
Effects of WMH and MCBP on cognition
Results
Demographics
Effects of WMH and MCBP on cognition
Discussion
Conclusions
|
dexmedetomidine , the pharmacologically active d - isomer of medetomidine , is a highly selective and specific 2-adrenoceptor agonist .
dexmedetomidine , by its central sympatholytic action , promotes haemodynamic stability when used as an adjuvant during general anaesthesia .
however , because of its sedative property , it is unknown if the recovery from anaesthesia would be delayed when used as continuous infusion .
the aim of this study was to assess the effect of intravenous dexmedetomidine on perioperative haemodynamics and also postoperative recovery in elective surgical procedures with continuous monitoring of depth of anaesthesia by entropy analysis .
after obtaining the approval from institutional ethical committee and written informed consent from patients , 60 patients with asa status i and ii , aged 1855 years , and undergoing elective surgical procedures were randomly allocated into two groups : group a ( control group ) : sevoflurane fentanyl and group b ( test group ) : sevoflurane dexmedetomidine .
patients older than 55 years , those with a history of psychiatric / neurological illness , hypertensive patients , morbidly obese patients , pregnant and nursing women , patients with known allergic reaction to any of the study medications , patients on recent use of sedatives or analgesics , and patients with significant laboratory abnormalities were excluded . on arrival in the operating room , patient 's heart rate , blood pressure and oxygen saturation were monitored by continuous electrocardiogram ( ecg ) , noninvasive blood pressure ( nibp ) monitor and pulse oximetry ( spo2 ) monitor , respectively .
depth of anaesthesia was evaluated by entropy ( state entropy , response entropy ) analysis ( datex - ohmeda s/5 avance workstation , ge healthcare , helsinki , finland ) .
glycopyrrolate 4 g / kg and ondansetron 0.15 mg/ kg intravenous were given as premedication in both the groups .
dexmedetomidine ( 2 ml diluted in 48 ml of saline ) in a dose of 1 g / kg over 10 min through infusion pump .
ten minutes after the administration of the study drug , anaesthesia was induced with thiopentone sodium 5 mg / kg intravenous slowly .
anaesthesia was maintained with sevoflurane to a maximum of 2.5% end tidal to maintain the heart rate and blood pressure within 20% of the baseline value and entropy value between 40 and 60 .
patients in the test group received dexmedetomidine continuous infusion between 0.2 and 0.8 g/ kg / h .
the anaesthesiologist was permitted to treat haemodynamic events , defined as heart rate and/or blood pressure more than 20% of baseline , in spite of increasing sevoflurane concentration to 2,5% and dexmedetomidine infusion to 0.8 g / kg , with incremental doses of metoprolol 25 mg , or fall in heart rate and blood pressure by more than 20% of baseline with ephedrine 5 mg intravenously . in group b , dexmedetomidine
diclofenac sodium 1 mg / kg ( max dose of 75 mg ) was given intravenously in both groups at the time of skin closure .
immediately after extubation , sedation was assessed with sedation score as follows : 1 , awake ; 2 , sleepy but arousable ; and 3 , sleepy difficulty to awake . in the postoperative period , if visual analogue score ( vas ) was > 6 , inj .
heart rate , blood pressure , spo2 and entropy were recorded continuously at predetermined time intervals as per the protocol .
recovery was assessed by modified aldrete 's and sedation score as follows : score 1 , awake ; score 2 , sleepy but arousable ; score 3 , sleepy difficult to arouse .
statistical analysis was conducted with epi info software ( version 3.5.3 , 2011 ) for windows statistical package using unpaired t - test for continuous variables with normal distribution . the nonparametric kruskal
wallis test was used for variables not normally distributed . for categorical variables , chi - square test and
a sample size of 30 patients per group was needed to detect an intergroup difference of at least 10% in blood pressure and heart rate with a power of 0.80 and of 0.05 .
the two groups were comparable regarding age , sex , weight and asa physical status .
the type of surgery and duration of anaesthesia ( p=0.27 ) were comparable between both groups .
pre - induction heart rate , systolic and diastolic blood pressure ( sbp and dbp , respectively ) were comparable between the two groups ( p>0.05 ) . at intubation
, there was 10% rise in pulse rate from baseline in the test group as compared to 17% rise in the control group ( p=0.01 ) .
intraoperatively , the test group showed significant fall in mean heart rate from baseline as compared to the control group at all time points .
the mean heart rate in the test group showed a 6% fall as compared to 3.7% rise in the control group from baseline .
post extubation , the test group showed significant fall in average pulse rate of 22.5% from baseline as compared to the control group which showed average 1% rise [ figure 1 ] .
changes in heart rate ( meanse ) : 5 and 10 min after the start of dexmedetomidine ; at induction ; at intubation ; at 5 , 10 , 15 , 30 , 45 and 60 min post intubation ; at extubation ; at 5 , 10 , 15 and 30 min post extubation ( da - dexmedetomidine administration ; pi - post intubation ; e - post extubation ) ten minutes after administration of the test drug , significant fall in sbp and dbp of 6% and 9% , respectively , from baseline as compared to the control group was observed ( p<0.05 ) . at intubation , both the groups showed rise in sbp and dbp .
the test group showed a rise of 6% versus 23% in sbp and 7% versus 20% in dbp as compared to the control group ( p<0.05 ) .
intraoperatively , a significant difference in the change of sbp and dbp from baseline was observed between the two groups at all time points till 60 min ( p<0.05 ) .
the test group showed an average of 8% fall in sbp and dbp as compared to 3.6% rise in the control group [ figure 2 ] .
post extubation at 30 min , a 12% fall in sbp was seen in the test group as compared to 0% in the control group ( p<0.05 ) , while change in diastolic pressure was not significantly different between the two groups .
changes in systolic and diastolic blood pressure ( meanse ) : 5 and 10 min after the start of dexmedetomidine ; at induction ; at intubation ; at 5 , 10 , 15 , 30 , 45 and 60 min post intubation ; at extubation ; at 5 , 10 , 15 and 30 min post extubation ( da - dexmedetomidine administration ; pi - post intubation ; e - post extubation ; sbp - systolic blood pressure ; dbp - diastolic blood pressure ) postoperatively , the test group showed significant sedation with a mean score of 1.430.50 at 2 h as compared to 1.00.0 of the control group ( p=0.00 ) . the recovery as assessed by modified aldrete 's score was significantly better in the control group ( 10.000.0 ) as compared to the test group ( 8.060.64 ) at 30 min post extubation ( p=0.00 ) . however , modified aldrete 's score significantly improved in the test group at the end of 2 h and was similar to that of control group .
postoperative analgesia as assessed by vas , showed significant analgesic effect of dexmedetomidine with a mean score of 5.700.60 as compared to 8.130.70 of the control group ( p=0.00 ) [ table 1 ] .
analgesia , sedation and recovery score post extubation bradycardia was observed in two patients in the dexmedetomidine group within 10 min post extubation , which promptly responded to inj .
after operation , none of the patients in either of the groups complained of any discomfort or of intraoperative awareness .
this prospective randomised study demonstrated that intraoperative infusion of dexmedetomidine attenuates stress responses to various noxious stimuli during surgery and maintains haemodynamic stability without the requirement of opioids with prolongation of recovery .
dexmedetomidine , a highly selective 2 agonist , has significant sympatholytic and haemodynamic stability property .
earlier studies have shown that dexmedetomidine attenuates stress response to intubation by decreasing central sympathetic outflow , thereby decreasing serum epinephrine and norepinephrine levels .
these findings are consistent with our study results which showed a significant decrease in change in heart rate , systolic blood pressure and diastolic blood pressure .
tanskanen et al . in their study showed that intraoperative infusion of dexmedetomidine at a rate of 0.4 g / kg / h maintains heart rate and blood pressure in acceptable range for a longer duration as compared to placebo group .
the decrease in heart rate and blood pressure is similar to the findings by feld et al . who compared dexmedetomidine with fentanyl in bariatric surgery .
thus , showing that dexmedetomidine by its sympatholytic activity attenuates various stress responses during surgery and maintains haemodynamic stability .
our study showed a significant attenuation of heart rate at the time of extubation in dexmedetomidine group .
use of haemodynamic end points for assessing the depth of anaesthesia in a study on sympatholytic drugs such as dexmedetomidine would be unreliable as there may be compromise in the depth of anaesthesia .
thus , use of electroencephalogram - dependent indices such as bispectral index and entropy to measure the depth of anaesthesia would be appropriate . in our study ,
adequate depth of anaesthesia was maintained throughout surgery with response and state entropy being maintained between 40 and 60 .
one of the most commonly encountered problems in postoperative anaesthetic care unit ( pacu ) is delayed recovery and longer discharge time of patients receiving perioperative dexmedetomidine infusion.[1315 ] similar findings were observed in our study . however , there was no difference between two groups at the end of 2 h. the delayed recovery might be due to the sedative property of dexmedetomidine .
however , unlike opioids , sedation with dexmedetomidine is not associated with respiratory depression . in our study , dexmedetomidine was discontinued 20 min prior to extubation , thus we could not establish the potential benefit of dexmedetomidine in attenuating haemodynamic response at the time of extubation and during postoperative period . also , recording the time taken to extubate after the completion of surgery would have established objectively the effect of dexmedetomidine on immediate recovery .
dexmedetomidine , when administered as a pre - anaesthetic medication and intraoperative infusion , attenuates stress response to various noxious stimuli and maintains haemodynamic stability . dexmedetomidine 's sedative property delays postoperative recovery , thus continuous monitoring is essential during the first few hours of postoperative period .
|
background : dexmedetomidine , an 2 agonist , when used as an adjuvant in general anaesthesia attenuates stress response to various noxious stimuli , maintains perioperative haemodynamic stability and provides sedation without significant respiratory depression postoperatively.methods:sixty patients were randomly divided into two groups of 30 each . in group a , fentanyl 2 g / kg and in group b dexmedetomidine
were given intravenously as loading dose of 1 g / kg over 10 min prior to induction .
after induction with thiopentone , in group b , dexmedetomidine was given as infusion at a dose of 0.20.8 g / kg .
sevoflurane was used as inhalation agent in both groups .
haemodynamic variables and entropy ( response entropy and state entropy ) were recorded continuously .
postoperative sedation and recovery were assessed by sedation score and modified aldrete 's score , respectively.results:dexmedetomidine significantly attenuates stress response at intubation with lesser increase in heart rate ( 10% vs. 17% ) , systolic blood pressure ( 6% vs. 23% ) and diastolic blood pressure ( 7% vs. 20% ) as compared to the control group ( p<0.05 ) .
intraoperatively , an average of 8% fall in systolic blood pressure and 8.16% fall in diastolic pressure in the test group as compared to 3.6% rise in systolic and 3.3% in diastolic pressure of the control group was observed .
postoperatively , the test group showed significant sedation at 2 h as compared to the control group ( p=0.00 ) and recovery was better in the control group for the first 2 h post extubation.conclusion:dexmedetomidine attenuates various stress responses during surgery and maintains the haemodynamic stability when used as an adjuvant in general anaesthesia . also , the sedative action of dexmedetomidine delays recovery for the first few hours post extubation .
|
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
|
craniosynostosis is a wonderfully descriptive term , meaning " the skull has fused bone " . however , it is also a fairly nonspecific clinical designation , encompassing multiple presentations ranging from isolated single suture involvement to multi - sutural fusions5 ) .
multi - sutural fusion can occur as isolated fusions or with associated anomalies that occur outside the skull .
epidemiologic studies have suggested that the incidence of craniosynostosis may be as high as 1 in 1700 live births612 ) .
for example , early theories regarding the pathogenesis of this condition , based on clinical observations and an experimental animal model , included intrauterine constraint811 ) . over the last quarter century , there have been many advances in the understanding of craniosynostosis , resulting in more rational management of the problems associated with this condition , both in its simple form and its syndromic manifestations315 ) .
these advances have included reduced emphasis on the technical aspects of this disease and greater emphasis on its pathology and natural history , along with more accurate analysis of the morphology of the craniofacial skeleton1310 ) .
more recently , genetic studies have identified the genetic loci of many craniosynostosis syndromes , as well has determining the downstream pathways associated with disease development .
recent clinical and genetic studies have identified multiple forms of human craniosynostosis , each associated with mutations within various growth factor signaling pathways .
knowledge gathered from these investigations may result in the future development of alternative strategies to enhance or perhaps even replace current approaches for the treatment of craniosynostosis .
craniosynostosis was first treated surgically in the late 1800s , using techniques such as fragmentation of the cranial vault and linear craniectomies .
these early procedures were associated with high rates of reossification and poor esthetic outcomes , mandating many subsequent procedures7 ) . at present , however , simple craniectomy is used only in patients with transient cranial decompression .
these early procedures have been supplanted by surgical remodeling of the affected area of the cranial vault and orbits .
surgery is generally performed at age 69 months to take full advantage of the regenerative capacity of the skull at this age .
three stages of growth have determined the approach to the management of craniosynostosis3 ) . during this time , cerebral growth is the greatest , and craniosynostosis may have detrimental effects on the developing brain . the original surgical approach consisted of excision of the premature suture or sutures by linear craniectomy , allowing the growing brain to expand .
if urgent decompression was required , fronto - orbital advancement and multiple suture excisions were performed , resulting in lateral and posterior expansion .
after the second year , cerebral growth slows ; nevertheless , severe craniostenosis may still lead to papilledema and potential visual failure .
the cranial capacity must therefore be expanded by large bilateral decompressive craniotomies , generous fronto - orbital advancement , or a combination of these procedures .
definitive facial surgery can be performed , beginning at age 10 years , although waiting until maturity may achieve better esthetic outcomes .
patients with crouzon syndrome who have proptosis and/or maxillary hypoplasia are especially likely to need surgical treatment1214 ) .
early surgical correction has been limited by a late diagnosis and the risks associated with intraoperative blood loss , which was less effectively managed in the past than currently13 ) . since the beginning of the 21st century
, many centers have tended to determine whether a less invasive procedure could be performed at an earlier stage with acceptable risk .
endoscopic linear craniectomy , with postoperative application of helmets and cranial remodeling through small skin incisions , has led to cosmetic results comparable to those of more invasive procedures if performed during the first 46 months of life , with acceptable blood loss and operative risks13 ) .
more recently , the combination of frontal advancement and posterior cranial enlargement during the first months of life has been found to protect both the ocular and posterior cranial structures .
currently , a free bone flap ( floating technique ) or springs are used to allow for cerebral growth until a rigid fronto - orbito - maxillary advancement can be performed13 ) .
the advent of osteodistraction has lowered the age for facio - maxillary advancement ( 35 years ) , and may avoid the necessity of repeating fronto - orbital procedures .
this has reduced the risks of dural tears and postoperative cerebrospinal fluid fistulas in a significant proportion of patients . as the procedures used to remodel the calvarial vault are extensive
although the mortality rate has been reported to be as high as 2.3% , most international studies had mortality rates of 1.5% to 2%7 ) .
most deaths were attributed to hemorrhagic complications , but various other causes have been reported , including air emboli , cerebral edema , and respiratory infections4 ) .
attention to intraoperative hemodynamics and careful postoperative intensive care unit monitoring are critical in minimizing overall morbidity and mortality rates .
during this time , cerebral growth is the greatest , and craniosynostosis may have detrimental effects on the developing brain . the original surgical approach consisted of excision of the premature suture or sutures by linear craniectomy , allowing the growing brain to expand .
if urgent decompression was required , fronto - orbital advancement and multiple suture excisions were performed , resulting in lateral and posterior expansion .
after the second year , cerebral growth slows ; nevertheless , severe craniostenosis may still lead to papilledema and potential visual failure .
the cranial capacity must therefore be expanded by large bilateral decompressive craniotomies , generous fronto - orbital advancement , or a combination of these procedures .
definitive facial surgery can be performed , beginning at age 10 years , although waiting until maturity may achieve better esthetic outcomes .
patients with crouzon syndrome who have proptosis and/or maxillary hypoplasia are especially likely to need surgical treatment1214 ) .
early surgical correction has been limited by a late diagnosis and the risks associated with intraoperative blood loss , which was less effectively managed in the past than currently13 ) . since the beginning of the 21st century , many centers have tended to determine whether a less invasive procedure could be performed at an earlier stage with acceptable risk .
endoscopic linear craniectomy , with postoperative application of helmets and cranial remodeling through small skin incisions , has led to cosmetic results comparable to those of more invasive procedures if performed during the first 46 months of life , with acceptable blood loss and operative risks13 ) .
more recently , the combination of frontal advancement and posterior cranial enlargement during the first months of life has been found to protect both the ocular and posterior cranial structures .
currently , a free bone flap ( floating technique ) or springs are used to allow for cerebral growth until a rigid fronto - orbito - maxillary advancement can be performed13 ) .
the advent of osteodistraction has lowered the age for facio - maxillary advancement ( 35 years ) , and may avoid the necessity of repeating fronto - orbital procedures .
this has reduced the risks of dural tears and postoperative cerebrospinal fluid fistulas in a significant proportion of patients . as the procedures used to remodel the calvarial vault are extensive
, complications can occur following surgery for craniosynostosis . although the mortality rate has been reported to be as high as 2.3% , most international studies had mortality rates of 1.5% to 2%7 ) .
most deaths were attributed to hemorrhagic complications , but various other causes have been reported , including air emboli , cerebral edema , and respiratory infections4 ) .
attention to intraoperative hemodynamics and careful postoperative intensive care unit monitoring are critical in minimizing overall morbidity and mortality rates .
the combination of early technical success with recent advances in treatment14 ) has indicated the necessity of multidisciplinary management .
the overall approach can be distilled into six principles9 ) : 1 ) care should be provided by multi - disciplinary teams ; 2 ) care should be a protocol - driven process , with all forms of care defined and delivered optimally ; 3 ) care should be longitudinal , as age , healing and growth processes ; 4 ) secure financial support is needed to implement such longitudinal care11 ) ; 5 ) competent professionals should be involved in ongoing education and training in teaching and research ; and 6 ) research should explore causes , treatment strategies , and treatment outcomes .
centers with the appropriate vision and infrastructure are necessary to optimize the care of patients with craniosynostosis , as well as to enhance scientific knowledge and education about this disease .
several investigations have evaluated the roles of various growth factors and cytokines in determining the fate of sutures .
fibroblast growth factors ( fgfs ) are particularly important , as mutations in their receptors have been implicated in many craniosynostosis syndromes .
mutations in three of the four known fgf receptors have been associated with premature pathologic suture fusions1617 ) .
recent advances in developmental biology and genetics have identified some of the events governing suture fate , highlighting multiple axes of cellular signaling with the potential for clinical manipulation . such knowledge and comprehension
may facilitate therapeutic translations , ultimately enhancing or perhaps even replacing contemporary modalities for treating craniosynostosis .
|
craniosynostosis has a varied clinical spectrum , ranging from isolated single suture involvement to multi - sutural fusions .
greater understanding of the pathogenesis of craniosynostosis has led to the development of practical treatment protocols .
three stages of growth have determined the approach to managing craniosynostosis : the early period , up to 12 months ; the intermediate period , from 1 to 10 years ; and the late period , beginning at 10 years .
this review discusses current surgical management and future perspectives in craniosynostosis .
|
INTRODUCTION
CURRENT SURGICAL TREATMENT
The early period, up to 12 months
The intermediate period, from 1 to 10 years
Late period, beginning at age 10 years
FUTURE PERSPECTIVES IN CRANIOSYNOSTOSIS
CONCLUSION
|
however studies have failed to establish a cause and effect relationship between barium retention in the appendix and subsequent development of the appendicitis .
it is still not advisable to perform appendectomy in such circumstances without the evidence of an acute episode .
we present one such patient who had features suggestive of right ureteric colic and presented with retained barium in the appendix .
a 28 years old male attended our outdoor with an 8 days old x - ray film showing tubular radio - opacity in the right iliac fossa region .
he had two episodes of severe pain right lower abdomen in past 2 months for which he underwent barium meal follow through .
although the report came out to be normal but again he had an episode of pain .
this time x - ray abdomen was not completely normal and the patient came to our opd for advice .
daysevents0barium meal follow through study4x - ray abdomen for repeat episode of pain abdomen12presented to our opd with x - ray filmroutine blood investigations , repeat x - ray abdomen ( fig .
2 ) done60x - ray abdomen - normal5 monthslost in follow up routine blood investigation were sent and found to be normal .
repeat x - ray abdomen confirmed that the findings were not an artifact ( fig .
1 ) . ultrasound abdomen was advised to rule out abnormalities in right urinary system or some obvious bowel pathology .
a contrast enhanced ct scan of the abdomen was then performed to rule out any bowel pathology especially in the appendix or near ileocaecal region .
it concluded that the bowel was normal but there was a tiny right renal calculi . an x - ray of abdomen performed 7 days later , showed persistence of the radiographic opacity ( fig .
we concluded that this is a case of retained barium in appendix with recurrent right ureteric colic because of passage of renal calculi .
symptoms got relieved on conservative therapy and subsequent usg showed no calculi in renal system .
the radio - opacity was not noticeable on x - ray abdomen , 2 months after barium study .
prophylactic appendectomy was not done in our case , and the patient did not developed signs of acute appendicitis till 5 months when he was lost in follow up .
daysevents0barium meal follow through study4x - ray abdomen for repeat episode of pain abdomen12presented to our opd with x - ray filmroutine blood investigations , repeat x - ray abdomen ( fig .
repeat x - ray abdomen confirmed that the findings were not an artifact ( fig .
ultrasound abdomen was advised to rule out abnormalities in right urinary system or some obvious bowel pathology .
a contrast enhanced ct scan of the abdomen was then performed to rule out any bowel pathology especially in the appendix or near ileocaecal region .
it concluded that the bowel was normal but there was a tiny right renal calculi .
an x - ray of abdomen performed 7 days later , showed persistence of the radiographic opacity ( fig .
based on above findings and investigation we concluded that this is a case of retained barium in appendix with recurrent right ureteric colic because of passage of renal calculi .
symptoms got relieved on conservative therapy and subsequent usg showed no calculi in renal system .
the radio - opacity was not noticeable on x - ray abdomen , 2 months after barium study .
prophylactic appendectomy was not done in our case , and the patient did not developed signs of acute appendicitis till 5 months when he was lost in follow up .
the appendix is visualized in 8090% of barium swallow or enema studies , and this is accepted as a reliable sign of a non - diseased appendix but post - examination only 10% of the patients retain barium in the appendix beyond 72 h. its prolonged retention in the appendix has been viewed as altered physiology or pathological .
although barium sulphate is inert and not harmful to the mucosa , cases have been reported where appendicitis developed long after barium studies and were tagged as barium induced appendicitis .
baroliths have been conclusively retrieved from appendectomy specimen , even after 3 months following barium study in patients with acute appendicitis . despite these case
reports studies have failed to prove the cause and effect relationship between barium study and acute appendicitis .
important in this regard is the work of maglinte et al . who studied thirty - one patients with retained barium in the appendix for longer than 72 h and followed them for over 1 year .
based on above we conclude that the decision to perform appendectomy should be based on the diagnosis of acute appendicitis and that the prolonged retention of barium in the appendix is not an indication for surgery . in our case since no signs or symptoms were suggestive of acute appendicitis , the active search for other causes were made .
finding of small renal calculi in right kidney suggested that the etiology could be ureteric colic .
if a person presents with retained barium in the appendix , no appendectomy should be performed in absence of findings suggestive of acute appendicitis .
if atypical presentation is found , then they should be appropriately further investigated . some unusual things like right ureteric colic ( as in our case ) ,
written informed consent was obtained from the patient for publication of this case report and accompanying images .
a copy of the written consent is available for review by the editor - in - chief of this journal on request .
dr mithilesh kumar sinha : data collections , data analysis and writing , dr rajan kumar sinha : writing .
|
introductionretention of barium in the vermiform appendix for more than 72 h following barium meal study is uncommon
. it may produce undue concern for increased risk for appendicitis and even prophylactic appendectomies have been performed for this.presentation of casewe encountered one such patient who presented with x - ray plate suggestive of tubular radio - opacity in the right iliac fossa region .
the patient had episodes of severe pain in the right lower abdomen for which he underwent barium meal study .
further evaluation and investigations established the diagnosis as , a case of recurrent right ureteric colic with retained barium in the appendix.discussionreview of the literature suggests that , in absence of acute appendicitis there is no role of appendectomy , even in a case of retained barium in the appendix.conclusionif atypical presentation is there , they should be appropriately further investigated .
|
Introduction
Presenting concerns
Clinical findings
Timeline
Diagnostic focus and assessment
Therapeutic focus and assessment
Follow-up and outcomes
Discussion
Conclusion
Conflict of interest
Funding
Ethical approval
Author contributions
|
the ability of mammals to respond to an inadequate o2 supply , commonly termed hypoxia , is crucial for their survival .
although a proper response to changed o2 tensions triggers adaptation , a number of pathological conditions or failures in the o2 response are associated with various diseases such as anemia , myocardial infarction , thrombosis , atherosclerosis , or cancer . when exposed to hypoxia or even anoxic conditions , mammalian organisms initiate a variety of responses in different organs , aiming to increase the delivery of oxygen to the tissues .
in addition to the switch from an aerobic to an anaerobic metabolism and the suppression of energy - using reactions , the carotid body chemoreceptor cells stimulate the brain stem center controlling the respiratory and cardiovascular systems to enhance ventilation , heart rate , and blood pressure ( reviewed by prabhakar1 ) .
in addition , neuroepithelial cells in the lung contribute to adjusting pulmonary perfusion and gas exchange .
moreover , organs and cells switch their gene expression profile : the kidneys produce erythropoietin , which increases red blood cell production in the bone marrow , and vascular cells produce vascular endothelial growth factor to promote angiogenesis and flow of enhanced blood volume ( reviewed by semenza2 ) . in addition to the expression of erythropoietin and vascular endothelial growth factor , the expression of more than 500 genes , products of which are involved in glycolysis , angiogenesis , erythropoiesis , cell death , and differentiation , is also changed on exposure to hypoxia ( reviewed by wenger and stiehl3 and semenza4 ) . in mammals ,
the hypoxia - dependent changes on the level of gene expression are mainly mediated by the -subunits of hypoxia - inducible transcription factors ( hifs ) .
hif -subunits are tightly regulated , and post - translational hydroxylations in response to hypoxia appear to be of major importance .
in addition to hypoxia , hif -subunits were also found to respond to various growth and coagulation factors , hormones , cytokines , or stress factors already under normoxia .
indeed , different kinases , among them glycogen synthase kinase 3 ( gsk-3 ) , have been identified to directly phosphorylate hif- proteins .
this review discusses the regulation of hif- by gsk-3 and compares it with hydroxylase - dependent hif- protein regulation .
in their active form , hifs are heterodimeric transcription factors consisting of an - and -subunit .
the hif -subunit represents the stable nuclear subunit primarily represented by the ubiquitously found arnt ( arylhydrocarbon receptor - nuclear translocator ) protein ; however , arnt2 or artn3 , although to a lesser extent , also appear to be able to take part in the formation of hif dimers ( reviewed by semenza5 ) .
so far , three -subunit proteins , hif-1 , hif-2 ( also known as epas,6 hlf,7 hrf,8 or mop29 ) , and hif-3 have been identified .
together , the different hif - and -subunits may give rise to the formation of several combinations of hif dimers.5,10 hif-1 and hif-2 are the best - studied hif- isoforms .
although they share structural and functional similarities , it appears that differences in the cell - type expression pattern , the target genes , the embryonic deletion phenotypes , and the effects on tumorigenesis exist between hif-1 and hif-2.1114 the function of hif-3 , from which several splice variants exist in humans,15,16 is largely unknown , although some human hif-3 variants and a mouse splice variant termed inhibitory pas protein ( ipas ) appear to act as negative regulators of the hypoxic response.1619 similar to the arnt proteins , the hif -proteins belong to the basic helix - loop - helix pas ( per - arnt - sim ) protein family . in particular , hif-1 and
hif-2 show the highest degree of sequence identity in the basic helix- loop - helix ( 85% ) , pas - a ( 68% ) , and pas - b ( 73% ) domains .
both also contain two nuclear localization sequences responsible for translocation to the nucleus under hypoxia ; they are localized in the n terminus ( amino acids 1733 in hif-1 and amino acids 150 in hif-2 ) and in the c terminus ( amino acids 718721 in hif-1 and amino acids 689870 in hif-2).20,21 with the exception of hif-3 , which does not contain a c - terminal transactivation domain ( c - tad),22,23 hif -subunits also contain n- and c - terminal transcriptional activation domains ( n - tad and c - tad ) . a unique oxygen - dependent degradation domain ( oddd , amino acids 401603 in hif-1 and amino acids 517682 in hif-2 ) overlaps n - tad .
the residues between n - tad and c - tad represent an inhibitory domain ( amino acids 604785 in hif-1 and amino acids
hif -subunit activation under hypoxia is mainly the result of an increased protein stability and coactivator recruitment , although transcriptional and translational mechanisms also were shown to be involved in hif -subunit activation.22,2630 as a result , hif- proteins accumulate , translocate to the nucleus , and dimerize with hif- to form a functional transcription factor.31 thus , in the presence of oxygen ( ie , normoxia ) , hif- proteins become degraded .
this is primarily achieved by oxygen - dependent hydroxylations at the oddd.32 under normoxia , prolyl hydroxylase domain proteins ( phds),33,34 in particular phd2 , hydroxylate two crucial residues in the oddd of hif -subunits ( p402 and p564 in hif-1 and p405 and p531 in hif-2).25,32,35 prolyl hydroxylation is required for binding the von hippel - lindau protein ( vhl),36,37 which represents the substrate recognition subunit of an e3 ubiquitin protein ligase consisting of elongin c , elongin b , ring box 1 , cullin 2 , and an e2 ubiquitin - conjugating enzyme ( figure 1 ) .
the prolyl hydroxylation and ubiquitination can be further promoted by the binding of phd2 to os938 and that of hif-1 , vhl , and elongin c to ssat2 , respectively.39,40 in addition to prolyl hydroxylation , a conserved asparagine residue ( n803 in hif-1 and n852 in hif-2 ) in the c - tad is hydroxylated by the factor - inhibiting hif in an oxygen - dependent manner .
this hydroxylation prevents interaction with the coactivator proteins cbp / p300.4144 thus , the major posttranslational modification appears to be the oxygen - dependent hydroxylation.36,37
in addition to hydroxylation , hif- transcriptional activity and protein stability appear also to be dynamically regulated by other posttranslational modifications such as acetylation , s - nitrosylation , sumoylation , and phosphorylation ( for review , see dimova and kietzmann45 ) .
phosphorylation appears to be of special importance under normoxic conditions , mediating the response of hif- to various growth and coagulation factors , hormones , cytokines , or stress factors ( reviewed by dimova et al46 ) under normoxia .
indeed , a panel of protein kinases is reported to be involved in hif-1 phosphorylation , either directly ( table 1 ) or indirectly.4752 although the individual action of certain kinases on hif-1 regulation was mainly studied in vitro ( table 1 ) , the in vivo mechanisms are likely much more complex .
at least the extent to which the kinases can be involved in hif- phosphorylation may vary according to the signal , cell type , or tissue . given the different developmental and/or differentiation status of a cell or tissue , the expression of various growth factors , their receptors , and respective signaling components and
thus , it seems not to be surprising that phosphorylation of hif- by different kinases or after modulation of signaling pathways may be a highly cell type - specific event .
although direct proof is currently lacking , it is plausible that the phosphorylation pattern of hif- in a certain cell may be explained by different layers of regulations that affect kinases depending on the cellular context .
in addition to being activated by a variety of extracellular signals , the pi3k / akt cascade appeared also to be regulated by hypoxia , thus integrating hypoxia signaling with extracellular signals affecting multiple cellular processes such as apoptosis , metabolism , cell proliferation , and cell growth ( for review , see braccini et al53 ) .
the pi3k / akt pathway is considered to control hif-1 within the cell via regulation of hif-1 protein synthesis and stability .
however , it appeared that hif- proteins are not directly phosphorylated by pkb / akt but , rather , by a pkb / akt target .
the pkb / akt targets hdm2,54,55 mammalian target of rapamycin ( mtor),56 and gsk-357 were shown to contribute to changes in hif- protein levels ; however , only gsk-3 was shown to do this directly , ie , by phosphorylating hif- proteins .
gsk-3 is a serine / threonine kinase that was first identified as a negative regulator of glycogen synthesis ; inhibition is achieved through phosphorylation of glycogen synthase.58,59 since its initial discovery , gsk-3 has been found to be involved in numerous signaling pathways initiated by diverse stimuli and to contribute to the regulation of cell proliferation , stem cell renewal , apoptosis , and development , which are processes often associated with hypoxia . because of these multiple involvements ,
dysregulation of gsk-3 has been implicated in the pathogenesis of human diseases , including type 2 diabetes , bipolar disorders , inflammation , alzheimer s disease , and cancer ( reviewed by frame and cohen,60 grimes and jope,61 and woodgett62 ) .
two isoforms , gsk-3 ( 51 kda ) and gsk-3 ( 47 kda ) , have been identified in mammals . despite their homology in the catalytic domain ( 98% ) , they significantly differ in their n- and c - terminal parts63,64 and do not have entirely overlapping roles in metabolism ( reviewed in force and woodgett65 ) .
moreover , gsk-3 ( gsk-3 ) homozygous knockout mice showed an embryonic lethal phenotype around day 16 because of hepatic apoptosis or a cardiac pattern defect,66,67 whereas homozygous gsk-3 ( gsk-3 ) knockout mice are viable and fertile.68,69 gsk-3 is a target of pkb / akt , which can phosphorylate both gsk-3 isoforms ( serine 21 of gsk-3 and serine 9 of gsk-3 ) , leading to an inhibition of gsk-3 activity.70 interestingly , these serine residues can also be phosphorylated by other kinases such as erk1/2,71 p70 ribosomal s6 kinase 1,72 camp ( cyclic adenosine monophosphate)-dependent protein kinase a ( pka),73 and protein kinase c ( pkc).74 in contrast , an autophosphorylation event leading to phosphorylation of tyrosine 279 in gsk-3 and of tyrosine 216 in gsk-3 increases gsk-3 activity.75,76 neurons seem to possess a spliced gsk-3 variant called gsk-32 that contains a 13 amino acid residue insert within the kinase domain , leading to reduced kinase activity.77,78 although gsk-3 is mostly known in the insulin field as a regulator of glycogen synthesis , it has been shown that early hypoxia enhanced pi3k / akt activity and increased hif-1 protein levels.57 similarly , hypoxia was capable of inhibiting gsk-3 by phosphorylation in different cell types , such as pc-12 ( rat pheochromocytoma cell line ) cells,79 ht1080 ( human fibrosarcoma cell line ) cells,80 and hepg2 ( human liver hepatocellular cell line ) cells,57 as well as in vivo.81 although this effect was not observed in other cell types , including some breast cancer cell lines,82 pc-3 prostate cancer cells,83 and 3t3 cells,84 it was considered to have a cell type - specific component .
however , the findings that gsk-3 inhibition57 and small interfering rna - mediated depletion induced hif-1 , whereas gsk-3 overexpression reduced hif-1 protein levels,85 suggested that hif-1 is a direct target of gsk-3. indeed , the oddd86 and three sites , s-551 , t-555 , and s-589 , located within the oddd overlapping the n - tad of hif-1 were found to be directly phosphorylated by gsk-3.85 another study reported five sites , t-498 , s-502 , s-505 , t-506 , and s-510 , within the n - tad of hif-1 as gsk-3 phosphorylation sites.87 the disparity of the different phosphorylation sites is difficult to explain , but the different oxygen concentrations ( 8% o2 compared with 2% o2 ) used in these studies may contribute to the differences .
it is possible that different oxygen levels may induce variable signaling pathways that have unequal effects on hif-1 and its ability to act as a substrate for gsk-3. another possibility could be the different cell types ( hepg2 compared with sk - ov-3 [ human ovarian cancer cells ] ) that were used in the studies . despite the differences in the phosphorylation sites , both studies demonstrated that the regulation of hif-1 by gsk-3 is independent of o2 , hydroxylation , and recruitment of the vhl - containing e3 ubiquitin ligase .
experiments with vhl - deficient cells showed that gsk-3-dependent hif-1 degradation occurred independent of vhl , indicating that the phosphorylation of hif-1 by gsk-3 target hif-1 for proteasomal degradation in an oxygen - independent manner.85 this suggested involvement and recruitment of another so - far - unknown e3 ubiquitin ligase to gsk-3-phosphorylated hif-1. indeed , two groups demonstrated that the f - box and wd protein fbw7 ( also known as hcdc4 in yeast , hsel10 in caenorhabditis elegans , or ago in drosophila ) acted as the substrate - recognition component of a multisubunit e3 ubiquitin ligase , which was crucial for the proteasomal degradation of gsk-3 phosphorylated hif-1.85,87 in this e3 ligase , fbw7 interacts with skp1 ( s - phase kinase - associated protein 1 ) , cul1 ( cullin 1 ) , and rbx1 , forming the so - called scf complex .
similar to vhl , fbw7 is considered to serve as a tumor suppressor , and three fbw7 isoforms ( fbw7 , fbw7 , and fbw7 ) are known to be produced by alternative splicing .
they are found in the nucleoplasm , cytoplasm , and nucleolus , respectively.88 in addition to hif-1 , fbw7 was shown to be involved in the degradation of various oncogenic proteins , including cyclin e,89 c - myc,90,91 c - jun,92,93 and notch.94 several studies have shown that loss of the fbw7 gene is associated with malignant transformation , especially in ovarian cells and t cells,95 in breast cancer cells,96 and later also in human colorectal cancers,97 which leads then to chromosomal instability and some types of malignancy .
furthermore , investigation of more than 1,500 human tumors revealed that approximately 6% of those tumors showed mutations in the fbw7 coding region .
specifically , cholangiocarcinomas ( 35% ) , t - cell acute lymphocytic leukemia ( 31% ) , and endometrial ( 9% ) , colon ( 9% ) , and stomach ( 6% ) cancer98 had the highest mutation rates .
strikingly , nearly half ( 43% ) of these were missense mutations that resulted in amino acid substitutions within the wd40 domain ( arg465 and arg479 ) , which are shared by all three fbw7 isoforms , suggesting that all fbw isoforms might collectively contribute to the tumor - suppressor function.98 with respect to hif-1 , all three fbw7 isoforms were able to induce hif-1 degradation , and the loss of the fbw7 wd domain abolishes gsk-3-initiated degradation , leading to higher hif-1 levels , which has been found to be associated with several tumors.99101 the finding that hif subunits can be targeted for degradation by two different e3 substrate recognition proteins indicates that the system is highly dynamic .
the human genome encodes nearly 100 deubiquitylating enzymes that are predicted to be active and that oppose the function of around 600 e3 ligases.102,103 similar to e3s , deubiquitylating enzymes have a central role in cell cycle regulation and dna damage response and , depending on the context , can act either as a tumor promoter or suppressor ( see references in love et al104 ) .
with respect to vhl , two different deubiqiutinating enzymes , vdu1 ( usp33 ) and vdu2 ( usp20 ) , were suggested to oppose the vhl - e3 ubiquitin ligase.105,106 later , it was shown that vdu2 but not vdu1 can interact with hif-1.107 experiments with cycloheximide and hypoxia showed that the half - life of hif-1 was significantly increased upon overexpression of vdu2 , whereas a catalytic inactive vdu2 c154a mutant had no effect .
in addition , it was shown that only vdu2 , not vdu1 , deubiquitinated hif-1 , resulting in the stabilization of hif-1 protein107 ( figure 1 ) .
experiments with gsk-3- and fbw7-deficient cells revealed that gsk-3- and fbw7-dependent hif-1 degradation can be antagonized by ubiquitin - specific protease 28 ( figure 1).99 these findings suggest that the gsk-3-dependent degradation of hif-1 is not limited by the presence of oxygen and is therefore independent of vhl .
together , these results demonstrate that hif-1 protein stability is regulated in a dynamic manner involving different ubiquitin ligases and deubiquitinases .
as such , the hydroxylation- and vhl - dependent ubiquitination and degradation of hif-1 under normoxia is opposed by the deubiquitinase vdu2 .
in contrast , the oxygen - independent but phosphorylation - dependent ubiquitination of hif-1 is counteracted by ubiquitin - specific protease 28-mediated deubiquitination .
the latter process allows the integration of the hif system into the cellular response to various physiologic and pathophysiologic signals independent of the oxygen tension .
the finding that gsk-3 is involved in the degradation of hif-1 indicated similarities with the destruction of -catenin in the canonical wnt signaling pathway . in this pathway ,
gsk-3 and -catenin are part of a destructive complex in which binding of gsk-3 and -catenin promotes phosphorylation of -catenin by gsk-3 , which requires priming phosphorylation by casein kinase 1 , -isoform .
the phosphorylated -catenin is recognized by the f - box / wd protein -trcp and subsequently ubiquitylated and targeted for proteasomal degradation ( for review , see cohen and frame108 and metcalfe and bienz109 ) .
when this phosphorylation event is blocked , -catenin accumulates and binds to the t - cell - specific transcription factor / lymphoid enhancer - binding factor 1 family of transcriptional activators to activate numerous target genes ( reviewed by reya and clevers110 ) contributing to embryonic development and adult tissue homeostasis ( reviewed by clevers111 ) .
similarly , gsk-3-mediated phosphorylation of hif-1 recruits fbw7 , and thus targets hif-1 for ubiquitylation and proteasomal degradation.99 those very similar scenarios imply interference or interconnection of both the wnt/-catenin and hypoxia / hif-1 signaling on the level of gsk-3 .
actually , crosstalk between the hypoxia and/or hif-1 and wnt/-catenin pathway was reported and appears to be quite complex because of controversial and likely cell / tissue / differentiation - stage specific data.112117 indeed , it was reported that hypoxia and/or hif-1 can inhibit wnt/-catenin signaling .
several mechanisms , such as binding of hif-1 to hard1 ( human arrest - defective-1 protein ) with subsequent interference with acetylation of -catenin,112 blocking processing and secretion of wnt proteins,113 down - regulating -catenin via p53-dependent activation of siah-1 ( seven in absentia homolog 1),118 or direct interaction between hif-1 and -catenin119,120 were proposed to contribute to these effects .
in contrast , hypoxia was also shown to activate wnt/-catenin signaling in undifferentiated cells and in vivo.115,117 in hypoxic embryonic stem cells , this occurred via hif-1-mediated expression of lymphoid enhancer - binding factor 1 and t - cell - specific transcription factor , followed subsequently by increased interaction of -catenin with lymphoid enhancer - binding factor 1/t - cell - specific transcription factor , and thus activating wnt/-catenin targets.115 in addition , hypoxia was able to activate -catenin via gsk-3 inactivation116,121 in different human cell lines such as ht-29 ( human colorectal adenocarcinoma cell line ) and hepg2 ; this activation contributed to an endothelial mesenchymal transition program , leading to significantly increased invasiveness,121 and in renal tubular cells , this process impaired wound healing.116 together , the reported findings indicate that complex interconnections between hypoxia and/or the hif-1 and wnt/-catenin pathway exist and that cell- , tissue- , and differentiation - specific aspects contribute to their functional consequences .
hypoxia and hifs play important roles in many critical aspects of physiological and pathological processes .
most solid cancers contain hypoxic areas , and clinical data demonstrate that overexpression of hif-1 is associated with an increased risk for patient mortality . in line ,
downregulation of hifs interferes with tumor growth , vascularization , invasion , and metastasis , as well as radiation and chemotherapy .
activation of multiple oncogenic pathways including growth factor signaling coupled with enhanced kinase signaling is a common event in tumors , thus making it likely that kinases are involved in the modulation of hif- function .
because regulation of hif- protein stability is critical for its activation , identification of kinases contributing to hif- stability may provide a link explaining normoxic hif- stabilization by extracellular stimuli . in light of this , dysregulation of gsk-3 is thought to underlie the pathogenesis of various diseases that are also associated with hypoxia and changed hif- levels , such as type 2 diabetes mellitus , alzheimer s disease , mood disorders , cardiovascular diseases , and cancer.122 thus , given that gsk-3 upstream regulation leads to inhibition of gsk-3 and hif-1 accumulation , this raises the question whether it is an option to target gsk-3 in those diseases and disregard the adverse effects . although research from the last decade has demonstrated that a number of kinase pathways contribute to hif-1 regulation , data for hif-2 or hif-3 are limited .
taking into consideration the overlapping , but different , roles of the hif- proteins , more knowledge about the phosphorylation - dependent regulation of hif-2 and hif-3 is necessary to better understand both already - observed general and different effects .
|
hypoxia - inducible factors ( hifs ) , consisting of - and -subunits , are critical regulators of the transcriptional response to hypoxia under both physiological and pathological conditions .
to a large extent , the protein stability and the recruitment of coactivators to the c - terminal transactivation domain of the hif -subunits determine overall hif activity .
the regulation of hif -subunit protein stability and coactivator recruitment is mainly achieved by oxygen - dependent posttranslational hydroxylation of conserved proline and asparagine residues , respectively . under hypoxia ,
the hydroxylation events are inhibited and hif -subunits stabilize , translocate to the nucleus , dimerize with the -subunits , and trigger a transcriptional response . however , under normal oxygen conditions , hif -subunits can be activated by various growth and coagulation factors , hormones , cytokines , or stress factors implicating the involvement of different kinase pathways in their regulation , thereby making hif--regulating kinases attractive therapeutic targets . from the kinases known to regulate hif -subunits , only a few phosphorylate hif- directly . here , we review the direct phosphorylation of hif- with an emphasis on the role of glycogen synthase kinase-3 and the consequences for hif-1 function .
|
Introduction
HIFs: basic aspects
Oxygen-dependent regulation of HIF -subunits: role of hydroxylation
Regulation of HIF -subunits by phosphorylation
GSK-3 and HIF- regulation
Interconnection among the GSK-3, hypoxia/HIF-, and Wnt/-catenin pathways
Conclusion
|
streptococcus pneumoniae remains a major cause of childhood morbidity and mortality worldwide , particularlyin lower income countries .
pneumococcal diseases are the leading source of vaccine preventable deaths , mostly due to community - acquired pneumonia ( cap ) , accounting for approximately 11% of all deaths in children under 5 years old ( 1 ) .
colonization of the nasopharynx is a necessary step along the path to pneumococcal disease ( pd ) ( 2 ) .
pneumococcal conjugate vaccines ( pcv ) reduce nasopharyngeal carriage of serotypes which included in the vaccine by conferring capsular - specific immunity .
experience from countries where conjugate vaccines have been introduced has shown rapid and sustained carriage reduction of vaccine serotypes ( vt ) following vaccination ( 3 ) .
the microorganism produces a plethora of virulence factors , including the polysaccharide capsule , several surface - located proteins , and the toxin pneumolysin ( 5 , 6 ) .
the polysaccharide capsule is highly efficient in protecting the bacteria from opsonophagocytosis ( 7 ) .
surface proteins of s. pneumoniae ( pneumococcus ) have been investigated for their role in pneumococcal pathogenicity and as candidate antigens for protein based vaccines ( 8) . among the surface - associated proteins ,
the pneumococcal surface protein a ( pspa ) andc ( pspc ) are the best characterised choline - binding proteins ( 6 , 9 ) .
pneumolysin is a cytoplasmic toxin released by autolysis of the cell and it is a very important virulence factor with multiple effects .
the major autolytic enzyme of the pneumococcus is lyta ( nacetylmuramoyl - l - alanine - amidase ) , which is responsible for the deoxycholate- and penicillin induced cell lysis in the stationary phase , having a great clinical importance ( 10 ) . the lyta - encoded major autolysin of s. pneumoniae is a member of a widely distributed group of cell wall - degrading enzymes located in the cell envelope and postulated to play roles in avariety of physiological functions ( 11 ) .
rrga is a virulence factor in a murine lung infection model and has a varied distribution among serotypes of s.pneumoniae ( 12 ) .
s. pneumoniae adherence was significantly enhanced by expression of an extracellular pilus composed of three subunits , rrga , rrgb and rrgc ( 13 ) .
some of the pneumococcal virulence factors are potential targets for protein- based pneumococcal vaccine production .
thus , in this study the presence of three genes were detected among the isolates of s. pneumoniae .
a total of 260 samples from nasopharynx of healthy children under 6 years old were taken carefully and transferred under cold condition to the laboratory .
these samples were collected from children attending day care centers in different geographical areas of mashhad , iran .
both plates were incubated at 37c in an atmosphere of 5% co2 for 24 hours .
the plates were examined and -haemolytic colonies suspected to be streptococcus pneumoniae were confirmed by optochin test and pcr for cpsa gene as described earlier ( 14 ) .
the optochin - susceptibility test was performed using a 6.5 mm diameter disc containing 5 mg optochin ( oxoeid ) in an atmosphere of 5% co2 .
the genomic dna of the bacterial isolates were extracted by dnaase tissue kit ( kiagen , tehran , iran ) .
the presence of rrga , pspc and lyta genes were detected among confirmed s. pneumoniae isolates by three single pcr assays .
oligonucleotides which were used as primers to amplify particular sequences of s. pneumoniae amplification conditions for lyta and pspc genes were : 94 c for 2 min , 25cycles of 94 c for 10 s , 58 c for 15 s , and 72c for1 min , followed by a final extension at 72 c for 5 min .
amplification conditions for rrga gene were : 95c for 2 min , 25cycles of 95c for 30 s , 51c for 30 s , and 72c for 90 s , followed by a final extension step at 72c for 5 min .
one amplicon from s. pneumoniae with the gene rrga was sequenced by macrogen company ( south korea ) .
sequences were examined for identity with published sequence data from national center for biotechnology information ( ncbi ) .
a total of 260 samples from nasopharynx of healthy children under 6 years old were taken carefully and transferred under cold condition to the laboratory .
these samples were collected from children attending day care centers in different geographical areas of mashhad , iran .
both plates were incubated at 37c in an atmosphere of 5% co2 for 24 hours .
the plates were examined and -haemolytic colonies suspected to be streptococcus pneumoniae were confirmed by optochin test and pcr for cpsa gene as described earlier ( 14 ) .
the optochin - susceptibility test was performed using a 6.5 mm diameter disc containing 5 mg optochin ( oxoeid ) in an atmosphere of 5% co2 .
the genomic dna of the bacterial isolates were extracted by dnaase tissue kit ( kiagen , tehran , iran ) .
the presence of rrga , pspc and lyta genes were detected among confirmed s. pneumoniae isolates by three single pcr assays .
amplification conditions for lyta and pspc genes were : 94 c for 2 min , 25cycles of 94 c for 10 s , 58 c for 15 s , and 72c for1 min , followed by a final extension at 72 c for 5 min .
amplification conditions for rrga gene were : 95c for 2 min , 25cycles of 95c for 30 s , 51c for 30 s , and 72c for 90 s , followed by a final extension step at 72c for 5 min .
one amplicon from s. pneumoniae with the gene rrga was sequenced by macrogen company ( south korea ) .
sequences were examined for identity with published sequence data from national center for biotechnology information ( ncbi ) .
among 260 nasopharanx samples from healthy children under 6 years , 59 isolates were confirmed as s. pneumoniae .
distribution of the lyta , rrga and pspc genes among isolates of s.pneumoniae were determined .
rrga and pspc were also found in17 ( 28.81% ) , and 2 ( 3.38% ) isolates respectively .
five patterns of these genes were seen among s. pneumoniae isolates : lyta ( n=33 , 55.93% ) , rrga ( n=2 , isolates , 3.38% ) , pspc ( n=1 1.69% ) , lyta+rrga ( n=16 , 27.11% ) and lyta+ rrga+ pspc ( n=1 , 1.69% ) .
the amplicon represented expected sequences with more than 90% identity with published data from ncbi ( genbank : ef 560634.1 ) .
the amplicon represented expected sequences with more than 90% identity with published data from ncbi ( genbank : ef 560634.1 ) .
it remains unclear , however , why some children develop invasive disease , whereas in the majority of cases , colonization remains asymptomatic , a combination of bacterial virulence and host factors may be responsible ( 18 ) .
we studied the s. pneumoniae isolates giving special reference to identification and distribution of virulence markers such as autolysin among the isolates from nasopharynx .
this exercise may help in understanding the factors contributing to the pathogenicity of s. pneumoniae .
further , there are numerous reports giving us increasing evidences on the role of lyta in pneumococcal pathogenesis suggesting that this might be more appropriate as vaccine antigen against s. pneumoniae infections .
the cpsa was used as a novel genetic marker specific for identification of s. pneumoniae and to differentiate it from the closely viridans group streptococci as well as other pneumococcus - like streptococci such as s. pseudopneumoniae ( 19 ) .
many virulence genes contribute to the colonization of s. pneumoniae ; however , our study only demonstrates this for pspc , rrga and lyta genes .
thus , synergistic effect and correlation of these virulence factors is necessary for our future work .
our results showed that most of the isolates had lyta gene ( 84.74% ) which has an important role in colonization .
this observation is in concordance with previous report which was showed that all of the unencapsulated isolates of s. pneumoniae were negative for lyta and psaa by pcr .
detection of lyta and psaa in six encapsulated isolates for which a serotype could be determined was negative ( 9 ) .
false negative results were obtained by the pcr assays for these two genes may be due to mutations or sequence variation . on the other hand , lyta is essentially a rather conserved gene displaying limited genetic variation ( 11 ) . in the study of whatmore ,
33 out of 62 isolates of s. pneumoniae were selected to represent a diverse range in terms of serotype , clinical association , and time and place of isolation .
autolysin which was found in all strains , might appear to be a suitable target virulence , and apparently highly conserved , for inclusion in a potential vaccine ( 11 ) .
the pneumococcal protein lyt a is the major autolysin of s. pneumoniae , it has an important function in pathogenesis by releasing pneumolysin and plays a fundamental biological role in bacterial lysis after exposure to certain antibiotics ( 19 ) .
it has been reported that the lyta gene has higher specificity than the pspc for identification of s. pneumoniae ( 15 , 20 ) . in our study , only 2 out of 59 isolates of s. pneumoniae ( 3.38% ) had pspc gene .
given the high sequence diversity of pspc , it is unlikely that pspc alone can be a vaccine antigen to provide protection from across different pneumococcal strains ( 16 ) .
it was showed that rrga is central in pilus - mediated adherence and disease , even in the absence of polymeric pilus production ( 13 ) .
however , it has been demonstrated that numerous protein virulence factors are involved in the pathogenesis of pneumococcal disease ( 21 ) .
hence , new vaccine designs are focused on the surface proteins ( e. g. , pspa and pspc ) , cytolysin , and pneumolysin ( 22 ) .
we concluded that the gene cpsa was specific and highly conserved among s. pneumoniae isolates which were colonized in nasopharynx . on the other hand
, we showed that lyta gene was the most frequent genes among the s. pneumoniae isolates , and combination of rrga , lyta was the most observed pattern.thus this should allow for appropriate screening of adhesin - based vaccines to prevent infections by streptococci .
|
background and objectives : many surface proteins are implicated in nasopharyngeal colonization and pathogenesis of streptococcus pneumoniae .
some of these factors are candidate antigens for protein based vaccines .
new vaccine designs focus on the surface proteins ( e. g. , pspa and pspc ) and also cytolysin , and pneumolysin . in this study ,
3 key virulence genes , lyta , pspc , and rrga , which encoded surface proteins , were detected among s. pneumoniae isolates.materials and methods : a total of 260 nasopharyngeal swabs were collected from healthy children under 6 years old attending day care centers in mashhad , iran .
isolates of s. pneumoniae were confirmed by optochin susceptibility and colony appearance and also by pcr for cpsa gene .
the presence of lyta , pspc , and rrga genes were also detected by pcr.results:a total of 59 isolates were confirmed as s. pneumoniae . among these isolates , 50 ( 84.74% ) , 19 ( 32.20% ) , and 2 ( 3.38% )
were positive for lyta , rrga , and pspc genes respectively .
the presence of these genes among s.pneumoniae isolates were as follows : 1 ) rrga , lyta , pspc ( 1 isolate ) , 2 ) rrga , lyta(17isolates ) , 3 ) pspc ( 2 isolate ) , 4 ) lyta ( 50 isolates).conclusion : cpsa gene was specific for detection of s. pneumoniae isolates which were colonized in nasopharynx .
the lyta gene was the most frequent gene among the s. pneumoniae isolates , and combination of rrga , lyta was the most observed pattern .
thus , it is important for future monitoring of vaccine formulation in our country .
|
INTRODUCTION
MATERIALS AND METHODS
Sampling.
Isolation.
DNA extraction.
Detection of virulence genes.
PCR assays.
Sequence analysis.
RESULTS
Sequence analysis.
DISCUSSION
CONCLUSION
|
medical costs in the united states have risen rapidly
and substantially , making health care expenditure one of
the most troubling economic and social problems for
the u.s.a .
the local , state and federal governments as
well as citizens are generally aware of the magnitude
and seriousness of the problems of health care costs to
individuals and their families , to communities , and to
the nation . according to the national coalition on
health care ( 1 ) , the u.s.a . has the highest health care
expenditures compared to other industrialized nations .
in fact , the u.s.a . annual health care expenditures
constitute a significant portion of the u.s . gross
domestic product ( gdp ) .
for instance , health care
expenditures in the u.s.a . reached $ 2 trillion in 2005
and are projected to reach $ 4 trillion or 20% of the gdp by 2015 ( 1 ) .
in addition to this health - care cost problem ,
the numbers of elderly adults and population are
growing along with increased longevity and more
demands for different health care services resulting
from changes in lifestyles . according to the u.s.a .
census bureau ( 2 ) , 35.9 million people were aged 65
and older as of july 2003 and this number is expected to
double within the next 25 years . by 2030 , almost 1-outof-
5 americans , about 72 million people , will be 65
years or older .
worldwide , the number of adults older
than 65 years is also expected to increase from 420
million in 2000 to 974 million by 2030 . apparently ,
there are two severe problems facing the u.s.a .
health
care system ( and perhaps those of other developed
countries ) : enormous health care expenditures and the
large growing number of elderly adults who need
frequent and more health examinations and monitoring ,
which in turn drive up the health care cost further .
while the sky - rocketing health care expenditures are
the result of various issues such as increased and uncontrollable charges from health care providers and
increased cost of medicine , it is believed that
implementing advanced technologies for medical
practices and health treatments not only will help
reduce medical costs , but will also improve the
treatment for patients from medical professionals . for
instance , the use of advanced robotic , wireless and
information technologies can assist surgeons to perform
remote surgeries quickly , accurately and safely on
individuals located far away from the surgeons ( 3 ) .
therefore , it is crucial that not only advanced health
care techniques and devices are developed and
deployed , but also that technologies potentially leading
to improvement in health care are introduced to the
health - care professionals and organizations .
technologies , in general , and sensing , imaging , and
wireless communications , in particular , have advanced
considerably in the past decade which in turn can affect
not only health care costs , but can also influence health
examinations and monitoring .
for instance , advances
in sensing , imaging , and wireless communications have
opened the door for possible wireless , fast , accurate and
remote monitoring , examination and diagnosis of the
inside of the human body .
these advances have led to
the development of advanced medical techniques and
equipment such as magnetic resonance imaging ( mri )
( 4 ) and endoscopic ultrasound ( 5 ) . in order to push
forward medical technologies , improvement in health
care techniques
to
that end , it is imperative that advances in technologies
be utilized wisely and properly for medical applications .
one of the most significantly advanced technologies
in the past decade is radio - frequency identification
( rfid ) , which has profound impact on various
applications from consumer to military applications .
rfid technology dates back to the 1940s as a military
technology for identifying aircraft in world war ii ( 6 ) .
the recently declining cost along with improved
sensitivity and durability of rfid systems has made
these systems increasingly interesting for the
distribution and retail industry .
in fact , rfid has been
identified as potential technology to replace the
currently dominant upc bar - code system ( 7 ) .
rfid is
very attractive for medical applications and if properly
utilized can potentially lead to breakthroughs in the way
medical examinations and monitoring of patients are
conducted . in this paper
, we present several ideas of using rfid
technology for personal health examination and
monitoring .
our main objective is not to present
specific rfid systems for these medical practices ,
which are outside the scope of our research and requires
significant collaborated efforts between different
disciplines , but to give ideas that can possibly lead to significant improvements in medical examinations and
monitoring of patients .
it is hoped that this paper will
provide medical professionals and students some
overview of what rfid technology can potentially do
for medical applications , effectively motivating and
facilitating them to utilize this valuable technology not
only for the improvement of patient treatments , but also
to possibly develop particular rfid devices , in
collaboration with other disciplines such as biomedical
and electrical engineering , for specific medical needs .
understanding the basics of rfid helps medical
professionals , such as medical doctors , not only to
properly utilize the technology for medical practices ,
but also to possibly improve it for specific medical
needs .
1 shows a general rfid system consisting
of a tag ( or transponder derived from
transmitter / responder ) and a reader ( or interrogator ) .
the basic objectives of a rfid system are to acquire ,
store , process , and report specific data or information
about objects at an appropriate time and location ,
determined by users or operators , via wireless means
.
detailed information on rfid as described in this
section can be found in the rfid handbook by klaus
finkenzeller ( 8) . in operation , the tag is attached to the object to be
sensed , monitored , imaged , or identified .
for instance ,
tags about the size of a large grain of rice are implanted
under the skin of a dog , cat , or other animal for
identification purposes ( 9 ) .
implantable tags of about
11 millimeters long and 1 millimeter in diameter can
also be placed under the skin of hands and arms in
humans for identification , physiological characteristics ,
health , nationality , etc .
particularly , as noted in
( 8) , alzheimer s patients , mentally ill patients and
people with communication difficulties could benefit
from implanted rfid chips for identification purposes .
data can be pre - programmed or written ( during
operation by the reader ) into the tag to provide
instructions for the tag to perform certain functions or identification of the object .
the tag acquires and stores certain data from the object
for various functions such as monitoring the object s
activities .
the tag is typically programmed so that it
automatically activates when it is within the
interrogation zone of readers .
however , the tag can also
be activated at any time by the user or operator .
the receiver includes circuits used for specific
functions , such as collecting temperature of the object ,
and is used to receive , store and possibly process
specific information of an object onto which the tag is
attached ( with help of dsp ) as well as receive
information sent by the reader .
not all tags
require transmitter , receiver , antenna or dsp , nor do
they conduct the same functions ; depending on the
applications , some of the components or functions are
not needed . for instance , in applications where only
data reading is needed , such as object identification , a
receiver is not contained in the tag .
a reader may include a communication device to
enable the transmission of retrieved data to other
systems , such as a pc and/or other portable devices like
pda for storing , processing and display or further
communication with other media such as the internet ,
from which the information can be retrieved by remote
offices .
the dsp controls the reader , processes and
stores retrieved information , and provides interface with
other possible communication devices for transmission
or reception of information .
all rfid functions are carried by radio waves at
various frequencies depending on the application .
these include low frequency band from 100 - 500 khz ,
intermediate frequency band from 10 - 15 mhz , and high
frequency bands from 850 - 950 mhz and 2.45 - 5.8 ghz
( 8) .
higher frequency
provides a broader choice of different types of antenna
( 13 ) , which is the main component influencing the
overall size of the rfid system .
higher frequency also
results in smaller antennas and hence smaller systems
( 8) . in applications involving media with substantial
losses , such as the inside of the human body , lower
frequency bands may be needed to facilitate the
transmission and reception of signals .
typical rfid
systems have practical read distances ranging from about 5 cm up to a few meters depending on the chosen
frequency and antenna ( 8) .
personal health care and treatment for patients
depends largely on the ability to assess their health
condition .
accurate and reliable assessment of health
condition is essential not only for personal health , but
also for the planning of health maintenance , including
treatment .
it is , indeed , one of the most important tasks
in health care and preventive medicine .
current
personal health examination and monitoring typically
involves data collection using devices and equipment
connecting to the body via wires
this kind of health examination
and monitoring is normally carried out periodically in
certain durations and thus may not represent an
optimum or completely desirable solution for patients
who are constantly in need of examination and
monitoring , such as those having severe problems . as
will be seen in the following , the use of rfid allows
personal health examination and monitoring to be
wirelessly conducted either periodically or
continuously , which should benefit medicine
significantly , not only in cost reduction but also in
enhanced treatment of patients , as compared to what is
available now .
2 illustrates an idea of personal health
examination and monitoring using the rfid technique .
it shows tags attached to the body s skin and readers
located externally to the body .
different tags may be
used for different functions , such as reading the lung s
temperature , measuring the blood flow , measuring the
heart rate , etc . ) .
the readers may be placed at various
locations for instance , at different places in the house
of the person being monitored or in a medical facility .
the tag performs specifically instructed functions and
transmits the data collected to the readers .
the tags can
also receive instructions from the readers to perform
additional functions or to modify some preprogrammed
functions , such as changing the time or duration of the
data collection period .
the readers transfer the received
data to a router , a pc or pda which is connected to the
internet .
the pc or pda is also used to store the data
and can analyze and display them in a useful format , such as heart - beat waveforms , for immediate use by
someone such as an in - house care taker .
the
information transmitted to the internet can be viewed by
different authorized remote offices such as a doctor s
office , an emergency facility , etc .
the reader can also
be configured as a mobile handheld device to be
operated by a medical professional , such as a nurse , to
read the patient s tag data on his / her visit to the doctor s
office .
information from this portable reader is
transmitted wirelessly to a pc for data storage ,
processing , diagnosis and display .
additional functions
such as data analysis and display can be built into this
portable reader to provide patient s information directly
to the medical professional without using a pc .
it
should be noted that medical professionals from remote
offices can also control the readers to act upon the tags
through commands transmitted to the readers via the
internet , as needed for proper actions .
another idea for personal health examination and
monitoring using the wireless rfid technique has tags
implanted onto certain body s parts and readers attached
to the body s skin . as described in the other idea
illustrated in fig .
3 , the tags perform various functions
on the human s parts and transmit the collected
information to the readers , which are connected to the
router , pc , or pda wirelessly .
this idea requires the use
of implantable tags and is thus more difficult to be
implemented .
however , it is more effective for
examination and monitoring because tags have direct
contact with the parts to be monitored and can
accommodate a wider range of monitoring than those
used in fig .
wider range of
monitoring is possible since more implantable devices
can be placed at different places in the body for
monitoring while only a limited number of parts can be
monitored using tags attached to the body s skin .
a
potential problem of this approach is the transmission of collected data from the implanted tags to the readers
partly or completely across the body , whose media have
very high loss ( 15 ) and hence attenuate substantially the
transmitted signals that carry the collected data .
these
signals may thus be too weak to be detected by the
readers .
another potential problem is the radiation
exposure inside the human body which might cause
other health issues .
this potential health effect indeed
needs extensive study before the deployment of
implantable tags .
this concern , however , is expected to
be less than that with cell phones if the radio signal
reaching the implantable tags is limited to much less
than that currently used by cell phones , which is
possible since the rfid range is only a fraction of what
is used for cell phones .
implantable tags should not
allow radiation exposure exceeding a safe level , nor put
patients at further risk for other problems such as
infections after they are approved by an authorized
government health organization such as the food and
drug administration ( fda ) , which regulates medical
devices in the u.s.a .
in fact , in 2004 , the fda
approved an rfid tag for implantation in humans as a
means for accessing a person s health records ( 17 ) .
implantable rfid chips designed for animal tagging
are now being used in humans ( 10,11 ) .
most
importantly , this approach should be used only for those
in dire conditions , such as patients in the situation that
have no other choices for monitoring purposes , due to
the need of patients operation and implantation .
implantable tags require special materials and
packaging for them not to cause detrimental effects to
the body upon implantation .
development of such tags
requires significant interdisciplinary collaboration
between different disciplines such as electrical
engineering , biomedical engineering , and medicine .
4 shows an application of rfid in examining and monitoring many patients simultaneously in a
medical facility .
each patient carries tags that are either
attached externally to the body or implanted inside the
body and can be identified with a unique i d
preprogrammed into his / her tags .
this arrangement not
only improves the examination and monitoring of
patients significantly but also substantially reduces the
use of the financial and personal resources , thus
offering a very efficient and cost - effective health
assessment and monitoring system .
although periodic personal health inspections and
monitoring are important , there is a significant interest
and need not only to examine but also to monitor health
condition continuously .
continuous monitoring of the
health of patients provides instantaneous information on
the state of patients , effectively and significantly
improving health care , especially for elderly patients or
those experiencing deteriorating health .
this can
effectively help medical professionals to respond
quickly and more efficiently in emergencies , helping
prevent many potentially catastrophic events .
additionally , continuous monitoring supplies valuable ,
complete and accurate health condition of the patients .
this information will allow medical professionals to
conduct long - term studies of the patients health
condition and gain invaluable knowledge not only of
the patients but also of particular illnesses .
the
information can also make feasible more accurate
prediction of the patients future health conditions
thus allowing optimum treatment plans to be made .
continuous monitoring of personal health conditions
wirelessly from a remote location further offers more
convenience , lower cost and facilitates improved care
for patients , particularly out - patients or those living in
rural areas that are inconvenient and difficult to access
to health care facilities , or in emergency situations .
it
should be particularly noted that the constant
monitoring of patient well - being does not take into
account the non - measurable aspects such as pain , but
the use of the proposed rfid techniques should not
duplicate the function of a nurse working with the patient for pain . as with any technology and its intended applications ,
the cost of the proposed rfid systems can be
approximately broken down into two categories : rfid
system development cost and implementation cost . for
system development cost , existing tags and readers
developed for consumer applications
consumer tags and
readers are relatively inexpensive - a tag can cost as low
as a few u.s .
cents ( 18 ) and a reader can cost around
100 u.s . dollars ( 19 ) .
specially designed implantable tags are needed in
one of our proposed ideas and , for these tags , new
research and development needs to be conducted .
we
expect that the cost of these implantable tags , once fully
developed and mass produced , should be very similar to
that of the existing tags .
the primary cost of developing
the proposed rfid systems lies in the integration and
testing of the rfid systems for the particular
application of personal health examination and
monitoring .
all these development costs , however , are
born by biomedical - equipment development companies
just as in the development of any other medical
equipment .
this cost mainly
consists of the cost for purchasing readers and tags , cost
of maintenance and for the rfid sensor networks to be
installed in medical facilities as proposed in fig .
the prices
of the readers and tags , as discussed earlier , are
inexpensive .
the rfid system is basically a wireless
device and its maintenance is also relatively
inexpensive .
the installation cost for rfid sensors in
a medical facility is very similar to the installation of
other in - building wireless networks such as wi - fi
hotspots for internet access ( 20 ) .
nevertheless , presently , the
actual cost for implementing the proposed rfid sensor
network is not available , as this system is new and has
not been implemented for medical practices .
a major concern with the use of rfid technology in
the medical field is its ethical aspects .
for instance , it may be possible to gather
sensitive data about a patient without consent through
reading of the tags at a distance without his or her
knowledge .
furthermore , tags can be used for nonconsent
surveillance of the patient or other purposes .
additionally , patient data transmitted through air or the
internet can also possibly be stolen without consent .
these privacy issues , however , may be completely or
partially resolved with proper encryption methods . in
general ,
the ethical concerns can possibly be addressed
with better and more secured technologies for rfid .
we have presented several ideas using rfid
technology , one of the most significantly developed and
advanced technologies in the past decade , for personal
health examination and monitoring .
these ideas
demonstrate that health examination and monitoring of
patients can be done wirelessly at any time and any
place without interfering with the patients normal
activities and should therefore be attractive once
developed and deployed for the medical field .
it is
believed that implementing the rfid technology would
not only help reduce the enormous and significantly
growing medical costs in the u.s.a . , but also help
improve the health treatment capability as well as
enhance the understanding of long - term personal health
and illness .
it is hoped that these ideas will serve as a
way to illustrate potential use and advantages of the
rfid technology for medical applications .
potential
applications and advantages of the rfid as well as
other advanced electronic technologies for medicine are
enormous and if properly developed and implemented ,
can have tremendous impacts on the medical field . in
essence , they allow us to see farther , wider and
clearer into patients bodies , which is one of the
ultimate objectives of medical professionals and most
desirable capabilities in medical treatments .
applications of these technologies to medicine seem to
be limited only by our imagination with creativity ,
numerous applications of these technologies in the
medical field can be derived .
|
this paper presents several possible applications of the radio - frequency identification
( rfid ) technology for personal health examination and monitoring .
one application involves using
rfid sensors external to the human body , while another one uses both internal and external rfid
sensors .
another application involves simultaneous assessment and monitoring of many patients in a
hospital setting using networks of rfid sensors . all the assessment and monitoring
are done wirelessly ,
either continuously or periodically in any interval , in which the sensors collect information on human
parts such as the lungs or heart and transmit this information to a router , pc or pda device connected
to the internet , from which patient s condition can be diagnosed and viewed by authorized medical
professionals in remote locations .
instantaneous information allows medical professionals to intervene
properly and in a timely fashion to prevent possible catastrophic effects to patients .
the continuously
assessed and monitored information provides medical professionals with more complete and long - term
studies of patients .
the proposed ideas promise to result in not only enhancement of the health
treatment quality but also in significant reduction of medical expenditure .
|
INTRODUCTION
FUNDAMENTALS OF RFID
PERSONAL HEALTH EXAMINATION AND
MONITORING USING RFID TECHNIQUE
CONCLUSION
|
carpal tunnel syndrome ( cts ) is the most common entrapment neuropathy1,2,3 , caused by compression and traction of the median nerve at the level
of the carpal tunnel , which is a cylindrical , inelastic cavity , delimitated by the carpal
bones and transverse carpal ligament4 , 5.the compression results in impaired nerve
conduction , paresthesia , and pain , which worsen at night and often wake the patient6 , 7 .
the treatment for cts can be broadly divided into surgical and non - surgical approaches .
according to the findings of several important randomized controlled trials ,
carpal tunnel - release surgery is
effective in 7075% of patients , but is relatively invasive and can be accompanied by
complications10 .
furthermore ,
recurrence after surgery , although uncommon , can be difficult to treat11 . moreover , surgical decompression leaves 8% of patients in
a worse condition than they were previously12 , and persistent pain in the scar or proximal palm 5 years after
numerous non - surgical , less - invasive treatment options are available , including oral
medication , splinting , exercise , corticosteroid injections , and mobilization
interventions14,15,16,17 .
only a few complications result from non - surgical approaches in
those with mild - to - moderate cts .
thus , non - surgical treatment might be indicated for
patients with cts when surgery is not desired or for any other reason not immediately
indicated18 . to the best of our
knowledge , surgical decompression or carpal tunnel release
. however , there is currently limited evidence indicating any benefit from
splinting , exercise , and mobilization15 , 16 .
this study aimed at prospectively
assessing the effectiveness of nocturnal splinting by using clinical scores and ncs .
of the 66 consecutive outpatients to our neurological clinic , 41 patients who met the
inclusion criteria were finally enrolled ( fig .
all individuals provided informed consent to participate in this study , and the
local ethics committee of the hospital approved this study .
the inclusion criteria of the
report of the quality standards subcommittee of the american academy of neurology were
implemented19 , whereby all the patients
must meet at least one of the first 2 items and one of the remaining 5 items below to be
included in the study:19 , 20 ( 1 ) numbness in the median nerve territory or the whole
hand ; ( 2 ) pain or hypesthesiain the hand ; ( 3 ) awakened from sleep by numbness or pain
mid - night or early in the morning ; ( 4 ) numbness relieved by shaking the hand and aggravated
by flexing the wrist , or numbness becoming more severe in winter than in summer ; ( 5 )
weakness of the hand ; ( 6 ) atrophy of the thenar muscle ; and ( 7 ) positive phalen or tinel
sign . in this study , patients with the following conditions were excluded : ( 1 ) clinical or / and
electrophysiological findings suggesting ulnar nerve lesion , cervical radiculopathy ,
polyneuropathy , systematic diseases ( e.g. , diabetes mellitus , hypothyroidism , and rheumatoid
arthritis ) , and stroke20 ; ( 2 ) cts caused
by wrist trauma or deformity20 ; ( 3 ) prior
treatment for cts through any approach , either surgical or non - surgical ; and ( 4 ) severe
weakness or atrophy of the hand , and no response of compound muscle action potentials ( cmap )
elicited from the muscle of the abductor pollicis brevis ( mapb ) on ncs .
the follow - up was
completed in 20 patients with splinting ( fig .
1 ) .
this study had a prospective design recommended by american association of neuromuscular
and electrodiagnostic medicine ( aanem)21 .
all the subjects enrolled in this study underwent electrophysiological detection after oral
informed consent was obtained .
electrophysiological detections were completed on keypoint 4
( detec , denmark ) by one examiner .
while ncs
was performed , surface electrodes were used for stimulating or recording . while recording
cmap , the median and ulnar nerves were both stimulated at the wrist .
while recording the
sensory nerve action potentials ( snap ) , the median and ulnar nerves were also stimulated at
the wrist , and ring electrodes were placed at digits 2 and 5 , respectively .
in addition , for
comparing the difference in distal sensory latency ( dsl ) , the median and ulnar nerves were
stimulated at the wrist , recording the snap at digit 4 .
the following parameters were
analyzed : the distal motor latency ( dml ) from the wrist to mapb or abductor digiti minimi ;
the sensory conduction velocity ( scv ) from the wrist to digits 2 , 5 , and 4 ; and the dsl .
needle electromyographic detection was performed to observe abnormal spontaneous activities
only when the amplitude of the cmap of mapb reduced . considering stevens ( aanem ) and our previous study20 , 22 , the following
electrodiagnostic criteria for cts were used in this study : ( 1 ) scv ( wristdigit 2 ) < 40.0
m / s or scv ( wristdigit 4 ) < 43.5 m / s ; dml ( wrist to mapb ) 3.7 ms ( distance 5.56.5 cm ) ;
or dsl 0.4 ms ; and ( 2 ) normal sensory or motor conduction in the ipsilateral ulnar
nerve .
symptom severity scale ( sss ) and functional status scale ( fss ) were used to evaluate the
patients at each hospital visit23 .
sss
consists of 11 questions with multiple - choice responses , which includes 6 critical domains
for the evaluation of cts : pain , paresthesia , numbness , weakness , nocturnal symptoms , and
over - all functional status .
fss consists of 8 functional activities , including writing ,
buttoning of clothes , household chores , and bathing and dressing .
each question was scored 1
point ( mildest / no difficulty with the activity ) to 5 points ( most severe / unable to perform
the activity ) . the overall score was calculated as the mean of the scores for all the
individual items .
items that were left unanswered and if not applicable , they were not
included in the calculation of the overall score23 .
all the subjects were instructed to make stereoplasm splints of approximately 912 cm
57 cm size by themselves and bring them to us for assessment .
subsequently , they were asked
to wear each splint on the dorsal and palmar surface of the hand , centered at the distal
wrist crease , immobilizing the wrist in the neutral posture at bedtime , keeping the fingers
relatively free.the wrists were fixed to ensure that they were neither too tight nor too
loose . meanwhile ,
the subjects were instructed to avoid flexing their wrists during any
daytime activities such as washing clothes , riding a bicycle , working on a computer , and
carrying heavy grocery bags .
the follow - up was completed in 20 patients ( 31 wrists ) with splinting .
all statistical analyses were performed using statistical package for social sciences
( spss ) for version 17.0 ( spss china , shanghai ) .
one - sample t - test was used to compare the values with known population means ,
and paired - sample t - test was used to compare paired measurement data . in bivariate
correlations , data of normality was analyzed by person correlations , else by spearman
correlations .
the shapiro - wilk test was used to test the normality of the measurement data .
values of p<0.05 were considered statistically significant ; * represents p<0.05 , * *
p<0.01 and * * * p<0.001 .
of the 41 patients who met the inclusion criteria , 90% were female , and the mean age of all
patients was approximately 50 years old .
the clinical data are presented in table 1table 1.clinical data of the subjectsall 41 patients20 patients with splintingage ( years)2072 ( 50.2 12.0)2672 ( 52.5 12.6)illness duration ( years)1.1 1.71.5 2.3male / female ( wrists)4 ( 5)/37 ( 59)1 ( 1)/19 ( 30)bilateral hands affected ( cases)2411right hand affected ( cases)114left hand affected ( cases)6 ( 4 left - handed)5 ( 4 left - handed)numbness ( or pain ) at night ( cases)216tinel sign ( wrists)1811phalen sign ( wrists)2014 . in 20 patients ( 31 wrists ) with cts treated by splinting , sss ( 1.77 0.38 , 1.55 0.38 ;
t=5.956 , p=0.000 ) and fss ( 1.53 0.31 , 1.40 0.27 ;
t=5.452 , p=0.000 ) decreased .
in addition , dml ( 4.53 1.25 , 4.14 0.76 ;
t=2.431 , p=0.021 ) shortened and dsl ( 1.24 0.61 , 0.97 0.60 ;
t=2.978 , p=0.006 ) decreased significantly after splinting ( table 2table 2.comparison of clinical scores and ncs findings of splinting in 20 patients ( 31
wrists)parametersbefore splintingafter splintingclinical scoressss1.77 0.381.55 0.38fss1.53 0.311.40 0.27parameters of ncsdml ( ms)4.53 1.25 4.14 0.76scv of wrist digit 2 ( m / s)42.02 8.29 42.12 7.58scv of wrist digit 4 ( m / s)38.20 6.72 38.51 6.42dsl ( ms)1.24
0.610.97 0.60ncs : nerve conduction studies ; sss : symptom severity scale ; fss : functional status
scale ; dml : distal motor latency ; scv : sensory conduction velocity ; dsl : sensory
latency difference between median and ulnar nerve*p<0.05 , * * p<0.01 , * * * p<0.001 ) .
there was no improvement in the clinical scores in 9 patients ( 14 wrists ,
45.8% ) after splinting .
there were significant correlations between sss and dml
( r=0.420 , p=0.019 ) , scv of wrist digit 2 ( r=0.425 ,
p=0.017 ) , and scv of wrist digit 4 ( r= 0.519 , p=0.003 ) .
no correlations
were noted between sss and dsl ( p>0.05 ) , and between fss and all the parameters of ncs
( p>0.05 ) ( table 3table 3.correlation of clinical scores with ncs findings in 20 patients treated by
splinting dml scv of wristdigit2 scv of wristdigit4dslsss0.420*0.425*0.519 * 0.189fss0.1920.1750.3190.124values represent correlation coefficients*p<0.05 ) .
ncs : nerve conduction studies ; sss : symptom severity scale ; fss : functional status
scale ; dml : distal motor latency ; scv : sensory conduction velocity ; dsl : sensory
latency difference between median and ulnar nerve * p<0.05 , * * p<0.01 , * * * p<0.001 values represent correlation coefficients
in this study , 90% of patients with cts were female , and the mean age was approximately
50 years , which is consistent with the epidemiological data3 .
studies have shown that females with the highest body mass index
( bmi ) are more likely to develop cts24 , 25 .
a bmi 30 kg / m almost doubled
the risk of cts , and when bmi was assessed as a continuous variable , the hazard ratio
increased approximately linearly with increasing bmi24 .
splinting is the most common method among the non - surgical treatments available for
cts7 . in this study ,
a self - administered
questionnaire was used to assess the severity of symptoms and functional status in cts , as
questionnaires are reproducible , internally consistent , valid , and responsive to clinical
change23 .
the questionnaire for
clinical evaluation was subjective , but it can be semi - quantitative .
we recommend that the
questionnaire should be used at least in the clinical research on cts , so that the outcome
of each researcher is relatively comparable . in this study ,
both sss and fss were 3 points ,
which suggested that the condition in most patients was not severe . in a previous study
, 89%
of patients with severe cts experienced recurrence of the symptoms within 1 year after
conservative treatment , whereas only 60% of patients with mild cts experienced
recurrence26 .
therefore , the
conservative approach is more successful in patients with mild nerve lesion than with severe
cts .
conservative management with splinting should be initiated in patients with cts with
only mild symptoms12 .
splinting is an acceptable method for patients in the early phases of cts , as it is simple
and inexpensive and can be used at home . in this study ,
sss and fss decreased significantly ,
dml shortened , and dsl decreased . moreover , 54.2% of patients with cts were effective after
neutral wrist nocturnal splinting .
neutral wrist splinting can reduce the pressure on the
median nerve and increase blood flow , especially when the wrist is held in a flexion
position at night27 . in a review
assessing the effectiveness of conservative therapy for cts ,
it was reported that splinting
is an effective therapy especially when used for the whole day28 .
no improvement was noted in the clinical scores of almost half the patients in this study .
this could be because the splints made by the patients may not have immobilized the wrist .
additionally , patients low compliance for the complex course of splinting may have
influenced the therapeutic effect , although we emphasized that the patients should follow
our guidelines strictly .
splinting
reduces latency , suggesting that the intervention may alter the underlying
pathophysiological course of cts4 .
moreover , there was no correlation between sss and dsl , and between fss and the parameters
of ncs .
thus , there was a weak correlation between the clinical scores and ncs , which is
consistent with the findings of a previous study29 .
therefore , the use of both clinical scores and ncs may allow us to
evaluate the therapeutic effect of splinting on cts through different aspects .
overall , conservative approaches such as splinting have a negligible incidence of serious
complications and should be used more widely12 . providing patients the facts about their conditions and
nevertheless , patients
with cts who do not show satisfactory improvement with non - surgical treatment should be
offered surgery30 .
the therapeutic effect of combination splinting and other conservative treatments on cts
were not compared , and the number of patients enrolled in this study was small , which were
the main limitations in this study .
moreover , patients made the splints by themselves , which
may cause a variation in the therapeutic effect among individuals , although the splints that
they made were checked . in conclusion ,
neutral wrist nocturnal splinting is effective in at least the short term in
patients with cts .
there is a weak correlation between the clinical scores and ncs , which
suggests that both approaches should be used to assess the therapeutic effect of treatment
on cts .
|
[ purpose ] to prospectively assess the effectiveness of neutral wrist nocturnal splinting
in patients with carpal tunnel syndrome ( cts ) by using clinical scores and nerve
conduction studies ( ncs ) .
[ subjects and methods ] forty - one patients enrolled in the study
were clinically evaluated by a symptom severity scale ( sss ) and functional status scale
( fss ) , and were electrophysiologically evaluated by conventional ncs ; distal motor latency
( dml ) , sensory conduction velocity ( scv ) , and difference in sensory latency between the
median and ulnar nerves ( dsl ) were measured .
subjects were treated with wrist splinting .
patients who showed no improvement in symptoms were treated with other conservative
treatments , the remaining patients continued to wear splints .
sss , fss , and ncs were
evaluated after splinting as well .
[ results ] the follow - up was completed in 20 patients
( 31 wrists ) with splinting .
sss and fss decreased , the dml shortened and dsl decreased
significantly after splinting for 3.03 1.16 months .
there were significant correlations
between sss and dml , scv of wrist digit 2 , and scv of wrist digit 4 .
no correlations were
found between sss and dsl , and fss and the parameters of ncs .
[ conclusion ] neutral wrist
nocturnal splinting is effective in at least short term for cts patients .
there is a weak
correlation between clinical scores and ncs , which suggests that both approaches should be
used to effectively assess the therapeutic effect of cts treatment .
|
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
|
the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig .
1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .
1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig .
1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .
these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .
1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .
the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .
the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .
lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .
the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .
however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .
lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case
gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .
lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .
also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .
unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .
one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .
2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation
. bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .
the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .
ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .
they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .
rajeswaran et al . solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,
there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .
their crystal structures as well as the structure of the native ribosome are available through the protein data bank ( pdb ) .
these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .
two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .
theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .
although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,
no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .
the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .
these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .
the methods applied in the calculations are described in the first section of the paper .
we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds .
four structures of clindamycin are available in the protein data bank ( as of may 2011 ) .
three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .
the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome clindamycin complexes of deinococcus radiodurans ( pdb code 1jzx ) and haloarcula marismortui ( pdb code 1yjn ) .
therefore , based on the known x - ray structures of clindamycin ( fig .
2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .
black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .
black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .
, we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig .
2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .
the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .
the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .
no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called exp - opt , were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .
to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .
therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,
the solvent is described by a dielectric constant , which was set to 80 in our work .
the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .
the positions of these point charges were obtained from the x - ray structures of the ribosome
the coordinates of residues within 10 of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .
next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .
the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .
nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .
shielding constants were calculated using the b3lyp / aug - pcs-1 method and the giao routine [ 2630 ] .
the chemical shifts of the i - th nuclei were calculated as \documentclass[12pt]{minimal }
\usepackage{amsmath }
\usepackage{wasysym }
\usepackage{amsfonts }
\usepackage{amssymb }
\usepackage{amsbsy }
\usepackage{mathrsfs }
\usepackage{upgreek }
\setlength{\oddsidemargin}{-69pt }
\begin{document}$$ \delta ( i ) = \sigma_i(x ) - \sigma_i(c ) , $ $ \end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .
spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .
the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .
the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .
data were analyzed using gabedit , and the visualizations were carried out with vmd and xdrawchem .
the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15 k calculated in the gas phase , taking solvent effects into account , are depicted in table 1 .
the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table 2 .
3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381 zpe323.26323.66332.14332.40305.59305.85 g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20 e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988 zpe323.29323.53332.19331.99305.35305.37 g8.050.00.230.05.320.0
e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224 zpe323.87324.10332.77332.56305.92305.94 g6.580.00.430.05.400.0 e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514 zpe329.99329.71339.94339.56312.95312.74 g5.550.01.070.03.860.0table 2selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .
our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o h , n
h , c h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .
because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .
first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0 kcal / mol .
the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,
no single conformer is favored at 298.15 k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .
the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980 ) and the shortest ihb distance , r(c19o9
h39c15 ) ihb distance in the less stable a conformer is 2.370 , and r(c15h39 ) = 0.970 is the most important component of the conformer s stability .
the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .
n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .
the values of the starting dihedrals were different in the a and b conformers , as shown in fig . 2 .
full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .
the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig .
both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .
table 1 confirms that solvation has a relatively small effect on the energy difference between conformers .
the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6 kcal / mol according to the pcm and point - ion models , respectively.fig .
4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .
natural population analysis is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section
analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .
the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .
such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .
the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table 3 . the selected orbital interactions ( with a stabilizing effect of over 8 kcal / mol )
are presented in fig . 5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .
5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor
acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2
is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .
other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .
additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .
the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,
the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .
to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .
therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .
the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .
in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .
it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .
topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .
it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .
however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .
the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .
a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .
a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .
in other words , generally , the laplacian of is positive when is locally reduced , and negative if it is locally concentrated .
according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch oc in the a conformer and oh o = c in the b conformer , and unconventional h - bonds of type oh x ( x = o , cl ) in both conformers ( fig . 6 ) . the numerical values for the electron density ( bcp ) and
figure 7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .
the general shape of the bcp curve is that of an exponential decay , as expected ( see fig .
7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7 h41c172.3220.01550.0164cp6c19o9
7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .
0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions
. however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .
this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .
the experimental c nmr chemical shifts of clindamycin fall within the interval 1690 pm .
the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure
, we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .
as usual , the central part of the molecule is the most interesting part to consider for this purpose .
the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185 ppm , (c h ) close to 70 ppm .
(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6 ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9
intramolecular hydrogen bond more than the a conformer ( 185.1 ppm ) is stabilized by the c19o9
these chemical shifts have also been shown to depend strongly on the local properties of the electron density .
the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .
finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and
its chemical shift is lower for the b conformer than for the a conformer of clindamycin .
the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as references
the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .
some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7 hz when moving from the a to the b conformer
. however , more interesting is the jh37o9 sscc transmitted through the c = o h o ihb , which is equal to 5.3 hz in the b conformer .
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum
the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15 k calculated in the gas phase , taking solvent effects into account , are depicted in table 1 .
the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table 2 .
3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381 zpe323.26323.66332.14332.40305.59305.85 g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20 e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988 zpe323.29323.53332.19331.99305.35305.37 g8.050.00.230.05.320.0
e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224 zpe323.87324.10332.77332.56305.92305.94 g6.580.00.430.05.400.0 e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514 zpe329.99329.71339.94339.56312.95312.74 g5.550.01.070.03.860.0table 2selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .
our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o h , n
h , c h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .
because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .
first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0 kcal / mol .
the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,
no single conformer is favored at 298.15 k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .
the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980 ) and the shortest ihb distance , r(c19o9
h39c15 ) ihb distance in the less stable a conformer is 2.370 , and r(c15h39 ) = 0.970 is the most important component of the conformer s stability .
the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .
n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .
the values of the starting dihedrals were different in the a and b conformers , as shown in fig . 2 .
full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .
the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig .
both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .
table 1 confirms that solvation has a relatively small effect on the energy difference between conformers .
the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6 kcal / mol according to the pcm and point - ion models , respectively.fig .
4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method
in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .
natural population analysis is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section
analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .
the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .
such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .
the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table 3 .
the selected orbital interactions ( with a stabilizing effect of over 8 kcal / mol ) are presented in fig .
5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .
5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor
acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2
is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .
other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .
additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .
the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize
, nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .
therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .
the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .
it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .
topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .
it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .
however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .
the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .
a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .
a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .
in other words , generally , the laplacian of is positive when is locally reduced , and negative if it is locally concentrated .
according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch oc in the a conformer and oh o = c in the b conformer , and unconventional h - bonds of type oh x ( x = o , cl ) in both conformers ( fig .
the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table 4 .
figure 7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .
the general shape of the bcp curve is that of an exponential decay , as expected ( see fig .
7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7 h41c172.3220.01550.0164cp6c19o9 h39c152.3700.01460.0131cp7c17cl28
7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .
bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .
0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .
however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .
this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .
the experimental c nmr chemical shifts of clindamycin fall within the interval 1690 pm .
the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as
chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .
the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185 ppm , (c h ) close to 70 ppm .
(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6 ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9
intramolecular hydrogen bond more than the a conformer ( 185.1 ppm ) is stabilized by the c19o9
these chemical shifts have also been shown to depend strongly on the local properties of the electron density .
the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .
finally , it is clear that (o ) can be classified into two groups . in the first group ,
the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as references
the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .
some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7 hz when moving from the a to the b conformer .
however , more interesting is the jh37o9 sscc transmitted through the c = o h o ihb , which is equal to 5.3 hz in the b conformer . this value is large enough to be measured experimentally.fig .
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum
we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .
internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,
we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin spin coupling constants to the conformation of clindamycin .
the two most stable conformers of clindamycin exhibit c = oh o intramolecular hydrogen bonds . according to nbo and aim analyses ,
the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water .
|
lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens .
this group of drugs inhibits the activity of the bacterial ribosome by binding to the 23s rna of the large ribosomal subunit and blocking protein synthesis .
currently , three x - ray structures of the ribosome in complex with clindamycin are available in the protein data bank , which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin . in this work , we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23s rna .
we studied three lincosamide antibiotics : clindamycin , lincomycin , and pirlimycin at the b3lyp level with the 6 - 31 g * * basis set .
the focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties .
for both functional conformers , we applied natural bond orbital ( nbo ) analysis and the atoms in molecules ( aim ) theory , and calculated the nmr parameters .
based on the results obtained , we were able to show that the structure with the intramolecular hydrogen bond c = o h o is the most stable conformer of clindamycin .
the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , was found to be the dominant factor influencing the high stability of this conformer.figuremolecular graph of more stable conformer of clindamycin .
|
Introduction
Computational details
Results and discussion
Conformational analysis
Natural bond analysis
Atoms in Molecules analysis
NMR chemical shifts
Conclusions
|
the increasing prevalence of obesity represents a major public health concern in many countries , and healthy eating can play a predominant role in prevention strategies . in this context , it is important to identify effective nutritional strategies to help individuals achieve or maintain a healthy weight by making better food choices .
one of these strategies consists in the regulation of food labeling , which includes the use of nutrition claims intended to provide a quick and easy way to identify foods with nutritional features of interest [ 24 ] . in this
regard , satiating properties of foods induced by specific nutrients such as fibres or proteins are of particular interest [ 5 , 6 ] .
indeed , there is convincing evidence that appetite sensations are important determinants of long - term energy intake [ 7 , 8 ] and that satiety - enhancing foods may provide benefit to consumers by facilitating appetite control and compliance with weight - management efforts .
furthermore , the regulation of nutrition claims related to satiety is currently evaluated and developed by health organizations , which clearly recommend that such claims are supported by strong scientific evidence [ 10 , 11 ] .
while nutritional information related to the satiating effects of foods might be relevant for weight control , less is known about consumers ' perception and understanding of such nutrition claims and how these claims can affect perceived appetite sensations . indeed ,
nutritional information conveyed by food labeling can be difficult to understand for many consumers , particularly for older adults , adolescents , or those with less education [ 12 , 13 ] .
nutrition claims can also be misunderstood , which may lead to misleading inferences or unexpected eating behaviors .
for example , a recent study showed that a low - fat - labeled candy was perceived healthier than a regular - labeled candy , independent of caloric information .
healthy was considered less fattening and that the amount eaten was 35% higher than when the snack was described as
unhealthy . with regard to appetite sensations , it has been demonstrated that a food considered as unhealthy is perceived to contribute more to weight gain and satiation , while imposed healthy eating can increase the sensation of hunger .
beliefs and expectations about the satiating power of a food can also affect perceived fullness , and this effect can persist as long as three hours after the meal .
another study showed that a meal presented as highly satiating , without having physiological satiating properties , can increase satiety potential more than a meal having physiological satiating properties , but presented as regular .
this suggests that the psychological effect of a satiety - related claim can be as effective as a physiological effect related to intrinsic satiety properties and highlights the need to better understand this possible psychological effect .
individual factors such as sex , weight , or restrained eating could also influence the perception of foods and modulate behavioral change .
as the perception of food can influence appetite sensations , it could be hypothesized that individual factors can also modulate the perception of appetite sensations .
since women generally consider themselves as being more knowledgeable about nutrition and having better eating habits and tend to pay more attention to nutritional information , it could be suggested that women would be more readily influenced by nutrition claims .
overweight / obese individuals and restrained eaters might also be more sensitive to nutrition claims related to healthy eating or weight loss since they can use it as a tool for weight management .
interestingly , one study showed that food products carrying satiety - related claims were generally not seen as a magic bullet to weight loss , but that restrained eaters were more prone to overinterpretation of these claims , that is to interpret it as directly delivering weight loss .
individual factors can thus affect perception of foods and perceived appetite sensations , but they can also act in a physiological way . indeed ,
some studies have associated higher levels of restraint with an orexigenic hormonal profile [ 24 , 25 ] , suggesting that restrained individuals could more easily feel hunger .
it has also been proposed that overweight / obese individuals are less connected to their internal signals than normal - weight individuals , making the detection of hunger and satiation more complex .
sex , weight , and restrained eating are thus variables of interest when studying appetite sensations , alone or in combination with nutrition claims . in summary
, current literature suggests that nutrition claims may influence perception of foods and appetite sensations and that this influence could be modulated by individual factors such as sex , weight , or restrained eating . in that context
, there is an urgent need to better understand the way nutrition claims should be used , taking into account possible side effects , and evaluate the relevance of some claims in a public health nutrition context .
this study addresses this issue by evaluating combined and separated effects of nutrition claims and individual factors on appetite sensations .
the main purpose of this study was to evaluate the impact of nutrition claims and individual factors on perceived appetite sensations .
the specific objectives were ( 1 ) to evaluate the effects of healthy , diet , or
claims on appetite sensations , ( 2 ) to evaluate if sex , weight , or restrained eating influence appetite sensations , and ( 3 ) to investigate if sex , weight , and restrained eating can modulate the effects of nutrition claims on appetite sensations . note that the hedonic
condition refers to ingredients generally considered as highly palatable and that the diet condition refers to satiating properties ( see section 2.1 for a complete description ) .
our corresponding hypotheses are that ( 1 ) the snack in the healthy condition will be less satiating than in the diet or hedonic conditions , ( 2 ) individual factors such as sex , weight , and restrained eating will significantly influence appetite sensations , and ( 3 ) the satiating effects of the diet and hedonic conditions will be more important in overweight / obese restrained women .
the study was conducted among 164 men and 188 women ( aged 1865 ) from quebec city , canada , who were invited to taste and rate an oatmeal - raisin snack in a blinded and ad libitum context .
exclusion criteria were personal condition or history of diseases that could affect food intake ( unstable weight in the last two months , pregnancy , breastfeeding , eating disorders , type 1 or 2 diabetes , and uncontrolled hypo- or hyperthyroidism ) , use of medication that might interfere with appetite or food taste ( antidepressants , antipsychotic , or corticosteroids ) , and food allergies for safety reasons .
participants were randomly assigned to an experimental condition in a three ( healthy versus diet versus hedonic ) by two ( restrained or not restrained ) by two ( normal - weight or overweight / obese ) by two ( men versus women ) factorial design , which means that the recruitment was based on these characteristics in order to obtain a balanced number of participants in each subgroup .
this design results in eight subgroups based on individual characteristics combined with the three experimental conditions tested , which leads to a total of 24 combinations for the analysis . the factorial distribution of participants is presented in table 1 .
initial randomization was performed according to self - reported weight and height and restraint score , but participants were reassigned according to their measured data if needed .
condition emphasized the favorable nutritional characteristics of the snack : the snack product that you have to taste today is a new high - fiber oatmeal snack made with healthy ingredients .
you have certainly heard that whole oatmeal is good for your health because it contains soluble fibers .
so , this new oatmeal snack is high in soluble fibers , as well as low in saturated fat and free from trans - fat .
the diet condition focused more on beneficial satiating properties for weight management : the food product that you have to taste today is a new healthy high - fiber snack .
you must have probably heard that whole - grain foods , like those that contain oat soluble fibers , are composed of complex carbohydrates which are digested slowly and which can delay the appearance of hunger .
this new healthy snack has been especially designed to satiate , because it is a source of fibers , which make it an interesting choice for any persons concerned to reach and to maintain a healthy weight . finally ,
condition underlined the sweet and pleasant taste of the snack using hedonic food words :
( these are ) new gourmet cookies made with fresh butter and old - fashioned brown sugar .
so , these new cookies are a great treat with a pleasant , sweet taste .
cookie was never mentioned in the two first conditions , and that we specifically chose to use an oatmeal - raisin rather than a chocolate snack in order to facilitate the blinded context of the experiment , as oatmeal and raisins can be considered as healthy ingredients or not depending on the food containing it .
indeed , this psychological manipulation was previously shown to be effective in changing the healthiness perception of food [ 16 , 28 ] . in spite of the description ,
the study was approved by the institutional review board of universit laval ( # 2009 - 117/26 - 05 - 2009 ) and was registered in the clinical-trials.gov registry ( nct01141140 ) .
participants came to the institute of nutrition and functional foods ( universit laval ) between 8:30 a.m. and 7 p.m. at a single occasion .
they had to be in a fasting state for at least two hours prior to their visit .
they were given a food description according to the experimental condition they were assigned and then had 10 minutes to taste the snack ad libitum in a private room .
participants stayed for approximately two hours after the taste - rating task to complete appetite sensations scales , questionnaires , and anthropometric measurements .
they were told the real goal of the study only at the end of the visit and were free to withdraw their participation at that moment .
appetite sensations are defined as follows : hunger refers to the physiological recognition of a need to eat , while desire to eat reflects the motivation to eat , fullness is a physical feeling that could be related to the degree of stomach filling , and satiety refers to the processes that inhibit further intake after an eating occasion has ended .
appetite sensations were evaluated with unipolar 150 mm visual analog scales ( vas ) ( adapted from hill and blundell ) .
these scales are anchored at the two ends with the extremes of the subjective feeling evaluated .
vas have been widely used in the past to evaluate appetite sensations and are considered reliable for single meal protocols [ 31 , 33 ] .
participants rated their appetite sensations before the test meal ( time = 10 ) as well as immediately after ( time = 0 ) and 20 , 40 , and 60 minutes later . the first measure ( time = 10 ) was taken immediately before the description of the snack .
four appetite sensations were measured : desire to eat ( how strong is your desire to eat ? ; very low desire to very high desire ) , hunger ( how hungry do you feel ? ; not at all hungry to extremely hungry ) , fullness ( how full do you feel ? ; not at all full to extremely full ) , and prospective food consumption ( pfc ) ( how much food do you think you could eat ? ; none at all to a large amount ) .
the vas values were calculated by measuring the distance in mm with a ruler from the left end of the scale to the mark drawn by the participant with a precision of 0.5 mm .
one - hour area under the curve ( auc ) was calculated from time zero to time 60 with the trapezoid method . as recommended by blundell et al .
the auc allows an overall view of the appetite sensation response , while the raw vas data allows comparisons at each time .
appreciation of the snack ( in general , how much do you appreciate the food you just tasted ?
) was evaluated with a unipolar 150 mm vas labeled from bottom to top with not at all , moderately , and enormously . a validated food frequency questionnaire ( ffq )
the restraint scale allowed the distinction between restrained eaters ( score 12 for men and 15 for women ) and unrestrained eaters ( score < 12 for men and < 15 for women ) .
the validity of the restraint scale for the measurement of restrained eating has been previously reported [ 3739 ] .
the food pleasure scale ( fps ) was also used to evaluate the pleasure associated with eating .
body mass index ( bmi ) was calculated from weight and height measured after the completion of all questionnaires .
participants were classified as normal - weight if they had a bmi under or equal to 25 kg / m and as overweight / obese if they had a bmi higher than 25 kg / m .
energy needs were estimated from the mean of ffq data and the harris - benedict formula , as previously performed in our research institute . with a mean cohen 's d estimate of 0.35 ,
power analyses for anovas testing main effects and interactions indicated that a sample size of 180 males and 180 females would allow the detection of significant differences with an alpha level of 0.02 and a power ( 1 error probability ) of 0.90 .
one - way analyses of variance ( anovas ) were used to assess differences in initial characteristics between experimental conditions . in line with the factorial design of the study , factorial repeated measures anovas were performed with mixed procedures for the analysis of the appetite vas ( desire to eat , hunger , fullness , and pfc ) , with time , experimental condition , sex , bmi , and restrained eating used as independent variables and tested in interactions .
age and initial appetite sensations ( time = 10 ) were included in the models as potential confounding factors , as they may affect appetite sensations [ 29 , 44 ] .
adjustments for energy needs and total amount eaten were also made to palliate to the fact that the amount eaten was ad libitum and therefore not adjusted for energy requirements ( as the study also aimed to measure the effect of claims on food intake ) .
time of the day was also added as a covariate in case the two hours fasting period was not long enough to standardize initial appetite sensations .
finally , appreciation of the snack and pleasure associated with eating were added as covariates as hedonic eating indicators .
similar procedures without repeated measures were used for the analysis of auc for all appetite sensations .
the slice option was used to investigate significant experimental conditions effects in each of the eight predetermined subgroups ( based on sex , weight , and restrained eating ) .
the structure of covariance matrix was taken into consideration for all statistical models to ensure an optimal fit to the data .
a p value < 0.05 was considered statistically significant for all tests , with bonferroni corrections made for all multiple comparisons .
all analyses were performed with statistical analysis software version 9.3 ( sas institute , cary , nc , usa ) .
a total of 352 participants were recruited and completed the study . from these participants , 11 ( 4 men and 7 women ) were excluded from the analysis because of missing values in covariates .
the remaining total of 341 participants ( 160 men and 181 women ) were thus included in the present study .
one missing value was also observed for the initial pfc sensation ( time = 10 ) , which led to the exclusion of one participant from the pfc analysis because of the necessity of this covariate in the statistical model .
no significant differences were found between conditions for any initial characteristic . as explained previously in section 2.6 ,
all the following results are adjusted for age , time of the day , energy needs , amount eaten , and initial appetite sensations .
note that only initial appetite sensations and total amount eaten contributed significantly to the models .
no main experimental condition effect was observed for any of the four appetite sensations evaluated , when expressed in both vas means ( f(2,306 ) = 1.14 for desire to eat , f(2,306 ) = 1.54 for hunger , f(2,306 ) = 0.66 for fullness , and f(2,305 ) = 0.68 for pfc ; p = 1.00 ) and auc ( f(2,306 ) = 2.99 for desire to eat , f(2,306 ) = 3.20 for hunger , f(2,306 ) = 1.06 for fullness , and f(2,301 ) = 0.4440 for pfc ; p = 1.00 ) .
mean values for all vas at each time point ( 10 , 0 , 20 , 40 , and 60 minutes ) as well as their related auc are presented in table 3 for each experimental condition .
appetite sensations were affected neither by bmi ( p = 1.00 for all vas values and auc ) , nor by restrained eating ( p > 0.96 and p > 0.33 for all vas values and auc , resp . ) .
however , a main effect of sex was noted for all appetite sensations , with men reporting higher vas values than women for desire to eat , hunger , and pfc ( p < 0.004 ) and lower vas values than women for fullness ( p = 0.008 ) . as presented in table 4 ,
cohen 's d effect sizes ( ess ) ranged from 0.08 to 0.52 , which represents small to moderate effects .
an effect size comprised between 0.2 and 0.49 represents a small effect size , between 0.5 and 0.79 , a moderate effect size , and 0.8 a large effect size .
no sex differences were observed for desire to eat and hunger at baseline , but fullness was higher and pfc lower in women in comparison to men .
note that men had significantly higher energy needs and ate significantly more than women ( data not shown ) .
as previously explained in section 2.6 , adjustments were made for these covariates for all results presented .
significant effects of nutrition claims on appetite sensations were observed in specific subgroups ( based on combinations of individual factors ) . among normal - weight unrestrained women , lower auc values for desire to eat ( es = 0.76 ) ,
hunger ( es = 1.03 ) , and pfc ( es = 0.72 ) were observed in the diet condition compared with the healthy condition ( table 5 ) .
however , significant differences between these two conditions were not observed at each time point . some differences between diet
and hedonic conditions and also between healthy and hedonic conditions were also noted among normal - weight unrestrained women .
in fact , hunger was lower in the diet condition than in the hedonic condition at time 0 and was higher in the healthy condition than in the hedonic condition at time 20 ( table 5 ) . among overweight / obese unrestrained men , a higher auc value for fullness
was observed in the diet condition than in the healthy and hedonic conditions ( ess of 1.19 and 0.99 , resp . ) ( table 6 ) .
no significant differences were found between other subgroups for any of the four appetite sensations .
the aim of this study was to evaluate the impact of food labeling and individual factors on perceived appetite sensations and more precisely to ( 1 ) evaluate the effects of healthy , diet , or
claims on appetite sensations , ( 2 ) evaluate if sex , weight , or restrained eating influence appetite sensations , and ( 3 ) investigate if sex , weight , and restrained eating can modulate the effects of nutrition claims on appetite sensations . associated results are that ( 1 ) claims did not affect appetite sensations , ( 2 ) sex had a significant influence on appetite sensations , but not weight and restrained eating , and ( 3 ) a significant interaction between claims and individual factors has been observed in normal - weight unrestrained women and overweight / obese unrestrained men .
as previously reported , the psychological manipulation was effective in changing the healthy perception of the snack , as the healthy condition was considered to be healthier than the diet condition , itself considered healthier than the
contrary to our first hypothesis , the three experimental conditions resulted in similar perceived appetite sensations , suggesting that a snack labeled with healthy characteristics is not necessarily perceived as less satiating than a snack labeled with satiating or hedonic properties .
this finding is in contradiction with finkelstein and fishbach who demonstrated that participants felt hungrier after eating a
healthy chocolate - raspberry bar than after eating the same bar described as yummy .
however , few details are given in this study about the composition of experimental groups , and initial hunger sensation was not measured and then not used as a covariate , as performed in the present study . as our statistical models showed that initial sensations are important covariates to consider in the evaluation of appetite sensations , differences in statistical adjustments between studies may explain the different results obtained .
our finding is also in contradiction with arguin et al . who demonstrated that highlighting the satiating properties of a meal increases satiety potential against a control condition .
the different types of food ( i.e. , snack versus meal ) used and the absence of a control condition ( i.e. , without any description ) in our study may explain why we did not observe such a difference .
it could also be explained by the fact that our diet condition was not only targeting satiating properties , but also weight control . as we did not measure the expected satiating power ( as performed previously for perceived healthiness )
in any case , the absence of an effect of nutrition claims on appetite sensations in our study implies neither a contraindication nor incentive to their use in a public health context . as hypothesized , sex had a significant influence on the perception of appetite sensations , regardless of nutrition claims , energy needs , and amount eaten ( men had significantly higher energy needs and ate significantly more than women ) .
indeed , men reported higher levels of desire to eat , hunger , and pfc and lower levels of fullness .
these results suggest that women could feel more easily satiated than men and could be less tempted to prospectively eat more food .
some studies have already reported similar findings [ 7 , 44 ] , while other studies found no sex differences or mixed results depending on the appetite sensation observed and experimental conditions ( ad libitum versus controlled ) .
the fact that baseline fullness and pfc were , respectively , higher and lower in women than in men is also of interest .
it suggests that these two sensations could differ between men and women even before food consumption .
sex differences in the perception of appetite sensations could be explained by differences in hormonal profiles .
these observations involve that public health nutrition interventions may not have the same effects in men and women and that individual or group interventions should be adapted in consequence . according to our findings
, women could be more responsive to an intuitive or mindful eating approach than men , that is , to eat in response to physical sensations such as hunger and satiety signals [ 4951 ] .
health at every size ( haes ) programs , endorsed by the association for size diversity and health , are an example of interventions addressing the recognition of appetite sensations .
other programs are also sex - specific , focusing more on body image for women and energy and body functions for men .
current literature shows that obese individuals have lower fasting ghrelin levels and that their ghrelin response to food intake is less pronounced than in normal - weight individuals .
this is in line with the proposition that overweight or obese individuals could be less sensitive to their internal appetite sensations .
however , we found no difference between normal - weight and overweight / obese individuals , which has been found in past literature .
another study proposes that hormonal response to food intake could be dependent of a bmi by sex interaction , suggesting that the study of bmi alone is not sufficient .
appetite - regulating hormones are not necessarily related to subjective appetite sensations , which could explain why we obtained different results as we only measured perceived appetite sensations and not appetite - regulating hormones .
also , we did not differentiate between overweight and obese individuals , our sample including more overweight ( n = 124 ) than obese ( n = 51 ) individuals . no main effect of restraint status on appetite sensations was observed .
this was unexpected as restrained eating has been previously associated with higher levels of perceived hunger .
it has also been already associated with higher levels of ghrelin [ 24 , 57 ] .
hooper et al . also demonstrated that a high degree of weight cycling was associated with an appetite - stimulating hormonal profile .
as mentioned previously , however , appetite - regulating hormones are not necessarily well related to subjective appetite sensations . on the other hand , other studies found no effect of restrained eating on hunger [ 44 , 55 ] .
restrained eating can be measured either by the restraint scale ( as performed in the present study ) or by the restraint scales included in the three - factor eating questionnaire ( tfeq ) or the dutch eating behavior questionnaire ( debq ) .
the restraint scale combines the measure of concern with dieting and weight fluctuation , whereas both tfeq and debq measure attempt to restrict eating , independent of weight fluctuation .
the choice of the questionnaire used to classify participants as restrained or not could then explain differences in results obtained between studies . as proposed earlier , it is also possible that restrained eating needs to be considered in interaction with sex or bmi to reveal significant effects .
for example , martins et al . showed that restrained eating in women could be associated with lower levels of hunger and higher levels of fullness .
even if weight status and restrained eating are important factors to consider in individual consultation , our results suggest that they do not necessarily need to be taken into account for the evaluation of appetite sensations . to our knowledge , this is the first study to explore the influence of nutrition claims on appetite sensations according to a combination of individual factors , namely , sex , weight , and restrained eating .
our hypothesis that overweight / obese restrained women would be more easily satiated in the diet and hedonic conditions in comparison with the healthy condition was not confirmed as a satiating effect of the diet condition was rather observed in normal - weight unrestrained women and overweight / obese unrestrained men , while no satiating effect of the hedonic condition was noted .
being more easily influenced by satiety - related claims could either indicate a greater susceptibility to external factors , or in contrary a better predisposition for the recognition of internal satiety signals .
an effect of nutrition claims was only noticed in individuals presenting a low level of cognitive restraint , which has already been associated with a lower susceptibility to external influences [ 20 , 56 ] . it is then more likely that the diet
this observation highlights the relevance of considering individual factors in interaction when studying appetite sensations .
our results also suggest that nutrition claims emphasizing satiating properties could be more effective in reducing perceived hunger than claims focusing on hedonic properties .
more precisely , it suggests a potential benefit of satiety - related claims , at least in certain subgroups of individuals .
first , it might be argued that the healthy and diet labels were not congruent with the food tasted , which could explain the absence of a main effect on appetite sensations .
however , this argument is not supported as the manipulation effectively changed the perception of food . on the other hand ,
no control condition ( i.e. , without any description ) was used , and the diet condition was not only targeting satiating properties but also weight management .
also , the amount eaten was not standardized , which however allowed participants to eat according to their physiological and psychological needs .
the use of many multiple comparisons and bonferroni corrections could have led to a lack of power , but it can also be argued that the probability of committing type 1 errors is really small .
this study also has strengths , such as a high number of participants recruited on the basis of a factorial design .
this is also the first study to evaluate the role played by specific combinations of individual factors in the study of the effect of nutrition claims on appetite sensations .
we investigated the impact of nutrition claims and individual factors on the perception of appetite sensations .
women felt more satiated then men regardless of nutrition claims , while bmi and restrained eating alone did not influence appetite sensations .
no adverse effect of nutrition claims on appetite sensations was observed , which suggests no contraindication to their use .
positive effects of satiety - related claims have been observed only in specific subgroups of individual . in a real - world setting , foods labeled with satiety - related claims would possess demonstrated physiological satiating properties and therefore would probably induce a physiological effect ( as opposed to a psychological effect in this study ) . as enhanced satiety may benefit dietary control or weight - management , it may thus be useful to inform consumers about these specific properties . to conclude , nutrition claims do not generally affect appetite sensations and the evaluation of their relevance
sex is an important individual factor to consider when studying appetite sensations , while weight and restrained eating should be considered in interaction for a better understanding of their effects .
more studies are needed to assess a significant and beneficial effect of nutrition claims on appetite sensations and eating behavior , and to better understand the contribution of individual factors .
|
nutrition claims may help people to adopt healthier eating habits , but little is known about the potential cognitive effects of such claims on appetite sensations .
the main purpose of this study was to evaluate the impact of nutrition claims and individual factors on perceived appetite sensations . according to a three ( healthy versus diet
( i.e. , satiating ) versus hedonic ) by two ( restrained or not restrained ) by two ( normal - weight or overweight / obese ) by two ( men versus women ) factorial design , 164 males and 188 females aged 1865 were invited to taste an oatmeal - raisin snack in a blinded and ad libitum context .
visual analog scales ( 150 mm ) were used to evaluate appetite sensations before and over 1 h after consumption period .
bmi and restraint scale were used to categorize participants according to their weight and restraint status .
no main condition effect was observed for any of the four appetite sensations .
however , subgroups analysis revealed significant differences among specific subgroups .
a main effect of sex was also observed for all appetite sensations with men reporting higher levels of desire to eat , hunger and prospective food consumption , and lower levels of fullness than women .
these findings highlight the importance of considering individual characteristics in interaction when studying appetite sensations .
|
1. Introduction
2. Material and Methods
3. Results
4. Discussion
5. Conclusions
|
the prevalence of diabetes in the u.s . has increased ( 1 ) , and cardiovascular ( cv ) complications remain the major cause of morbidity and mortality in persons with type 2 diabetes , contributing substantially to increased health care costs ( 2 ) . as individuals attempt to manage their diabetes , there is increasing recognition that comorbid behavioral problems , including both depressive symptoms and stress , are associated with poor glycemic control , poor lifestyle behaviors , and increased health services utilization ( 310 ) .
we ( 11 ) and others ( 3,4 ) have shown that these behavioral challenges are associated with inadequate medication adherence , which may be associated with adverse outcomes .
similarly , poor meta - bolic control may worsen depressive symptoms ( 5 ) , and the relationship appears to be bidirectional ( 8) . the prevalence of these comorbidities and their potential to worsen disease management have led some to develop interventions designed to address comorbid depressive symptoms or stress in persons with diabetes ( 1214 ) .
while many studies have documented the cross - sectional presence of these comorbidities and the effect on glycemic control in subjects with diabetes , only a limited number of studies have examined the potential impact on cv outcomes .
most of these studies have been limited to patients with depression recruited from established health care settings , and the true population - level impact is unknown .
likewise , there has not been adequate comparison of individuals with and without diabetes who have concurrent depressive symptoms and/or elevated levels of perceived stress to examine the unique consequences of behavioral and medical comorbidity .
the objective of the present report was to examine the association between the presence of depressive symptoms and/or increased levels of perceived stress , determined at baseline , and risk of incident cv events over 5 years of follow - up in a major national cohort study that used population - based sampling methods , collecting data in the home both in subjects with and without diabetes .
the description of the population for this analysis has previously been reported ( 15 ) . in short ,
the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the stroke belt
it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s .
( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites .
the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults .
the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months .
the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) .
the study also included an in - home visit by a trained health professional for data collection .
participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation .
the present analysis included all members of the cohort who had complete follow - up data .
a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview .
height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) .
all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) .
atrial fibrillation was determined to be present if there was a self - reported history ( asked as has a doctor or other health professional ever told you that you had atrial fibrillation ? ) or if characteristic findings were present on the ecg as defined in the protocol .
left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol .
hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) .
diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin each obtained during the home visit .
the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes .
the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) .
the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) .
these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms .
subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms .
perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress .
it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded .
subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores .
the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death .
living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality .
a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease .
medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke ,
each event was adjudicated via medical record review by a neurologist - led medical review team .
stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification .
after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) .
specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) .
participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index .
for information surrounding the circumstances of participant death , proxies or next of kin were interviewed . additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports
were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) .
data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes .
additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) .
also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) .
smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker .
the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 .
follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up .
the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) . the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress .
age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models .
these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) .
the description of the population for this analysis has previously been reported ( 15 ) . in short ,
the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the stroke belt
it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s .
( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites .
the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults .
the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months .
the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) .
the study also included an in - home visit by a trained health professional for data collection .
participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation .
the present analysis included all members of the cohort who had complete follow - up data .
a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview .
height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) .
all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) .
atrial fibrillation was determined to be present if there was a self - reported history ( asked as has a doctor or other health professional ever told you that you had atrial fibrillation ? ) or if characteristic findings were present on the ecg as defined in the protocol .
left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol .
hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) .
diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin each obtained during the home visit .
the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes .
the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) .
the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) .
these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms .
subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms .
perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress .
it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded .
subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores .
the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death .
living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality .
a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease . for each positive response , the date and time of each event were recorded .
medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke ,
each event was adjudicated via medical record review by a neurologist - led medical review team .
stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification .
after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) .
specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) .
participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index . for information surrounding the circumstances of participant death , proxies or next of kin
additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) .
data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes .
additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) .
also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) .
smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker .
the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 .
follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up .
the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) .
the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress .
age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and
subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models .
these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) .
the current study included a total of 22,003 subjects , of whom 42% were black , 58% female , and 56% living in the southeastern u.s . stroke belt . among all subjects at baseline , 18.6% subjects ( n = 4,090 ) had diabetes , 10% ( n = 2,202 ) reported increased depressive symptoms , and 28% ( n = 6,132 ) reported elevated levels of stress .
subjects with diabetes were more likely to report either elevated depressive symptoms or stress or both than were subjects without diabetes ( 36.8% vs. 29.5% ; p < 0.001 ) .
detailed demographic characteristics for the study group are given in table 1 , separated into those with and without diabetes and stratified by the presence of behavioral comorbidities . among subjects with diabetes ,
those reporting either increased depressive symptoms or stress or the combination were more likely to be women , black , live in the stroke belt , and have limited income .
subjects reporting either elevated depressive symptoms or stress or both had a higher prevalence of elevated baseline hs - crp values . across each category of behavioral comorbidity ( i.e. , none , either elevated depressive symptoms or stress , both elevated ) , subjects with diabetes had a higher prevalence of elevated baseline hs - crp values , higher mean systolic bp , and greater prevalence of statin use than those without diabetes .
baseline characteristics of regards participants with or without diabetes , by depressive symptom / stress category * p value for comparison of subjects with diabetes vs. subjects with no diabetes .
table 2 provides age - adjusted incidence rates per 1,000 person - years of follow - up for each of the three independent cv outcomes , broken out by the presence of behavioral comorbidities in those with and without diabetes . with respect to individual cv outcomes ,
the pattern of age - adjusted incidence rates was approximately twofold higher in those with diabetes than in those without diabetes across the range of comorbid behavioral illness . among individuals with diabetes and one or both behavioral comorbidities , age - adjusted incidence rates were highest for acute chd , followed by cv death , and stroke . among those without diabetes but with one or more behavioral comorbidities , incidence rates were again highest for acute chd but were followed by stroke and cv death .
event numbers , age - adjusted incidence rates per 1,000 person - years , and crude and adjusted hrs for stroke , acute chd , and cv disease death for individuals with stress or depressive symptoms and with stress and depressive symptoms compared with those with neither , for individuals with and without diabetes crude model adjusted for depressive symptom and stress category .
model 1 adjusted for crude model plus demographic factors ( race , sex , age , region ) .
model 2 adjusted for crude model plus demographic factors plus social and economic factors ( income , education , health insurance ) .
full model adjusted for crude model plus demographic factors , social and economic factors , and risk factors ( bmi , total cholesterol , hs - crp , atrial fibrillation , left ventricular hypertrophy , systolic blood pressure , statin use , current cigarette smoking ) .
table 2 also provides the results of a series of crude and progressively adjusted hazard ratio ( hr ) models for each of the three independent cv outcomes , demonstrating the progressive impact of comorbidity on acute chd and cv death among subjects with diabetes , relative to those without diabetes , even in fully adjusted models that accounted for demographic , socioeconomic , and cv risk factors . unlike the pattern for acute chd and cv death ,
the magnitude of the hrs for stroke was very similar in subjects with one versus both behavioral comorbidities and diabetes .
figure 1 illustrates this significant and progressive pattern of worsening fully adjusted hrs for cv death among those with behavioral comorbidities , relative to those with no behavioral comorbidities , in subjects with diabetes and in comparison with those who do not have diabetes . among individuals with diabetes , the presence of a single behavioral comorbidity either increased depressive symptoms or stress increased the risk of cv death by 53% relative to individuals with diabetes but without either behavioral comorbidity . among individuals with diabetes , the presence of both behavioral comorbidities increased depressive symptoms and stress
increased the risk of cv death by 115% relative to individuals with neither behavioral comorbidity , even after adjustment for a wide range of demographic and cv risk factors .
while there was a pattern of increasing risk of cv death among those with one or both behavioral comorbidities ( vs. neither ) in subjects without diabetes , the differences were substantially smaller ( 12% and 27% increases , respectively ) and were not statistically significant .
consequences of comorbid diabetes and elevated depressive symptoms and/or stress on cv death during follow - up among participants in the regards study .
this article joins a growing body of literature that demonstrates a compelling pattern of augmented risk for cv events or cv death associated with comorbid behavioral illness among individuals with diabetes that far exceeds that observed in individuals with these same illnesses but without diabetes .
while several studies have shown that formally diagnosed depression and/or depressive symptoms in subjects who are patients in an existing health care system are associated with incident chd , this is among the first studies to compare the impact of these behavioral comorbidities on cv outcomes and mortality in a population - based sample of subjects with versus without diabetes in the same study .
it is the first study to compare the effects of both depressive symptoms and stress together and separately in subjects with versus without diabetes , obtained by population - based sampling rather than recruitment in established health care settings .
further , the regards study has a large sample size ; includes oversampling of african americans in the stroke belt region , allowing important racial comparisons ; and also includes rich data obtained in the subject s home environment .
the study is novel in that , as a community - sampled study , it provides more precise estimates of the population burden and racial disparities associated with these comorbidities and their cv consequences than other studies in which patients were recruited in health care settings .
the study provides a much larger sample of african americans and illustrates the greater prevalence of comorbidities among african american women in particular , suggesting the need for more careful screening .
further , cv outcomes and cv death were carefully adjudicated events in the current study ; by contrast , no adjudication process occurred in many other studies .
our findings suggest that the adverse consequences observed in individuals with behavioral comorbidities and diabetes are best understood in a larger context or spectrum of linkages between physical and behavioral illness and subsequent outcomes .
elevated levels of stress and depressive symptoms , alone and together , were associated with a pattern of increased hrs for acute coronary disease and cv death in adjusted models both in subjects with and in subjects without diabetes , achieving statistical significance in those with diabetes .
together this pattern suggests the potential for progressive impact of single and multiple behavioral comorbidities on adverse cv outcomes and mortality that is of greatest concern in subjects with comorbid diabetes .
there was a significant pattern of increasing hrs for acute chd and cv death in those with diabetes and with one or both behavioral comorbidities ( none , stress or depressive symptoms alone , or both together ) .
while diabetes has long been known to increase cv outcomes , our findings suggest that , relative to those individuals with diabetes who have no behavioral comorbidity , those with either depressive symptoms or elevated levels of stress as well as those with both together have progressively increased risks for acute chd events and cv mortality during 5 years of follow - up , even when other risk factors are controlled for .
in particular , behavioral comorbidities such as stress or depressive symptoms are not usually screened for in busy primary care practices , and many patients are reluctant to share these symptoms with busy primary care providers .
further , many individuals are reluctant to seek care from mental health providers . among the demographic group in this study shown to have the highest prevalence of comorbid behavioral disease and diabetes ( i.e. , black females with limited income in the southeastern u.s . ) , there may also be distrust of the traditional health care delivery system . as a result , these comorbidities may go unrecognized and unmanaged .
this study therefore provides strong evidence for adverse consequences associated with these comorbidities and suggests the need for more careful research to identify optimal screening and treatment strategies that work in busy primary care practices or other community settings .
interestingly , while the presence of any behavioral comorbidity resulted in important increases in the hr for stroke , the increased hrs for one versus both behavioral comorbidities in subjects with diabetes were the same in fully adjusted models .
this may suggest that a different mechanism is operative in the increased relationship of behavioral comorbidities and stroke .
these findings build on and expand an established literature that links the cross - sectional and longitudinal presence of stress , depressive symptoms , and/or established depression with cv outcomes and/or mortality including in individuals with comorbid diabetes ( 10,2832 ) .
because our study measured these symptoms at baseline and then followed patients longitudinally , it suggests the potential for the chronic influence of these behavioral comorbidities in subjects with diabetes to across time lead to adverse cv outcomes .
indeed , the chronic nature of depressive symptoms and stress in individuals with type 2 diabetes has been described by fisher et al .
( 10,33 ) , who called for a more careful understanding of the conjoint physical and emotional burden in these patients .
further , these authors join our findings in calling for integrating the screening and management of the emotional side of diabetes into regular diabetes care . in addition , hamer et al .
( 34 ) demonstrated an association between stress and cv events with hr of 1.5 and with the suggestion that behavioral processes explained the largest proportion of the variance in the hazards model .
this raises an important question regarding the mechanism(s ) through which behavioral comorbidities result in increased cv events and/or cv death .
an early review by plante ( 35 ) suggested that stress may lead to depressive symptoms that are subsequently associated with accelerated atherosclerosis , endothelial dysfunction , inflammatory reactions , and interstitial disturbances that may predispose the subject to premature cv disease or death . in the current study ,
hs - crp levels were elevated in subjects with diabetes relative to those without diabetes , but among subjects with diabetes , there was a clearly progressive pattern of higher hs - crp levels among those with either versus both behavioral comorbidities .
these elevated hs - crp levels may suggest a pattern of worsening atherosclerotic disease in subjects with diabetes with one or both behavioral comorbidities relative to those with no comorbidity . a recent article by pizzi et al .
( 36 ) supports this and shows that depressive symptoms are associated with progressive longitudinal increases in carotid intima - media thickness relative to those who did not report these symptoms .
this progressive pattern of atherosclerosis associated with these symptoms or potentially other behavioral problems might be expected to be more common in subjects with diabetes and therefore contribute to adverse cv outcomes .
behavioral comorbidities may also interfere with self - care behaviors . among individuals with diabetes , a study by bonnet et al .
( 37 ) showed that those with anxiety or depressive symptoms were less likely to engage in healthy behaviors including physical activity , proper eating behaviors , and avoidance of smoking than were those without anxiety or depressive symptoms .
similarly , a meta - analysis by gonzalez et al . ( 3 ) that included studies of individuals with depressive symptoms showed that those with depressive symptoms or depression were more likely to be nonadherent with medications as well as with the larger treatment regimen .
( 38 ) suggest a stronger relationship between diabetes - related distress and glycemic control than between depressive symptoms and glycemic control , and recent data by this group ( 39 ) show that specific reductions in regimen - related distress can result in improvements in medication adherence , physical activity , and glycemic control , all of which may influence cv risk .
the scope of the implications from these important associations with cv outcomes should be considered in the context of the recent second diabetes attitudes , wishes and needs ( dawn2 ) study , which shows that these behavioral comorbidities know no geographic or ethnic boundaries and are highly prevalent in subjects with diabetes in multiple countries around the world ( 6 ) .
this suggests the need for additional research to understand the mechanisms by which these behavioral comorbidities are associated with cv outcomes in subjects with diabetes as well as to understand innovative intervention strategies that will not only improve the behavioral comorbidities but will also decrease the risk for cv outcomes .
clearly , more research is needed to understand whether the effective treatment of depressive symptoms and stress can lower cvd events in individuals with diabetes . and ,
as noted above , more work needs to be done to establish appropriate screening strategies for behavioral comorbidities that can be effectively disseminated in busy primary care settings where most individuals with diabetes are managed .
this study is a prospective cohort study and not a randomized clinical trial , and the incident outcomes should be understood as associations with baseline characteristics without clear evidence of causality .
by design , included only non - hispanic white and black subjects ; extrapolation to other ethnic groups or to other nations should not be undertaken .
the current study did not include a measure of diabetes duration , which may have influenced the risk of cv events .
the study included data on a large number of cv risk factors that were used to help minimize the influence of confounders ; however , there may have been other unmeasured risk factors that were associated with adverse cv outcomes .
the presence of elevated stress or depressive symptoms in community - dwelling subjects with diabetes was associated with an increased risk for acute chd or cv death .
moreover , subjects with diabetes who reported both stress and depressive symptoms together had the greatest risk for acute chd and cv death .
the presence of either or both behavioral comorbidities in subjects with diabetes was also associated with increased hrs for stroke .
elevated stress and/or depressive symptoms in subjects without diabetes resulted in increased hrs in fully adjusted models that were much smaller in magnitude and not statistically significant .
these findings demonstrate the persistent disparities and negative cv impact of these comorbidities at the population level and suggest the need for more careful integration of behavioral screening and management in primary care settings , where most patients with type 2 diabetes are managed .
|
objectiveto evaluate the impact of comorbid depressive symptoms and/or stress on adverse cardiovascular ( cv ) outcomes in individuals with diabetes compared with those without diabetes.research design and methodsinvestigators examined the relationship between baseline depressive symptoms and/or stress in adults with and without diabetes and physician - adjudicated incident cv outcomes including stroke , myocardial infarction / acute coronary heart disease , and cv death over a median follow - up of 5.95 years in the national regards cohort study.resultssubjects included 22,003 adults ( 4,090 with diabetes ) ( mean age 64 years , 58% female , 42% black , and 56% living in the southeastern stroke belt ) .
elevated stress and/or depressive symptoms were more common in subjects with diabetes ( 36.8% vs. 29.5% ; p < 0.001 )
. in fully adjusted models , reporting either elevated stress or depressive symptoms was associated with a significantly increased incidence of stroke ( hr 1.57 [ 95% ci 1.05 , 2.33 ] vs. 1.01 [ 0.79 , 1.30 ] ) and cv death ( 1.53 [ 1.08 , 2.17 ] vs. 1.12 [ 0.90 , 1.38 ] ) in subjects with diabetes but not in those without diabetes . the combination of both elevated stress and depressive symptoms in subjects with diabetes was associated with a higher incidence of cv death ( 2.15 [ 1.33 , 3.47 ] ) than either behavioral comorbidity alone ( 1.53 [ 1.08 , 2.17 ] ) and higher than in those with both elevated stress and depressive symptoms but without diabetes ( 1.27 [ 0.86 , 1.88]).conclusionscomorbid stress and/or depressive symptoms are common in individuals with diabetes and together are associated with progressively increased risks for adverse cv outcomes .
|
Introduction
Research Design and Methods
Study Design, Cohort Description, and Medication Information
Physical Examination and Laboratory Measures
Diabetes, Depressive Symptoms, and Stress Measures
CV Outcome Measures and Death
Measurement of Potential Confounders
Analysis
Results
Conclusions
|
takotsubo cardiomyopathy is characterized by transient left ventricular ( lv ) regional wall motion abnormalities and usually involves apical segments in the absence of significant coronary artery stenosis . in the recent years , several cases on atypical forms of transient lv ballooning syndrome have been reported .
the pathophysiological mechanisms remain unclear , however , the catecholamine excess and increased sympathetic activity are likely to play a pivotal role in triggering this syndrome.1 - 3 ) in this report , we describe an unusual case of a 38-year - old woman who had pulmonary embolism ( pe ) and reverse takotsubo cardiomyopathy .
pe has been listed as stressors of stress - induced cardiomyopathy,2)3 ) because the pain and the decreased perfusion within the lung related to pe probably cause a release of catecholamines.4)5 ) however , it is uncommon to present reverse types of stress - induced cardiomyopathy in the setting of pe for our patient .
a 38-year - old woman with no history of cardiac diseases or cardiac risk factors was referred to our emergency department because of chest discomforts , arrhythmia and shortness of breath after the surgery . a few days before she had fallen off the ladder and underwent surgery for right lateral malleolar fracture under spinal anesthesia .
physical examination revealed vital signs as follows : blood pressure 90/60 mm hg , heart rate 75 beats / min , body temperature 36.7 , respiratory rate 22/min and oxygen saturation 88% on room air .
the electrocardiogram ( ecg ) recording showed sinus rhythm , st - segment depression in v3 through v5 , and there were no typical features of ecg abnormalities associated with pe such as sinus tachycardia , s1q3t3 pattern , complete and incomplete rbbb , and t wave inversion ( fig .
chest radiography showed diffuse increased bronchovascular lung markings with mild congestion and edema , d - dimer was elevated to 1572 ng / ml ( normal reference range 0 - 243 ng / ml ) and altered results of the arterial blood gas analysis ( ph 7.42/pco2 25.8 mm hg / po2 69.7 mm hg / hco3 16.4 mmol / l ) aroused suspicion of pe .
it was confirmed by computed tomography and thus , therapy with heparin infusion was initiated ( fig .
other laboratory findings indicated white blood cell counts of 10000/mm ; hemoglobin 9.7 g / dl ; platelet count 275000/mm ; c - reactive protein 0.0 mg / dl ; alanine aminotransferase 18 u / l ; aspartate aminotransferase 39 u / l ; total bilirubin 0.46 mg / dl ; and serum creatinine 0.7 mg / dl .
the cardiac enzyme levels were elevated with a peak level of creatine kinase - mb isoform 27 ng / ml ( normal reference range 0 - 3.6 ng / ml ) and troponin i 5.30 ng / ml ( normal reference range 0 - 0.1 ng / ml ) .
transthoracic echocardiography showed hypokinesia of mid / base segments of lv with hypercontraction of apical segments and reduced ejection fractions estimated at 47% with no significant valvular dysfunctions .
right ventricular systolic dysfunction or dilated right ventricle was not found , and yet an estimated systolic pulmonary artery pressure increased mildly to 43 mm hg on the assumption of right arterial pressure of 10 mm hg ( tricuspid regurgitation peak velocity : 2.87 m / s ) ( fig .
coronary angiography was immediately performed within an hour of admission and ruled out obstructive atherosclerotic diseases .
she was managed with medical therapy using -blocker and diuretics.2)3)6)7 ) angiotensin converting enzyme inhibitors was not indicated because of mild hypotensions . after the medical treatment , the patient was presented free of symptoms for the following few days .
transthoracic echocardiography was undergone 1 week after her admission and showed improvements in ejection fractions of 58% with no wall motion abnormalities ( fig .
the patient was discharged in good clinical conditions and remained well after 3 months of follow - up .
takotsubo cardiomyopathy , also known as apical ballooning syndrome , is generally characterized as severe , reversible lv dysfunctions of apical segments .
variants , the non - apical ballooning syndrome , have been recognized as reversed , mid - ventricular , and localized type based on the involvement of the left ventricular.8 ) our case is consistent with the reverse type , hyperdynamic apex and akinesia of the mid / base segments of lv .
compared to typical lv ballooning syndrome , this atypical form of transient lv ballooning syndromes have different characteristics of patients.6)8 ) hahn et al.6 ) described that patients with atypical transient lv ballooning syndrome were younger with the mean age of 36 and had fewer coronary risk factors such as hypertensions , diabetes , and smoking habits .
also , t wave inversion in an ecg was found as less similar to our patient .
however , according to the recent reviews and many published clinical cases , the excessive catecholamine and exaggerated sympathetic activities are greatly accountable.1 - 3)9 - 11 ) differences in anatomical location and density of cardiac adrenergic receptors and the degree of sympathetic activity may explain the wall motion abnormalities as seen in the typical and reverse types.8)11)12 ) clinical presentations and transient nature of lv wall motion abnormalities in reverse type are similar to those of classic takotsubo cardiomyopathy which could indicate a possibility of sharing pathophysiological mechanisms , and yet , none of related evidences has been revealed .
estrogens , which have a protective effect on cathecholamine - induced toxicity , appear to have influences on the preponderances of postmenopausal women toward takotsubo cardiomyopathy.1)2)13 ) however , further researches for the interaction of catecholamines and estrogen are necessary to clarify the underlying mechanisms of stress - induced cardiomyopathy at a younger age without estrogen deficiency , and the specific reasons for rare presentations of men with physiologically estrogen - deficient .
takotsubo cardiomyopathy has been described with a wide range of emotional or physical stressful triggers .
herein , we report a middle - aged woman with inverted takotsubo cardiomyopathy in the setting of pe , which is a rare coexistence for this association .
the exact mechanisms of the relation between pe and atypical takotsubo cardiomyopathy are not clear . but increased catecholamine levels during severe pain and perfusion defect within the lung related to pe seem to result in the development of lv wall motion abnormalities.4)5 ) in this case , it is difficult to detect pe and reverse takotsubo cardiomyopathy independently , due to the fact that both clinical features mimic acute coronary syndromes . despite of the poor sensitivity and specificity of ecg abnormalities to diagnose pe and inverse takotsubo cardiomyopathy , there are several frequent features including sinus tachycardia , both complete and incomplete rbbb , s1q3t3 pattern , and st - segment elevation , non specific t wave abnormalities , respectively.2)4 - 6 ) a transthoracic echocardiography ( tte ) provide the evidence of pe such as right ventricular systolic dysfunctions and pulmonary hypertensions,4)5 ) and stress - induced cardiomyopathy may have right ventricular involvement which is associated with more severe impairments in lv systolic functions.14 ) thus , it is important to carry out ecg promptly , tte and coronary angiography based on a high degree of clinical suspicions .
further research is needed to elucidate this relationship and different pathophysiological mechanisms for various ventricular morphologies of stress - induced cardiomyopathy .
diagnosing pe especially in a patient with characteristics of acute coronary syndrome such as stressinduced cardiomyopathy can be difficult .
it needs to be highlighted that pe should be considered as a potential stressor once the reverse takotsubo syndrome is suspected .
|
as the use of early coronary angiography and echocardiography become widely available in the setting of acute coronary syndrome , the gradual increase for variant forms of transient left ventricular ( lv ) apical ballooning syndrome have been recognized .
this syndrome usually occurs in women and is frequently elicited by an intense emotional , psychological , and physical event . while the patients ' characteristics between typical and non - typical lv ballooning syndrome seem to differ , the presentation , clinical features , and reversibility of lv wall motion abnormalities are similar .
we present a middle - aged woman who experienced inverted takotsubo cardiomyopathy triggered by pulmonary embolism . to the best of our knowledge
, this case is particularly unique and is rarely reported in the disease entity .
|
Introduction
Case
Discussion
|
the head and neck posture of an individual can influence soft - tissue relationships in the
cervical and shoulder region1 , 2 .
a common concern in the modern workplace is upper extremity
disorders arising from overhead work , which is associated with neck and shoulder disorders
and pain3 .
long - term overhead working
postures result in strain and fatigue of the shoulder muscles because arm elevation is
associated with shoulder muscle fatigue4 , 5 .
previous studies have focused on risk
factor analysis and the development of therapeutic exercises for overhead work - related
disorders rather than prevention6 , 7 .
some studies have been performed on
postural ergonomic interventions including working techniques for overhead work6 , 7 .
however , we found that few studies have focused on protective ergonomic devices for overhead
workers .
therefore , this study investigated a new neck support tying ( nst ) method that used
a thera - band for the prevention of neck and shoulder pain in workers performing overhead
work .
the new nst method supports the neck during hyperextension and prevents excessive
upward rotation of the scapula during overhead work .
the purpose of the present study was to
investigate the effect of this nst method on cervical rom and shoulder pain after overhead
work .
the subjects were divided into two groups as follows : a control group consisting of 7 males
without nst , and a nst group consisting of 7 males with nst .
the initial cervical rom and
initial ppts of the ut and mt were not significantly different between the two groups .
the
initial values of cervical flexion , extension , and right and left lateral flexion in the
control group were 63.44.2 , 72.86.0 , 53.92.9 , and 51.35.6 degrees , respectively .
the
initial values for cervical flexion , extension , and right and left lateral flexion in the
nst group were 62.35.1 , 72.53.9 , 53.33.0 , and 52.22.4 degrees , respectively .
all
participants gave their informed , written consent according to the protocol approved by the
human ethics committee of the yonsei university faculty of health science .
this study
examined a new nst method that uses a thera - band for the prevention of neck and shoulder
pain in workers performing overhead work . for the nst method
, we used the grey thera - band
( 60 cm length ) which was applied as follows .
the midpoint of the thera - band supported the
posterior aspect of the neck , and both ends of the thera - band were passed under both
axillae , and tied behind the back .
the nst provided support for neck hyperextension and
prevented excessive upward scapular rotation during overhead work .
cervical flexion ,
extension , and right and left lateral flexion were measured with a cervical range of motion
( crom ) instrument ( performance attainment associates , st .
a dolorimeter pressure algometer ( fabrication enterprises , white plains ,
ny , usa ) was used to measure the pressure pain threshold ( ppt ) of the right side upper
trapezius ( ut ) and the lower trapezius ( lt ) muscles .
a 1-cm rubber plate
delivers pressure from the probe to the body , and the pressure is read from a needle gauge .
all subjects performed one
trial of overhead work with their arms over their heads for 15 min .
the overhead work was
performed at a height of 25 cm above each subject 's head .
differences in cervical rom and ppt between the nst and control groups
after the overhead work were tested with the independent t - test using the spss statistical
package ( version 18.0 ; spss , chicago , il , usa ) .
the cervical flexion , extension , and lateral flexion angles of the nst group were
significantly larger than those of the control group ( p < 0.05 ) .
the cervical flexion ,
extension , and right and left lateral flexion of control group were 50.48.2 , 64.711.3 ,
41.77.9 , 43.29.2 degrees , respectively .
the cervical flexion , extension , and right and
left lateral flexion of nst group were 61.511.2 , 69.46.9 , 48.75.6 , 49.86.7 degrees ,
respectively .
the ppt of ut of the nst group ( 7.21.8 lb ) was significantly higher than
those of the control group ( 6.32.0 lb ) ( p < 0.05 ) .
the ppt of mt of the nst group
( 5.81.4 lb ) was significantly higher than those of the control group ( 5.01.2 lb ) ( p <
0.05 ) .
repeated and sustained working with elevated arms is known to lead to neck and shoulder
pain8 .
this study proposed a new neck
support tying method using thera - band and investigated its effect on cervical rom and
shoulder pain after overhead work .
reductions in rom have implications for the safety and
efficiency of functional activities , and lead to a loss of corrective or protective
reactions1 , 9 .
rom losses can occur from inactivity and structural changes of the
tissues in the cervical spine , and result in an increase in connective - tissue density ,
shortening of collagen tissue , and muscle fibrosis1 ,
9 . in this study , the cervical flexion ,
extension , and lateral flexion angles of the nst group
shoulder forward flexion with scapular upward rotation requires the activation of the upper
trapezius , and overstretches the middle trapezius through scapular protraction3 , 5 , 7 .
the ppts of the ut and mt were
significantly lower in the nst group than those of the control group .
these results indicate
that the nst supported the neck and prevented excessive scapular elevation and upward
rotation during overhead work .
the thera - band , which provides varied resistance through the
range of movement , has been used for rehabilitation in combination with therapeutic
exercise10 .
it is light and portable ,
has low resistance , and can be adjusted to accommodate various situations11 .
the nst method prevented rom reduction
and pain in the cervical and shoulder regions .
the nst method can be easily and simply
applied using a thera - band and is also inexpensive .
we suggest that industrial workers could
use the nst method when performing overhead work .
|
[ purpose ] this study proposed a new neck support tying ( nst ) method using thera - band for
the prevention of neck and shoulder pain in workers doing overhead work .
the purpose of
this study was to investigate the effect of the new nst method using thera - band on
cervical rom and shoulder pain after overhead work .
[ subjects ] fourteen male subjects were
recruited .
[ methods ] this study measured the cervical rom and pressure pain threshold
( ppt ) of the upper and middle trapezius ( ut and mt ) muscles after the control and nst
groups had performed overhead work .
[ results ] the cervical flexion , extension , and lateral
flexion angles of the nst group were significantly larger than those of the control group .
the ppts of ut and mt of the nst group were significantly higher than those of the control
group [ conclusion ] the nst prevented rom reduction and pain in the cervical and shoulder
regions .
|
INTRODUCTION
SUBJECTS AND METHODS
RESULTS
DISCUSSION
|
the number of people surviving into old age is increasing , and it is a global phenomenon affecting developing and developed countries .
disability is defined as a restriction in the ability to perform normal activities of daily living , and it helps to quantify the impact of disease or injury .
disability is particularly a useful concept in assessing the health of elderly people , because they have several diseases occurring simultaneously with varied severity and impacts on their daily lives .
gill and colleagues have reported the powerful effects of disability on individual well - being , the need for informal help and health care , as well as long - term care needs and costs . on the basis of this , the epidemiology of elderly disability can not be overemphasized .
the process of disability represents a distinct phase in the life of many elderly persons .
functional limitation is associated with loss of independence and with increased need for both formal and informal care [ 4 , 5 ] .
the prevalence of physical disability in elderly persons with functional limitation are , therefore , important for policy development on care of the elderly be it formal or informal care .
contrary to the developed nations , most developing nations , specifically those in sub - sahara africa , lack reliable data to formulate policy on aging even though the populations of their elderly persons ( aged 60 years and above ) are increasing even more than developed nations .
the pattern and profile of disability that is obtained among the elderly in developed countries differs from those in developing nations .
for example , life expectancy in nigeria is currently about 48 years for elderly men and 50 years for elderly women . in the developed countries ,
the available data suggest that life expectancy at birth was around 35 to 40 years in the mid-1700s , rose to about 45 to 50 by the mid-1800s , so that by the middle of the twentieth century , it was approximately 66 to 67 years probably because of that rapid improvements that began at the end of the nineteenth century . physical disability and functional limitation are common among older people , leading to adverse consequences such as dependency and institutionalization .
older people 's ability to function independently is important , as physical disability and functional limitation have profound public health implications with increased utilization of health care and a need for supportive services and long - term care . due to economic hardship and extreme limitation of well - paid job opportunities in rural areas , most children and caregivers of the elderly have migrated to towns and cities for greener pasture thus abandoning the elderly with their disabilities .
this underscores the need to study the prevalence of physical disability and functional limitation especially in a nigerian rural underserved population of older adults .
several studies on physical disability and functional limitation have been reported in developed countries .
however , data are very scanty for developing countries especially in rural nigeria . apart from demographic factors like female gender and increasing age
[ 12 , 13 ] , social variables like smoking and alcohol consumption , low - income earning , low education , and urban dwelling have been associated with increased risk of disability among elderly persons [ 12 , 14 ] . in many respects , these correlates of disability
the objective of this study is , therefore , to determine the prevalence and factors associated with physical disability and functional limitation among elderly nigerian .
the study subjects were elderly rural dwellers aged 60 years and above from abadam , guzamala , and mobbar local government areas ( lgas ) in borno state , north - eastern nigeria ; and the study was conducted between march and august 2010 .
elderly people who were non - nigerians and those who were temporary residents ( lived in the community less than 24 months ) were excluded from the study .
the study population was identified from 2006 community census list which was obtained from the lga population census unit .
homes of the participants and primary health centers in each selected local government areas served as the venues of interview and clinical assessments , respectively . in order to reduce or avoid missed opportunity ,
period of interview was in the morning and evening , while the clinical assessment was between 8 am to 4 pm daily in the health centers .
the interview and clinical assessment were conducted by seven community health officers and three nurses mostly kanuri indigenes that were very fluent in kanuri and hausa languages and were trained as research assistants in all aspects of the study such as questionnaire administration and clinical assessment .
the questionnaire was pretested in another kanuri community to avoid ambiguity and easy administration during the survey .
information on the following relevant health - related variables was obtained : presence of chronic conditions , depressive symptomatology , height and weight measurement , cognitive function , and presenting visual acuity .
questions about the presence of chronic diseases include have you ever been managed as a patient for diseases like hypertension , heart attack , diabetes , epilepsy , stroke , arthritis , pneumonia , and asthma ?
chronic conditions were coded a , b , and c. hypertension and heart attack belongs to code a , diabetes belongs to code
b , while arthritis , pneumonia , and asthma were coded c .
depressive symptomatology was assessed using the short version of the geriatric depression scale 15 item ( gds ) .
body mass index ( bmi ) was calculated as weight in kilogram divided by square of height in meter expressed as kg / m in the result and table .
ecaq has been shown to be a valid tool for assessment of cognitive impairment among older people living in developing countries .
a score of 0 to 4 indicates probable cognitive impairment , 5 to 6 borderline , and 7 and above as normal .
visual acuity was assessed at designated primary health care centers using a standard metric snellen chart of alphabets e type at 6 meters .
participants ' self reporting or presenting visual acuity ( pva ) was ascertained with wearing of habitual optical corrective glasses ( spectacles ) .
the world health organization defines mild or moderate visual impairment as pva of less than 6/18 but equal to or better than 3/60 .
face - to - face interview was used to collect data on sociodemographic variables like gender , age , education level , marital status , social support , monthly income , ownership of motor vehicle , ownership of modern house , living arrangement , and ethnicity .
this is a population - based cross sectional study and multistage sampling technique was used .
the three lgas were purposively chosen but randomly selected using simple random sampling technique from the available rural lgas and subjects were allocated to each lga proportionately according to population of the elderly in the lgas .
cluster sampling technique was used in the final selection of all participants ( total sampling ) in each lga that met the inclusion criteria .
two physical function measures were used in the assessment : performance - based functional limitation and self reported physical disability .
the tinetti performance oriented mobility assessment tool ( tpomat ) was used and is a measure of functional limitation that assesses older people 's gait and balance abilities [ 19 , 20 ] . in conducting the balance assessment ,
participants ' balance abilities were assessed by performing maneuvers such as sitting in chair , rising from chair , immediate standing balance ( first three to five seconds after standing ) , further standing balance , balance with eyes closed , turning balance , ability to withstand displacement when slightly pushed from the sternum , neck turning , one leg standing balance , back extension , reaching up , bending down , and finally sitting down . for gait assessment , participants were asked to stand with the examiner in an obstacle - free walkway .
participants used their usual walking aid and were asked to walk down the walkway at their usual pace .
participants ' gaits were observed for initiation of gait , step height , step length , step symmetry , step continuity , path deviation , trunk stability , walk stance , and turning while walking . for maneuvers such as path deviation , trunk stability , and walk stance , the examiner walked behind the participants and for others next to the participants .
participants were then asked to walk back at a more rapid than usual but safe pace using usual walking aids .
level of performance of each activity was rated 0 - 1 , where a score of 0 meant inability to perform the activity and a score of 1 meant ability to perform the activity .
the maximum score for the gait component was 12 points and for the balance component it was 16 points .
participants with scores of less than 12 ( for gait ) or less than 16 ( for balance ) were defined as having performance - based functional limitation . in order to test for physical disability ,
barthel index is an assessment of patients ' level of independence in activities of daily living ( adl ) .
the ten adl items assessed were feeding , bathing , dressing , grooming , toileting , bladder control , bowel control , transferring from bed to chair , walking , and stair climbing . for this study , the operational definition of physical disability is needing help in one or more of these adl activities . in order to compare this study with others
, physical disability was also determined using the six katz index of independence in adl items related to self care ( feeding , dressing , bathing , toileting , transferring , and walking ) [ 22 , 23 ] and five items related to self - care ( feeding , dressing , bathing , toileting , and transferring ) .
following collection , collation , and editing , data were entered into a computerized data base .
clustering of data and sampling probability weights were taken into consideration during the analysis . in order to allow comparison with prevalence of adl disability reported in other studies , we estimated the prevalence of ten , six , and five items adl dependence . in this study ,
the prevalence for disability was also aged standardized population using the indirect standardization method for proper comparison with studies elsewhere .
indirect standardization method was also used for comparing prevalence of physical disability and functional limitation across the three ethnic groups ( kanuris , fulanis , and hausa ) , and in this study , kanuris group was used as the standard population . as a result of the high prevalence of physical disability and functional limitation , prevalence ratio ( pr )
all analyses were carried out using spss log binomial regression and poisson regression with strong and reliable variance .
two sets of univariate analyses were performed using chi - square tests : firstly , analysis was done to identify associations between the ten items physical disability and health related and sociodemographic variables ; secondly , similar analysis was done with functional limitation .
then , multivariate poisson regression with robust variance was performed to test which of the health - related variables and sociodemographic variables were independently associated with physical disability and functional limitation .
variables with clinical and statistical significance ( p value of < .05 in the univariate analysis ) were regarded as being significant .
participants with cognitive impairment identified through scores of less than five based on the elderly cognitive assessment questionnaire ( ecaq ) , were removed from the analysis ( n = 43 ) .
individual medical conditions ( such as stroke , diabetes , arthritis , etc . ) were used as explanatory variables in the analysis to find out their association with disability as well as functional limitation . due to very high illiteracy level in the study areas , informed ( verbal ) consent was obtained from all participants .
of the 1905 questionnaires distributed to elderly people who satisfied the inclusion criteria , 1824 respondents completed the questionnaire and were also available for physical examination , giving a response rate of 95.7% .
reasons for nonresponse include lack of interest ( 25% ) , competing issues ( 60% ) , sickness ( 5% ) , and traveling ( 10% ) .
the age of respondents ranged from 60 to 87 yrs ( with mean age of 69 7 years sd ) . of the elderly men , 31.2% were within 6064 yrs age group , 28.6% were 6569 years , 23.7% were 7074 yrs , and
34.6% were within 6064 yrs age group , 26.1% were 6569 years , 21.1% were 7074 yrs , and 18.2% were 70 years and above .
there were more elderly women [ 977 ( 53.3% ) ] than elderly men [ 847 ( 46.7% ) ] .
illiteracy level was 85% and of those that were literate , 87% had primary education and 11% had secondary education and 3% had tertiary education .
of all the elderly women , only 4.6% had secondary education , 15.3% had primary education and 80.1% had no formal education .
kanuri ( 84.% ) constitutes the major ethnic group , while fulani and hausa represent 8.8% and 7.2% , respectively
. marital status of elderly men showed 86.3% married , 10.8% widowed , 1.8% divorced , and 1.1% single .
of the elderly women , 45.7% were married , 38.5% were widowed , 11.1% were divorced , and 4.7% were single . more than four - fifth ( 88.4% ) of the respondents live with other family members , while 11.6% lived alone . in both sexes ,
more than two - thirds had normal cognitive function , 92.1% in elderly men and 81.2% in elderly women .
elderly women ( 12.6% ) are two times borderline cognitive impaired compared with elderly men ( 6.1% ) . also ,
elderly women ( 5.4% ) are about three times probably cognitive impaired compared with elderly men ( 1.8% ) .
more elderly women ( 24% ) had more than one chronic disease than elderly men ( 15.5% ) .
elderly women ( 25.1% ) are slightly more depressed than elderly men ( 22.7% ) .
elderly women ( 16.4% ) are also more overweight than elderly men ( 12.5% ) .
more than three - quarters ( 78.5% ) earned less than one hundred american dollars per month and less than 5% of the participants earned more than three hundred american dollars per month ( table 1 ) .
more than one - quarter ( 28.3% ) of the respondents signified interest for assistance in at least one of the 10 adls in the barthel index .
the prevalence of disability based on at least one item of the six adl scale was 15.7% and prevalence of disability based on at least one item in the five adl scale was 12.1% .
the overall prevalence of disability ( 10 items adl , 6 items adl , and 5 items adl ) and functional limitation increased with advancing age .
the prevalence of needing help in at least one of the ten adls of the barthel index increased from 11.7 in those aged 6064 years , to 98.9% of those aged 75 years and older ( table 2 ) .
the prevalence of functional limitation rose from 12.4% in those aged 60 to 64 years to 98.3% in the 75 years and above age group ( table 2 ) .
in all the participants , the prevalence of both self - reported physical disability and objective measurement of functional limitation was higher in elderly women than in elderly men ( table 2 ) . among the three ethnic groups
, kanuris had the highest prevalence of physical disability ( 10 items adl , 6 items adl , and 5 items adl ) compared to the fulanis and hausa ( figure 1 ) .
however , the prevalence of functional limitation was almost similar across all ethnic groups . the correlations between performance - based functional limitation and self - reported physical disability among the different ethnic groups showed thus that the correlation coefficient for kanuris , hausas , and fulanis were 0.52 , 0.48 , and 0.23 , respectively .
elderly persons with levels of mid - arm circumference indicative of severe malnutrition had an increased risk of physical disability and functional limitation ( table 3 ) . among the four groups of monthly level of income , respondents that earned less than 100 us$/month had the highest prevalence of physical disability ( 10 items adl , 6 items adl , and 5 items adl ) .
the prevalence of functional limitation rose from 0.6% in those that earned more than 300 us$/month to 33.2% in those that earned less 100 us$ /month .
with increase in age , elderly women with low education self - reported one or more chronic diseases than their elderly men counterparts .
this study also showed that having depressive symptomatology and presence of visual impairment ( mild - to - moderate blindness ) were found to be more associated with physical disability .
furthermore , being at risk of isolation was associated with functional limitation ( table 4 ) . in table 4 , all explanatory variables except ethnicity used in the univariate analysis were significantly associated with functional limitation .
the multivariate associations between physical disability and functional limitation , and sociodemographic and health - related variables are shown in table 5 .
table 5 shows a significant independent associations between physical disability and advanced age , ( 75 years : prevalence ratio ( pr ) 22.2 ; 95% ci 14.536.8 ) , presence of diabetes ( pr 6.1 ; 95% ci 4.37.1 ) , and visual impairment ( blindness : pr 6.6 ; 95% ci 3.611.9 ) .
the independent variables found to be associated with functional limitation include advanced age , ( 75 years : pr 10.5 ; 95% ci 5.416.4 ) , female gender ( pr 9.3 ; 95% ci 3.718.3 ) , presence of arthritis ( pr 5.2 ; 95% ci 3.56.8 ) , and having depressive symptomatology ( pr 6.4 ; 95% ci 4.79.2 ) .
in contrast to physical disability , functional limitation represents an outcome that is free from external factors or environmental influences .
this adds clarity to the understanding of the dynamics of the pathway from disease to disability .
the study population was randomly selected from geographically defined rural communities , and a high response rate was recorded .
the prevalence rates of physical disability for all the three adl scales and functional limitations were less than one - third and increased with age .
the increase was observed to be higher in elderly women than elderly men and among kanuris than among fulanis and hausas .
advance in age , presence of diabetes , stroke , depressive symptomatology , and visual impairment were independent variables found to be more associated with physical disability .
furthermore , advance in age , female gender , arthritis , and depressive symptomatology were also significantly associated with functional limitation .
as previously stated , studies on physical disability among elderly people in nigeria is very scarce ; therefore , comparison of findings in this study with studies elsewhere is limited by definition criteria , differences in the way disability was measured , and sample characteristics .
studies in which the scope of definition of disability was not wide ; for example , in which disability is based on any level of difficulty in the performance of daily tasks , the prevalence of disability is expected to be high and probably higher than that from this study . in addition , studies that exclude institutionalized elderly persons , the range of disability will also be different .
institutional care of the elderly is very rare in nigeria , and this study includes the entire range of functional limitations obtainable in rural population and thus excludes elderly in institutional care . despite the scenario explained above , the rates of disability and functional limitation are comparable to levels reported among elderly persons in malaysia . using the 10 items barthel index
, this study revealed that more than one fifth ( 28.3% ) of the elderly aged 60 years and above were dependent in at least one adl .
this finding is similar to that reported by previous studies [ 27 , 28 ] .
however , a study in singapore found much lower disability prevalence than this study .
comparison with studies in other countries is difficult due to use of different adl measurements ; however , narrowing adl disability to receiving help for at least one of five adl items ( eating , bathing , dressing , transferring , and toileting ) or six adl items ( walking , eating , bathing , dressing , transferring , and toileting ) allows reasonable comparison across some studies .
elderly women in this study have more functional disability than elderly men . also , increase in age was found to be associated with increasing rates of disability .
all these variables are often associated with the occurrence of disability as reported in the literature . according to the six - item adl index ,
the prevalence of physical disability for people aged 65 in this study was 21.4% ( table 2 ) , which is moderately higher than the united states ' national long term care survey , which is 13% ( 7.9% when age - standardized to the nigerian population sample ) .
however , the prevalence of six - item adl disability among people age 60 years and above in the caribbean , and among people age 75 years and above in latin america and the caribbean , appears to be similar to the prevalence rates in this study . using the five - item adl index , for people aged 65 years and above
this appears to be much higher when compared to the findings from developed countries , for example 6% in canada ( 3.2% when age standardized ) , 10% in france ( 5.5% when age standardized ) , 14% in italy ( 7.3% when age standardized ) , and 11% in sweden ( 5.5% when age standardized ) .
disability prevalence rates in this study appear comparable to rates in other developing countries ; for example , among people aged 65 and older , the prevalence of 5 items adl disability in malaysia was 16% ( 14.8% when age standardized ) and 10% in srilanka , ( 14.4% when age standardized ) .
this is almost similar to 12% in india ( 9.3% when age standardized ) and 11% in malaysia ( 10.5% when age standardized ) .
however , the prevalence rate in this study is higher than 8% from shanghai , china .
differences in criterion definition and sample profile may account for disparity between this finding and that of china
. the following may be other possible reasons why nigerian 's disability prevalence rate is different from those reported from the developed countries : sample for this study was drawn from rural community , high illiteracy level ( 85% ) , the low educational levels in elderly cohorts ( 90.4% had no formal education or low education level ) , a figure higher than the developed country , and the prevailing sociodemographic differences between nigeria and the wealthy industrialized nations .
since disability in adl is one of the main reasons for institutionalization in the developed nations , this may be another reason why prevalence of physical disability in elderly people in developed countries is lower compared with this finding .
the african tradition , particularly nigerian tradition , dictates that elderly people should be taken care of by their family members .
the institutional care of the elderly is very rare in nigeria , thus majority of the elderly people in nigeria live with their spouse or other family members .
self - report or performance - based measures are useful in assessing functional limitation . however , performance based measurements offers more information , because they help to identify important physical parameters involved in performing daily activity tasks .
comparison of prevalence of functional limitation across studies is difficult due to differences in concept and measurement of functional limitation used .
using the tinetti performance - oriented mobility assessment tool , the overall prevalence of functional limitation among nigerian aged 75 years and above was 51.5% ( table 2 ) .
this is very close to a previous study that reported 48% but much higher compared with that reported among the italian aged 75 years and above which was 21% .
this study shows a higher prevalence of physical disability and functional limitation among elderly women than elderly men in all age groups and the higher the age the wider the gender difference .
previous studies [ 39 , 40 ] have also reported higher levels of physical disability and functional limitation in elderly women than elderly men . cumulative effect of pregnancy and childbearing , poor / lack of education , and poor health care may be responsible for higher physical disability and functional limitation seen in elderly women .
poor or lack of education may be associated with low income and poverty , poor standard of living , unhealthy lifestyle behavior , malnutrition , and less frequent use of health and medical care services .
this study showed that more elderly women ( 80.1% ) than elderly men ( 41.6 ) had no formal education ; this is in conformity with the literature that low socioeconomic status is associated with physical disability .
chronic disease like obesity was found to be commoner in elderly women than elderly men ; this finding is similar to malaysia study .
in addition , underweight is higher in elderly men than elderly women , this is also similar to malaysia study .
sedentary life style among elderly women and carbohydrate as the main food consumption could be the reason for overweight commoner in elderly women than elderly men . in this study ,
the bmi values ( 20 to 24.9 ) associated with optimum physical function coincide with values associated with lowest risk of morbidity similar to previous studies in chinese populations relating bmi to health outcomes [ 42 , 43 ] and mobility decline .
this observation is not unexpected , given the close inverse relationship between walking speed and health - related outcomes in well - functioning older people [ 45 , 46 ] .
the findings from this study emphasize yet another adverse effect of obesity in the elderly apart from increased risk of various diseases that of functional limitation .
for example , more than one - quarter ( 28.3% ) of the respondents signified interest for assistance in at least one of the 10 adls in the barthel index ( table 2 ) .
the request for help for some of the activities of daily living suggests that muscle function may be adversely affected and may partly account for functional limitations .
the association between grip strength and appendicular muscle mass emphasizes this point . in obese elderly people ,
an exercise component must be included in the treatment regimens to maintain or increase lean muscle mass and bone mineral density .
such regimens have been shown to result in a reduction in fat mass without changes in fat - free mass , increase physical performance , and improve quality of life . concerning the effect of ethnicity on prevalence of physical disability
, this study showed that hausas had the highest prevalence of self - reported physical disability followed by fulanis and kanuris .
the observed differences among the ethnic groups may be attributed to different types of occupations as similarly expressed in a previous study . in other words ,
this study revealed that hausas and fulanis are more commonly involved in very stressful and laborious manual occupations .
specifically , the hausas and fulanis are usually engaged in farming , tree felling , and truck pushing , while the fulanis are usually engaged in nomadic activities .
this study showed a significant association between functional limitations and advanced age , female gender , stroke , arthritis , and depressive symptomatology , and it is similar to findings from a previous study .
living alone , poor social support , being overweight or underweight , presence of diabetes mellitus , stroke , and visual impairment were not associated with functional limitation .
this finding is also similar to previous findings for advanced age , female gender , presence of arthritis and depressive symptomatology [ 49 , 50 ] being associated with functional limitation .
this study is limited by involving only elderly people in rural community , excluding elders from institutions as well drawing of inferences between health - related variables and physical disability or functional limitation .
other limitations include study design ( cross - sectional design ) , which does not allow determination of direction of causality despite the presence of associations .
in addition , the study design does not also include information on duration of disability thus making it difficult to confirm that some of the disabilities were or were not transient in nature .
this study , however , has a number of strengths ; this is one of the very few studies to assess the prevalence and correlates of performance - based functional limitation among elderly nigerians .
validated measures of disability that conform with theories of aging were used , for example , in the nagi model of disablement ; functional limitation takes priority before ( precedes ) disability .
relationship between malnutrition and disability has also been reported by a previous study in rural malawi , where chilima and ismail observed a relationship between undernutrition and handgrip strength , psychomotor speed and coordination , and mobility and ability to carry out activities of daily living independently .
disability in old age is an important indicator of any community population health , as elderly people usually have more than one illness , and the functional impacts of combined conditions provide a better measure of health than do diagnostic categories . in addition , in developing countries , access to physicians
this study has shown that the overall pattern of disability in nigeria has the relationships similar with studies elsewhere .
of particular note is that physical disability and functional limitation is common in nigerian elderly . though the prevalent rates of physical disability and functional limitation is higher than that obtainable in developed countries but similar and comparable to partner developing countries .
more importantly , elderly women , especially those with advanced age , chronic diseases , depressive symptomatology , and visual impairments showed greater risk of disability and functional limitation compared with elderly men .
this finding is indispensable when considering those to focus for appropriate prevention and intervention strategies like physical exercise , health education , and home visits of high - risk individuals in community - dwelling nigerians .
|
background .
the number of people surviving into old age is increasing , and it has now become a global phenomenon .
studies on the prevalence and correlates of physical disability and functional limitation among elderly nigerians are scanty . methodology .
this is a community - based cross - sectional study conducted in 3 local government areas ( lgas ) in nigeria , using a multistage sampling technique .
functional limitations of 1824 elderly persons were tested using tinetti performance - oriented mobility assessment tool ( tpomat ) and self - reported activities of daily living ( adl ) .
adl disability of ten , six , and five basic items were compared . results .
the prevalence ratios ( prs ) of physical disability using the ten , six , and five basic adl items were 28.3 ( 95% ci 25.231 .
5 ) , 15.7 ( 95% ci 13.419.8 ) , and 12.1 ( 95% ci 9.815.3 ) , respectively , while functional limitation was 22.5 ( 95% ci 18.124.4 ) . increased risk of disability was independently associated with female gender pr 3.6 ( 95% ci 1.57.4 ) , advanced age 75 years ; pr 22.2 ( 95% ci 14.5 , 36.8 ) , arthritis pr 3.7 ( 95% ci 2.64.6 ) , stroke pr 4.8 ( 95% ci 3.77.9 ) and diabetes pr 6.1 ( 95% ci 4.37.1 ) .
conclusions .
the findings from this study are pointers to unmet needs of the elderly disabled nigerians .
|
1. Introduction
2. Methodology
3. Results
4. Discussion
5. Conclusion
|
breast cancer is a leading cause of death and disability among women , especially young women , in low- and middle - income countries . though incidence and overall mortality rates continue to be lower than in most high - income countries , case fatality rates from breast cancer are very high .
these high case fatality rates are likely due to a lack of awareness of the benefits of detection and treatment and a scarcity of adequate facilities for detection and diagnosis , as well as poor access to primary treatment .
remarkable improvements have been achieved in the probability of survival for women diagnosed with breast cancer in the usa as compared to 60 years ago .
early detection through the use of mammography , high - quality surgery , and adjuvant therapies including chemotherapy and targeted therapies , such as hormonal therapy and , more recently the her2-directed agent trastuzumab , can be credited for much of the recent improvement in outcome for women with breast cancer in the usa . however ,
even prior to the routine use of mammography or adjuvant therapy , significant improvements were made in breast cancer survival , and these can be traced to relatively low - cost interventions that are still in use in high - income countries .
understanding which healthcare interventions were available and how they resulted in improvements in the probability of survival could be important , especially for designing programs in resource - constrained settings where breast cancer case fatality is high and many of the most costly and technology - intensive diagnostic and therapeutic options are not available .
in many developing countries , the incidence of breast cancer is now rising sharply due to changes in reproductive factors , lifestyle , and increased life expectancy . today , more than half of incident cases occur in the developing world [ 14 , 15 ] .
combined with still high case - fatality rates , this means that mortality from breast cancer is a leading cause of death among adult women in developing countries , as well as in the developed world . in mexico , for example
, breast cancer is now the second leading cause of death among women aged 30 to 54 and the leading cause of tumor - related death among adult women of all ages . the high probability of dying from breast cancer the case fatality rate , which is approximated by the ratio of mortality to income across the developing world further reflects the inequities in early detection and access to treatment [ 1 , 17 ] . the number of deaths as a percentage of incident cases in 2008 was 48% in low - income , 40% in low - middle - income , and 38% in high - middle - income countries , while it was 24% in high - income countries according to the most recent globocan / iarc data .
available evidence on stage at diagnosis , though scarce , indicate that a very high proportion of cases in the developing world are detected in late stages [ 1 , 3 ] .
( table 1 ) in many underserved populations , a majority of women present with advanced disease ; the figure is as high as 78% in black women in south africa .
in contrast , in the united states the majority of cases are detected in localized stages of the disease ( stages i and ii ) , a third is regionally advanced ( stage iii ) , and only 5% are distant - stage metastatic ( stage iv ) .
many reasons are given for the advanced stage at presentation and resultant poor survival rates in low- and middle - income countries : the stigma of breast cancer and the associated societal implications of its treatments ( especially mastectomy ) discourage women from seeking care early on ; lack of knowledge about breast health ; scant options for early detection due to limited access to routine care and examinations ; and lack of access to mammography and to affordable , high - quality treatment options .
in the short term , mammography and other expensive and technologically complicated resources and therapies will not likely be available to many of the world 's women . though we must continue to work at all levels to bring diagnostics and therapeutics with a proven impact on outcomes to these women as soon as possible ,
there are ways closer at hand to improve the immediate outlook for women in these settings .
figure 1 shows the incidence and mortality rates for breast cancer in the usa between 1940 and 2000 . from the late 1940s ,
thus mortality - to - incidence ratios decreased dramatically , even before the generalized use of mammography or adjuvant chemotherapy and antiestrogen therapy that commenced in the mid- to late 1970s .
table 2 presents the ratio of mortality over incidence , as an approximation of the case - fatality rate , in 5-year increments between 1950 and 1975 . between 1950 and 1975
incidence nearly doubled , increasing from 66.6/100,000 women to 119.2/100,000 , while mortality remained relatively constant , 28/100,000 and 31.6/100,000 , respectively .
thus , during this time period , the ratio of mortality over incidence ( an approximation of the case - fatality rate ) fell from 0.42 to 0.27 representing a 36% decline : this suggests that more women were surviving their cancers in 1975 as compared to 1950 and is true for both whites and blacks .
further , the reduction in case - fatality rates is at least as large as the improvement evidenced since the introduction of mammography and adjuvant therapy .
these findings suggest considerable room for reducing the high mortality - to - incidence ratio found in many developing countries even without mammography or adjuvant therapy .
the increases in incidence and survival for breast cancer in the usa between 1950 and 1975 can not be attributed only to detection of in situ cancers that would not have progressed .
the proportion of in situ cases in known - stage cases in the connecticut tumor registry in that period was very small and increased from only 0.3% in 19501954 to 1.9% in 19701974 , and it was largely unaffected by improved reporting and a reduction in unknown - stage tumors .
thus , the reduction in the mortality - to - incidence ratio must largely reflect outcomes for patients with invasive cancers . from 1940 to 1970 ,
regional and advanced stages fell from 58% to 54% between 19401944 and 19501954 , and to 45% in 19701974 . the period from 1940 to 1974 was a time in the usa when evidence - based medicine became more widespread , and healthcare became more generally available , including increased use of routine gynecologic and general physical examination .
for example , the american cancer society began promoting self - examination for breast cancer in 1950 and routine screening by cervical cytology starting in 1952 .
further , the era of oral contraceptives in the 1960s contributed to greater interactions between healthy women and their healthcare providers .
authors who analyzed data prior to 1974 assign the improvements in survival to more effective breast education programs , increased breast cancer awareness , detection of tumors palpable with self or breast - clinical examination , and better diagnostics [ 19 , 20 ] .
thus , the increase in survival rates in the usa prior to 1975 strongly suggests potential to improve breast cancer outcomes in developing countries more quickly than we will be able to make routine mammography and adjuvant therapy available .
recent studies , showing breast physical examination and breast self - examination to be unhelpful in reducing stage at diagnosis [ 2326 ] , have considered only developed countries or urbanized areas of developing countries where routine healthcare is generally available , breast cancer awareness and education are high , and mammography is more routinely accessible . these data , and hence the findings , are likely to be less applicable to a population where breast cancer education and awareness are low , access to the healthcare system severely restricted , and the vast majority of patients present with advanced disease .
while reducing the incidence of breast cancer is an ideal goal , the options for achieving this are limited and longer term , particularly for the developing world .
healthy lifestyle , including limiting alcohol consumption , maintenance of ideal body weight , regular physical activity , and avoidance of postmenopausal hormone replacement therapy , can have an important impact on breast cancer incidence [ 15 , 27 ] .
every effort should be made to limit these risk factors and thus breast cancer risk . yet ,
even with strong efforts aimed at prevention , the incidence of breast cancer is likely to increase in most developing countries due to changes in reproductive patterns including later first pregnancies , reductions in parity , and shorter duration of lactation ; as well as , declines in physical activity and increased life expectancy .
earlier detection and timely , adequate surgery would likely result in substantial improvements in survival in much of the developing world .
education about breast cancer , advocacy around curability , and increased coverage of basic healthcare including skilled breast physical examinations could produce improvement in survival rates as occurred in the usa between 1950 and 1975 .
education efforts need to address the reality that many women , particularly those with less income and education , may not seek care when they feel a breast mass , because they are unaware of what it represents , are concerned about the stigma of cancer and being rejected by their community and their partners , fear the potential loss of the breast , or believe there are no effective therapies for the disease especially if all the women they have known with breast cancer died . hiv a stigma - laden disease , that if untreated is universally fatal
by contrast , it has been demonstrated that by combining education , with better and more accessible healthcare facilities , trained medical personnel , and effective therapy , patients do seek and comply with treatment and benefit from it [ 2830 ] .
the ability to provide adequate affordable access to physical exams by healthcare workers is not a trivial obstacle .
an essential first element is the existence of a functioning primary care system staffed by providers trusted by their community . while many countries continue to battle with a weak primary infrastructure , examples , such as the oportunidades program and seguro popular in mexico and partners in health in rural africa and haiti ,
provide important lessons for strengthening primary healthcare including , and often especially , interventions to improve the health of women [ 16 , 31 , 32 ] .
these interventions are essential parts of overall health system strengthening and can help with the prevention and treatment of many diseases in addition to breast cancer .
clinical breast exams do not need to be performed by physicians or nurses . in settings
where community healthcare workers have learned to care for patients with diseases as complex as hiv , multidrug resistant tuberculosis , and malaria , they could be trained to effectively perform breast exams .
large - bore core needle biopsy is a reliable method to obtain tissue for diagnosis and can be performed by trained personal in relatively simple ambulatory settings .
pathology services must be available to process the specimens but can be located regionally or outsourced globally . in many developing countries ,
surgery is available in regional centers , although additional training of surgeons in appropriate techniques may be needed , and women will require financial support and transportation . where radiation therapy is not available , as is the case in many low - income countries
given the high proportion of hormone receptor positive cancers , tamoxifen can be effectively combined with surgery .
unlike many treatments for breast cancer , generic tamoxifen is low cost , taken orally , and in the vast majority of patients is well tolerated and does not generate unmanageable side effects or require additional medications or care to control symptoms .
options exist to greatly expand low - cost alternatives for earlier detection and treatment of breast cancer in developing countries .
guidelines have been developed and have been stratified according to the resources available in specific countries and health systems [ 3336 ] .
many of the basic interventions focus on education , awareness building , the health of women , and expanding capacity at the primary and community healthcare levels , and thus and also contribute to overall health system strengthening .
education to improve breast health awareness , breast self - examination , and clinical breast exam are relatively inexpensive and can be incorporated into existing primary health infrastructures .
surgery and hormone therapy based on tamoxifen are cost effective , especially with early detection , and implementable in poor - resource settings .
focusing on providing these interventions in locations where they do not currently exist could dramatically improve survival . in no way
does this abrogate the responsibility to eventually provide resources such as mammography , adjuvant chemotherapy , and advanced targeted therapies such as trastuzumab in these settings .
however , great benefit can emerge from basic breast cancer education and awareness , integrating breast exams into primary healthcare infrastructure , and adequate surgery combined with tamoxifen .
implementation of these interventions should proceed as quickly as possible , while the more complex and costly interventions , such as mammography , are being made more available .
the provision of better primary healthcare , education , and better medical outcomes will provide a solid foundation for reducing stigma and fear that will make more effective the introduction of complex technologies , such as mammography or adjuvant therapy . there is no reason not to immediately strive for the implementation of basic interventions for breast cancer care and control in all settings .
all authors have completed the unified competing interest form at http://www.icmje.org/coi_disclosure.pdf ( available on request from the corresponding author ) and declare that all authors had : ( 1 ) no financial support for the submitted work from anyone other than their employer , ( 2 ) no financial relationships with commercial entities that might have an interest in the submitted work , ( 3 ) no spouses , partners , or children with relationships with commercial entities that might have an interest in the submitted work , ( 4 ) no nonfinancial interests that may be relevant to the submitted work .
|
breast cancer survival in the usa has continually improved over the last six decades and has largely been accredited to the use of mammography , advanced surgical procedures , and adjuvant therapies .
data indicate , however , that there were substantial improvements in survival in the usa even prior to these technological and diagnostic advances , suggesting important opportunities for early detection and treatment in low- and middle - income countries where these options are often unavailable and/or unaffordable .
thus , while continuing to strive for increased access to more advanced technology , improving survival in these settings should be more immediately achievable through increased awareness of breast cancer and of the potential for successful treatment , a high - quality primary care system without economic or cultural barriers to access , and a well - functioning referral system for basic surgical and hormonal treatment .
|
1. Introduction
2. Breast Cancer in Low- and Middle-Income Countries
3. Opportunities to Improve Breast Cancer Outcomes for Women in Developing Countries
4. Explaining Improved Breast Cancer Survival Rates in the USA Prior to 1975
5. Next Steps to Improve Breast Cancer Survival in Low- and Middle-Income Countries
6. Conclusions
Competing Interest Statement
|
radiculopathy and myelopathy from degenerative , inflammatory , and traumatic processes have been successfully treated with anterior cervical discectomy and fusion ( acdf ) . despite clinical success with fusion treatment , there are concerns regarding the long - term effects of fusion on the cervical spine .
. reported on the incidence of radiculopathy and myelopathy at adjacent segment to a cervical fusion and found that 25% of acdf patients will have symptomatic adjacent segment deterioration ( asd ) within 10 years of acdf at a rate of 2.9% each year .
it is believed that although some changes in asd occur naturally , the effects of fusion disturb the biomechanics , most likely increasing the incidence of asd . in vitro studies demonstrated that intradiscal pressures , shear strains , and motion increased in upper and lower segments adjacent to fusion levels [ 47 ] .
recently , artificial cervical disc replacement ( acdr ) has become the alternative to fusion , with the potential to preserve the motion of the instrumented level and to prevent overload of the adjacent levels and subsequent asd .
acdr has been proven to be beneficial in terms of avoiding the deleterious effects of fusion .
however , indications for acdr are more stringent and hypermobility of the operative levels may occur , which lead to the limitation of multilevel acdr .
clinical studies regarding hybrid combinations of fusion and non - fusion have been reported , with improved total motion and earlier recovery and return to work [ 1215 ] .
biomechanical studies of 2-level hybrid acdf and acdr have demonstrated advantages of the hybrid in reducing compensatory adjacent motion and reduced internal stresses in the construct [ 4,1621 ] . in a clinical study , kang el al .
reported that the hybrid construct is a safe and effective alternative for 3-level cervical disk disease .
the hypothesis was that the motion response of disc replacement adjacent to fusion was comparable to a stand - alone disc replacement , and the non - operated segments in a hybrid construct would experience significantly less motion than with a fusion .
this study is a progression to 3-level from the 1- and 2-level biomechanical studies because 3 levels of fusion are common and hybrid combinations either performed initially or for revision of asd are more common today .
the objective of the present study was to compare the 3-level acdf to combinations of acdr and acdf with displacement controlled kinematics at instrumented and adjacent levels .
eighteen fresh adult human cadaveric cervical spines ( c2-t1 ; age range , 5273 years ) were used for biomechanical testing .
all cervical spines were evaluated for bone mineral density ( bmd ) using dual - energy x - ray absorptiometry scanning and measured bmd values ranged from 0.53 to 0.72 g / cm .
cervical spines with degenerative diseases or traumatic pathology were excluded by anteroposterior and lateral screening radiographs before biomechanical testing .
once harvested , all cervical specimens were immediately conserved in plastic bags and frozen at 20c . in preparation for biomechanical testing ,
the required spines were thawed at 4c for 12 hours . at room temperature on the testing day
, the proximal vertebra ( c2 ) and distal vertebra ( t1 ) were separately mounted in a cylindrical container using a low - fusion point ( 72c ) alloy .
and then , the c2 container was attached to the upper fixture while the t1 container was mounted to the lower testing platform .
markers made of 4 plexiglas motion detectors were fixed to the anterior aspects of each vertebra from c2 to t1 .
the 3-dimensional motion range of each vertebra was obtained with an optoelectronics measurement system ( optotrak , northern digital inc . ,
waterloo , ontario , canada ) capable of capturing the motion curve of the markers .
biomechanical tests were performed under displacement control by an mts machine ( cmt6104 ; mts systems ( china ) corp . ,
shenzhen , china ) which can replicate physiologic motion with displacement control ( figure 1 ) .
the flexion extension axis of each tested spine was placed eccentric to the load axis of the actuator .
the bending moments were limited to the upper bound of physiological human bending ( 4.5 nm ) .
an angular displacement transducer was assembled to measure the global rotation of the cervical spine ( c2-t1 ) .
a displacement transducer was used to measure the changes in moment arm length between the upper container and the load axis of the mts machine .
the spine specimens were tested by application of a 50 n preload in flexion - extension and lateral bending circumstances . during the biomechanical tests ,
after analysis of the intact spine , each specimen was sequentially reconstructed at c3-c6 3-level ) motion segments .
the conditions were as follows ( figure 2 ) : acdr , acdf , acdr , or 3-level disc plate disc ( 3dpd ) ; acdf , acdr , acdf or 3-level plate disc plate ( 3pdp ) ; three - level acdf or 3-level plate ( 3p ) . prior to biomechanical tests ,
measurements included vertebral motion , applied load , and moment . in the biomechanical tests ,
the acdr was a titanium - ceramic alloy cervical disc ( prestige lp cervical disc , medtronic sofamor danek usa , inc . ) .
the arthrodesis was performed using an interbody cage ( telamon tm , medtronic sofamor danek usa , inc . )
combined with an anterior cervical plating ( acp ) system ( doc cervical plate , depuy spine , inc . , raynham , ma , usa ) ( figure 3 ) .
this biomechanical protocol limited motion to 20 degrees in flexion and extension as well as 15 degrees in lateral bending and axial rotation .
the ratio of segmental rom to the total c2-t1 rom was applied to evaluate the operation effect by normalization method .
one - way anova ( p<0.05 ) was used to determine the statistical differences in both c3-c6 motion and adjacent level motion under the 3-level anterior plate fusion ( 3p ) , the 3pdp , and 3dpd conditions .
eighteen fresh adult human cadaveric cervical spines ( c2-t1 ; age range , 5273 years ) were used for biomechanical testing .
all cervical spines were evaluated for bone mineral density ( bmd ) using dual - energy x - ray absorptiometry scanning and measured bmd values ranged from 0.53 to 0.72 g / cm .
cervical spines with degenerative diseases or traumatic pathology were excluded by anteroposterior and lateral screening radiographs before biomechanical testing .
once harvested , all cervical specimens were immediately conserved in plastic bags and frozen at 20c . in preparation for biomechanical testing ,
the required spines were thawed at 4c for 12 hours . at room temperature on the testing day
, the proximal vertebra ( c2 ) and distal vertebra ( t1 ) were separately mounted in a cylindrical container using a low - fusion point ( 72c ) alloy .
and then , the c2 container was attached to the upper fixture while the t1 container was mounted to the lower testing platform .
markers made of 4 plexiglas motion detectors were fixed to the anterior aspects of each vertebra from c2 to t1 .
the 3-dimensional motion range of each vertebra was obtained with an optoelectronics measurement system ( optotrak , northern digital inc . ,
waterloo , ontario , canada ) capable of capturing the motion curve of the markers .
biomechanical tests were performed under displacement control by an mts machine ( cmt6104 ; mts systems ( china ) corp . ,
shenzhen , china ) which can replicate physiologic motion with displacement control ( figure 1 ) .
the flexion extension axis of each tested spine was placed eccentric to the load axis of the actuator .
the bending moments were limited to the upper bound of physiological human bending ( 4.5 nm ) .
an angular displacement transducer was assembled to measure the global rotation of the cervical spine ( c2-t1 ) .
a displacement transducer was used to measure the changes in moment arm length between the upper container and the load axis of the mts machine .
the spine specimens were tested by application of a 50 n preload in flexion - extension and lateral bending circumstances . during the biomechanical tests ,
after analysis of the intact spine , each specimen was sequentially reconstructed at c3-c6 3-level ) motion segments .
the conditions were as follows ( figure 2 ) : acdr , acdf , acdr , or 3-level disc plate disc ( 3dpd ) ; acdf , acdr , acdf or 3-level plate disc plate ( 3pdp ) ; three - level acdf or 3-level plate ( 3p ) . prior to biomechanical tests , positioning of implants
measurements included vertebral motion , applied load , and moment . in the biomechanical tests ,
the acdr was a titanium - ceramic alloy cervical disc ( prestige lp cervical disc , medtronic sofamor danek usa , inc . ) .
the arthrodesis was performed using an interbody cage ( telamon tm , medtronic sofamor danek usa , inc . )
combined with an anterior cervical plating ( acp ) system ( doc cervical plate , depuy spine , inc . , raynham , ma , usa ) ( figure 3 ) .
this biomechanical protocol limited motion to 20 degrees in flexion and extension as well as 15 degrees in lateral bending and axial rotation .
the ratio of segmental rom to the total c2-t1 rom was applied to evaluate the operation effect by normalization method .
one - way anova ( p<0.05 ) was used to determine the statistical differences in both c3-c6 motion and adjacent level motion under the 3-level anterior plate fusion ( 3p ) , the 3pdp , and 3dpd conditions .
as expected , 3-level arthrodesis resulted in significant reduction of rom at the three instrumented levels in all 6 loading conditions ( flexion , extension , left bending , right bending , left rotation , and right rotation ) . compared to intact spines , almost 80% of motion
was successfully restricted at c3-c6 fusion levels in flexion , extension , and lateral bending , as well as 65% in axial rotation . for hybrid constructs , 3dpd
construct resulted in slight increase at the 3 instrumented levels in extension , lateral bending , and axial rotation compared to intact ( p>0.05 ; maximal variation of + 7% ) .
however , the 3dpd condition resulted in a slight decrease at c3-c6 in flexion ( p>0.05 ; maximal variation of 9% ) . as another 3-level hybrid construct ,
3pdp construct resulted in significant decrease of rom at the c3-c6 instrumented levels in all 6 loading conditions except for left rotation ( mean variation of 21% ; maximal variation of 38% ) .
although 3pdp and 3p conditions produced significant motion decrease at the 3 instrumented levels , there was significant difference within the instrumented levels between 3pdp and 3p conditions in all 6 loading conditions ( p<0.05 ) .
on the other hand , there were also significant differences within the c3c6 levels between 3dp d and 3pdp conditions in all 6 loading conditions except for flexion and left rotation ( p<0.05 ) ( figures 4 , 5 ) .
for each instrumented level , 3dpd and 3pdp hybrid constructs caused significant reduction of rom in all 6 loading conditions at the arthrodesis level compared to intact ( p<0.05 ) and produced motion increase at the arthroplasty level .
for the 3dpd hybrid construct , implantation of upper - level ( c3-c4 ) acdr resulted in significant increase of rom only in right rotation ( p<0.05 ; maximal variation of + 36% ) , while implantation of lower - level ( c5-c6 ) acdr resulted in significant increase of rom in all 6 loading conditions except for flexion ( p<0.05 ; maximal variation of + 41% ) .
for 3pdp hybrid construct , implantation of middle - level ( c3-c4 ) acdr resulted in significant increase of rom in extension , left rotation , and right rotation compared to intact ( p<0.05 ; maximal variation of + 67% ) but produced motion increase in flexion , left bending , and right bending , without significant difference ( p>0.05 ) ( figure 6 , table 1 ) .
as suspected , 3-level arthrodesis resulted in an increased contribution of upper and lower adjacent levels .
significant changes were noted at the lower adjacent level in all 6 loading conditions as well as upper adjacent level in flexion and left bending ( p<0.05 ; maximal variation of + 197% ) .
concerning 3dpd hybrid construct , significant changes of motion increase were only noted at lower adjacent level in left bending , right bending , and right rotation ( p<0.05 ; maximal variation of + 57% ) .
importantly , there was a significant decrease toward normal in adjacent segment motion adjacent to an acdr .
concerning 3pdp hybrid construct , significant changes of motion increase were noted at both upper and lower adjacent levels and the largest motion increase was noticed at lower adjacent level in extension ( p<0.05 ; maximal variation of + 79% ) ( figures 4 , 5 ) .
as expected , 3-level arthrodesis resulted in significant reduction of rom at the three instrumented levels in all 6 loading conditions ( flexion , extension , left bending , right bending , left rotation , and right rotation ) . compared to intact spines , almost 80% of motion
was successfully restricted at c3-c6 fusion levels in flexion , extension , and lateral bending , as well as 65% in axial rotation . for hybrid constructs , 3dpd
construct resulted in slight increase at the 3 instrumented levels in extension , lateral bending , and axial rotation compared to intact ( p>0.05 ; maximal variation of + 7% ) .
however , the 3dpd condition resulted in a slight decrease at c3-c6 in flexion ( p>0.05 ; maximal variation of 9% ) . as another 3-level hybrid construct ,
3pdp construct resulted in significant decrease of rom at the c3-c6 instrumented levels in all 6 loading conditions except for left rotation ( mean variation of 21% ; maximal variation of 38% ) .
although 3pdp and 3p conditions produced significant motion decrease at the 3 instrumented levels , there was significant difference within the instrumented levels between 3pdp and 3p conditions in all 6 loading conditions ( p<0.05 ) .
on the other hand , there were also significant differences within the c3c6 levels between 3dp d and 3pdp conditions in all 6 loading conditions except for flexion and left rotation ( p<0.05 ) ( figures 4 , 5 ) .
for each instrumented level , 3dpd and 3pdp hybrid constructs caused significant reduction of rom in all 6 loading conditions at the arthrodesis level compared to intact ( p<0.05 ) and produced motion increase at the arthroplasty level .
for the 3dpd hybrid construct , implantation of upper - level ( c3-c4 ) acdr resulted in significant increase of rom only in right rotation ( p<0.05 ; maximal variation of + 36% ) , while implantation of lower - level ( c5-c6 ) acdr resulted in significant increase of rom in all 6 loading conditions except for flexion ( p<0.05 ; maximal variation of + 41% ) .
for 3pdp hybrid construct , implantation of middle - level ( c3-c4 ) acdr resulted in significant increase of rom in extension , left rotation , and right rotation compared to intact ( p<0.05 ; maximal variation of + 67% ) but produced motion increase in flexion , left bending , and right bending , without significant difference ( p>0.05 ) ( figure 6 , table 1 ) .
as suspected , 3-level arthrodesis resulted in an increased contribution of upper and lower adjacent levels .
significant changes were noted at the lower adjacent level in all 6 loading conditions as well as upper adjacent level in flexion and left bending ( p<0.05 ; maximal variation of + 197% ) .
concerning 3dpd hybrid construct , significant changes of motion increase were only noted at lower adjacent level in left bending , right bending , and right rotation ( p<0.05 ; maximal variation of + 57% ) .
importantly , there was a significant decrease toward normal in adjacent segment motion adjacent to an acdr .
concerning 3pdp hybrid construct , significant changes of motion increase were noted at both upper and lower adjacent levels and the largest motion increase was noticed at lower adjacent level in extension ( p<0.05 ; maximal variation of + 79% ) ( figures 4 , 5 ) .
this study demonstrated that the motion response of an acdr adjacent to a fusion maintains normal motion at the acdr level and normalizes adjacent segment motion in 3-level hybrid constructs . in longer fusions , the intradiscal pressures and the compensatory hyper - mobility
this study confirms the work of lee et al . and others [ 4,1321 ] that a longer fusion affects all adjacent levels with hyper - mobility .
this study shows that an acdr normalized the adjacent level motion in a 3-level construct .
acdr is the accepted alternative to anterior cervical fusion for single - level disc disease .
favorable outcomes and the prospect of a lower incidence of adjacent - level disease have encouraged surgeons to expand current acdr indications to multilevel disc disease .
however , the evidence in multilevel acdr is not as well established as its role in single - level disease , and some levels may be too degenerative for acdr . with that in mind , authors are reporting combinations of fusion and arthroplasty as an alternative to multi - level acdf or acdr .
described 2-level , 3-level , and 4-level hybrid surgery results as safe and reliable without revision .
shin el al . compared hybrid construct to acdf in 2 levels , with improved ndi , pain , return of motion , and reduced asd .
kang el al . confirmed these findings in 3-level hybrid treatments , with improvement in recovery of total motion and maintenance of adjacent segment motion .
jia et al . , in a systematic review of 8 biomechanical and 7 clinical papers , found a paucity of quality evidence to support hybrid surgeries and recommended prospective randomized trials .
previous biomechanical studies have investigated the operative- and adjacent - level kinematic properties of 2-level acdf and combined acdr / acdf .
acdf plus acdr had less severe biomechanical effects on adjacent levels when compared to 2-level acdf procedure .
reported that the acdf at c6-c7 and acdr at c5-c6 produced increased segmental motion at the arthroplasty level , particularly in axial rotation and flexion - extension .
barrey et al . analyzed the biomechanics of acdr placed above acdf and found similar kinematics to single - level acdr and adjacent to a previously implanted acdr .
compared the biomechanics of 2-level acdf and hybrid ; the hybrid construct had a better biomechanical performance than the fusion and the hybrid avoided excessive increase of adjacent level motion and loads .
. found that the location of the fusion ( cephalad or caudad ) did not affect the behavior of the disc replacement .
biomechanical studies investigating the kinematic properties of 3-level hybrid arthroplasty - arthrodesis reconstruction are limited . however , multilevel cervical surgeries , such as 3-level anterior surgeries , are more common in clinical practice .
hanai et al . reported that there was excellent clinical success and a 100% union rate for 3-level and 4-level cervical corpectomy and autograft strut graft reconstruction .
. revealed that the likelihood of pseudarthrosis was 10% for 1-level surgery , 44% for 2-level surgery , and 54% for 3-level surgery .
one study investigated acdr as a promising treatment for symptomatic adjacent level after prior 2-level cervical fusion .
characterized acdr kinematics above 2-level fusion and found that acdr placed adjacent to a 2-level fusion was subjected to a more challenging biomechanical environment as compared to a stand - alone acdr .
two groups reported both 2-level and 3-level hybrid surgeries are comparable to acdf and acdr in terms of safety and feasibility with a minimum follow - up of 2 years .
. stated that hybrid surgeries may be an alternative to acdf for 3-level cervical disease due to the equivalent or improved early clinical outcomes , better overall c2-c7 range of motion , and less impact at adjacent levels .
we found that rom in instrumented levels after 3p condition was systematically reduced in all planes .
in contrast , the 3dpd condition appeared to preserve motion at adjacent intact levels to the acdr segments .
compared to 3dpd condition , rom in instrumented levels after 3pdp condition was slightly reduced but the difference was not significant .
in addition , we did not observe an abnormal increase of motion for 3dpd and 3pdp conditions . at the adjacent levels ,
3p condition resulted in increased adjacent segment motion , especially in the caudal adjacent segment , which may potentially result in accelerated adjacent segment degeneration . on the contrary , and as expected , although 3dpd and 3pdp conditions did not fully restore motion within the instrumented levels , they did not demonstrate hyper - mobility , and in fact induced only minimal changes in rom at adjacent levels .
this confirmed the finding that the hybrid construct could avoid large motion increase in adjacent levels and clinically may reduce the risk of adjacent segment degeneration .
the 3dpd construct did produce a smaller motion change in adjacent segments compared with 3pdp construct .
this study has the limitations of any cadaveric biomechanical study of cervical fusion in that it allows only observation of the immediate effects of the intervention .
it did not remove all motion in the fusion segments and can not represent the long - term effect of increasing stiffness as the fusion progresses .
these biomechanical findings suggest that while hybrid conditions failed to restore normal motion within the construct , they significantly normalized motion in adjacent segments compared with the 3-level acdf condition .
the artificial disc in 3-level constructs has biomechanical advantages compared to fusion in normalizing motion .
|
backgroundthe ideal surgical approach for cervical disk disease remains controversial , especially for multilevel cervical disease .
the purpose of this study was to investigate the biomechanics of the cervical spine after 3-level hybrid surgery compared with 3-level anterior cervical discectomy and fusion ( acdf).material / methodseighteen human cadaveric spines ( c2-t1 ) were evaluated under displacement - input protocol . after intact testing , a simulated hybrid construct or fusion construct was created between c3 to c6 and tested in the following 3 conditions : 3-level disc plate disc ( 3dpd ) , 3-level plate disc plate ( 3pdp ) , and 3-level plate ( 3p).resultscompared to intact , almost 65~80% of motion was successfully restricted at c3-c6 fusion levels ( p<0.05 ) .
3dpd construct resulted in slight increase at the 3 instrumented levels ( p>0.05 ) .
3pdp construct resulted in significant decrease of rom at c3-c6 levels less than 3p ( p<0.05 ) . both 3dpd and 3pdp caused significant reduction of rom at the arthrodesis level and produced motion increase at the arthroplasty level .
for adjacent levels , 3p resulted in markedly increased contribution of both upper and lower adjacent levels ( p<0.05 ) .
significant motion increases lower than 3p were only noted at partly adjacent levels in some conditions for 3dpd and 3pdp ( p<0.05).conclusionsacdf eliminated motion within the construct and greatly increased adjacent motion . artificial cervical disc replacement normalized motion of its segment and adjacent segments .
while hybrid conditions failed to restore normal motion within the construct , they significantly normalized motion in adjacent segments compared with the 3-level acdf condition .
the artificial disc in 3-level constructs has biomechanical advantages compared to fusion in normalizing motion .
|
Background
Material and Methods
Specimen preparation
Biomechanical tests protocol
Reconstruction procedures
Data and statistical analysis
Results
Motion changes at the three instrumented levels
More normal motion with ACDR within construct
Motion changes at adjacent levels
Discussion
Conclusions
|
cutaneous leishmaniasis ( cl ) is endemic in more than 70 countries in the world ( 1 ) .
the available drugs for leishmaniasis treatment are expensive and toxic , cause to severe side effects and there is an increasing incidence of drug resistance ( 2 , 3 ) .
therefore there is a need for an effective vaccine to control and prevent from this disease .
however , in spite of intensive efforts during the past decades , only a few first vaccines generation of whole killed leishmania reached to phase 3 of clinical trials .
it seems that the reason of this failure is mainly due to lack of an appropriate adjuvant ( 2 ) .
an inoculation of live virulent parasites known as leishmanization ( lz ) was practiced in several countries including iran and uzbekistan , due to safety concerns and difficulties in standardization of the injected parasites , lz practice was stopped in different countries except in uzbekistan ( 5 , 6 ) .
a large number of adjutants and delivery systems like bcg , g - csf , il-12 , cpg oligonucleotides , plga microspheres and liposomes have been used to potentiate the immune response against leishmania antigens in animal models ( 3 , 4 , 6 - 8 ) .
mycobacterium bovis -bacillus calmette - gurin ( bcg ) has been used in field trial as an immunoadjuvant against different forms of leishmaniasis .
vaccination with autoclaved l. major ( alm ) mixed with bcg was found to be safe but did not induce significant protective immune response in healthy volunteers against cutaneous leishmaniasis ( 11 , 12 ) ; furthermore in several studies m. bovis bcg inoculation induced autoimmune reactions ( 13 ) .
the only vaccine adjuvant that is approved by fda is alum ( aluminum - based mineral salt ) ( 14 )
. the major limitations of alum is its poor inducer to elicit cell - mediated immunity and t helper 1 ( th1 ) responses that are required to protect against intracellular pathogens ( 15 , 16 ) .
opioids have significant role in the modulation of th 1/th 2 balances ( 17 ) .
naltrexone ( ntx ) , as opioid antagonist , is capable to block -opioid receptors and reduces the positive reinforcing effects of opioids ( 19 , 20 ) .
naltrexone can enhance lymphocyte proliferation and shift the immune response toward a th1 pattern ( 21 ) .
furthermore , that ntx reduce viral replication and inhibit tumor growth ( 19 , 22 , 23 ) .
ntx is a long - acting opioid receptor antagonist that is approved by the fda as a prescription drug and is widely used for treating alcohol and opiate addiction ( 20 ) . in the current study
, we tested the immunogenicity potential of naltrexone alone or in mixture with alum as an adjuvant against heat - killed l. major promastigotes in the susceptible balb / c mice .
female balb / c mice ( 68 weeks old ) were purchased from the razi vaccine and serum research institute of iran .
experiments were performed in accordance with the animal care and use protocol of urmia university of medical sciences .
the l. major strain mrho / ir/75/er was provided from school of public health , university of tehran .
promastigotes of l. major were harvested from stationary growth phase by centrifugation ( 2000 g , 20 min , 4c ) , washed 3 times with cold pbs ( ph 7.2 ) and homogenized with glass - glass homogenizer followed by autoclaving .
total protein content of the autoclaved antigen was determined by bradford method ( each dose containing 40 g / ml of protein parasite ) .
female balb / c mice were divided into five groups ( 10 mice per group ) .
each group was divided to two subgroups , five mice in each subgroup : one subgroup was evaluated for lymphocyte proliferation and shift the immune response to th1/th2 and the second subgroup was assessed for delayed type hypersensitivity ( dth ) and challenge by the parasite.the alum ntx mixture was prepared by thoroughly mixing 50 l of pbs containing neltrexone ( sigma , germany ) at a concentration of 6 mg/ kg with 50 l of alum ( aluminum phosphate , sigma ) .
balb / c mice were immunized subcutaneously three times at 2-week intervals with one of the followings : group vac ( 50 l ag + 100 l pbs /mouse ) , group al vac ( 50 l ag + 50 l al + 50 l pbs /mouse ) , group ntx vac ( 50 l ag + 50 l ntx + 50 l pbs /mouse ) , group al - ntx vac ( 50 l ag + 50 l al + 50 l ntx /mouse ) and control mice received 150 l of pbs .
two weeks after the last immunization , the spleens were aseptically removed ( five mice from each group ) and separately homogenized in incomplete rpmi 1640 ( gibco - brl ) .
erythrocytes were lysed with ammonium chloride ( 0.9% ) and the splenocytes were washed three times with incomplete rpmi 1640 .
the cell concentration was adjusted to 1 10 cells / ml in complete rpmi ( ccm ) containing 10% fbs ( gibco - brl ) , 2 mm l - glutamine , 100 g / ml streptomycin and 100 iu / ml penicillin .
one hundred microliters of diluted cell suspensions were dispensed into 96-well flat - bottom culture plates .
five l of the antigen suspension was added to each well and the volume was adjusted to 200 l with ccm .
control wells were made with 100 l of diluted cell suspension from the same mouse and 100 l of ccm . each mouse s splenocytes were plated in duplicate .
lymphocyte proliferation was measured by an mtt assay ( thiazolyl blue tetrazolium bromide , sigma , germany ) after 48 hours incubation in 37c and 5% co2 .
spleen cells were removed and cultured as above and after 72 h of culture the levels of cytokines ifn- and il-5 in the culture supernatant were measured by commercial kit ( elisa pro kit for mouse ifn- il-5 , mabtech ) .
leishmania major promastigotes were collected at the stationary phase by centrifugation , washed three times with cold pbs and diluted with pbs to have 1 10 promastigotes per ml .
three weeks after the last immunization , dth was assessed by sc injection of 50 l from this promastigote crude lysate into left footpad . as a control ,
four weeks after last immunization , all groups ( 5 mice per group ) were challenged subcutaneously in the tail base of the mice with 1 10 stationary phase promastigotes . lesion development and survival rates of the vaccinated and control mice were weekly monitored after the parasite challenge .
the l. major strain mrho / ir/75/er was provided from school of public health , university of tehran .
promastigotes of l. major were harvested from stationary growth phase by centrifugation ( 2000 g , 20 min , 4c ) , washed 3 times with cold pbs ( ph 7.2 ) and homogenized with glass - glass homogenizer followed by autoclaving .
total protein content of the autoclaved antigen was determined by bradford method ( each dose containing 40 g / ml of protein parasite ) .
female balb / c mice were divided into five groups ( 10 mice per group ) .
each group was divided to two subgroups , five mice in each subgroup : one subgroup was evaluated for lymphocyte proliferation and shift the immune response to th1/th2 and the second subgroup was assessed for delayed type hypersensitivity ( dth ) and challenge by the parasite.the alum ntx mixture was prepared by thoroughly mixing 50 l of pbs containing neltrexone ( sigma , germany ) at a concentration of 6 mg/ kg with 50 l of alum ( aluminum phosphate , sigma ) .
balb / c mice were immunized subcutaneously three times at 2-week intervals with one of the followings : group vac ( 50 l ag + 100 l pbs /mouse ) , group al vac ( 50 l ag + 50 l al + 50 l pbs /mouse ) , group ntx vac ( 50 l ag + 50 l ntx + 50 l pbs /mouse ) , group al - ntx vac ( 50 l ag + 50 l al + 50 l ntx /mouse ) and control mice received 150 l of pbs .
two weeks after the last immunization , the spleens were aseptically removed ( five mice from each group ) and separately homogenized in incomplete rpmi 1640 ( gibco - brl ) .
erythrocytes were lysed with ammonium chloride ( 0.9% ) and the splenocytes were washed three times with incomplete rpmi 1640 .
the cell concentration was adjusted to 1 10 cells / ml in complete rpmi ( ccm ) containing 10% fbs ( gibco - brl ) , 2 mm l - glutamine , 100 g / ml streptomycin and 100 iu / ml penicillin .
one hundred microliters of diluted cell suspensions were dispensed into 96-well flat - bottom culture plates .
five l of the antigen suspension was added to each well and the volume was adjusted to 200 l with ccm .
control wells were made with 100 l of diluted cell suspension from the same mouse and 100 l of ccm . each mouse s splenocytes were plated in duplicate .
lymphocyte proliferation was measured by an mtt assay ( thiazolyl blue tetrazolium bromide , sigma , germany ) after 48 hours incubation in 37c and 5% co2 .
spleen cells were removed and cultured as above and after 72 h of culture the levels of cytokines ifn- and il-5 in the culture supernatant were measured by commercial kit ( elisa pro kit for mouse ifn- il-5 , mabtech ) .
leishmania major promastigotes were collected at the stationary phase by centrifugation , washed three times with cold pbs and diluted with pbs to have 1 10 promastigotes per ml .
three weeks after the last immunization , dth was assessed by sc injection of 50 l from this promastigote crude lysate into left footpad . as a control ,
four weeks after last immunization , all groups ( 5 mice per group ) were challenged subcutaneously in the tail base of the mice with 1 10 stationary phase promastigotes .
lesion development and survival rates of the vaccinated and control mice were weekly monitored after the parasite challenge .
1 , the mean level of mtt in the mice immunized with al ntx vac and ntx vac induced significantly higher lymphocyte proliferation comparing to the control group ( p < 0.001and p < 0.01 , respectively ) .
spleen cell proliferation with al ntx vac ( p < 0.001 ) was significantly higher than al- vac and vac groups .
furthermore lymphocyte proliferation was significantly higher in mice immunized with the ntx vac compared with mice that received antigen alone ( p < 0.05 ) .
there was no significant difference in lymphocyte proliferation between other groups . as it is shown in fig .
2 , the supernatant of splenocytes of mice immunized with al ntx vac or ntx vac showed significantly the highest ( p < 0.01 ) level of ifn- compared with the other groups .
the mice of ntx- vac group produced the highest amounts of il-5 but ifn- to il-5 ratio was higher in al - ntx- vac group , none of them proofed as significant ( table 1 ) . to assess cell - mediated responses to parasite antigens in vivo
the results of dth showed that different groups of immunized mice induced a stronger dth response than pbs control group ( fig 3 ) ; however , the difference was not statistically significant except for the al ntx vac group ( p < 0 .
stationary phase promastigotes of l.major were injected subcutaneously at the tail base of 5 mice from each group , an injection site was analyzed over a period of 10 weeks .
there was no significant difference between immunized and control groups of mice , during 10 weeks after challenge with l. major ( data not shown ) .
1 , the mean level of mtt in the mice immunized with al ntx vac and ntx vac induced significantly higher lymphocyte proliferation comparing to the control group ( p < 0.001and p < 0.01 , respectively ) .
spleen cell proliferation with al ntx vac ( p < 0.001 ) was significantly higher than al- vac and vac groups .
furthermore lymphocyte proliferation was significantly higher in mice immunized with the ntx vac compared with mice that received antigen alone ( p < 0.05 ) .
2 , the supernatant of splenocytes of mice immunized with al ntx vac or ntx vac showed significantly the highest ( p < 0.01 ) level of ifn- compared with the other groups .
the mice of ntx- vac group produced the highest amounts of il-5 but ifn- to il-5 ratio was higher in al - ntx- vac group , none of them proofed as significant ( table 1 ) .
to assess cell - mediated responses to parasite antigens in vivo , the dth reaction was measured 72 h post infection .
the results of dth showed that different groups of immunized mice induced a stronger dth response than pbs control group ( fig 3 ) ; however , the difference was not statistically significant except for the al ntx vac group ( p < 0 . 01 ) .
stationary phase promastigotes of l.major were injected subcutaneously at the tail base of 5 mice from each group , an injection site was analyzed over a period of 10 weeks .
there was no significant difference between immunized and control groups of mice , during 10 weeks after challenge with l. major ( data not shown ) .
in this study , alum naltrexone as a th1 immunostimulatory adjuvant has been used for the first time with autoclaved l. major to elicit a protective immune response challenge with the parasite in susceptible balb / c mice . our selection to test naltrexone in this paper was based on reports of the literature indicating that has been effective in shifting immune responses to some antigens from a th2 response to th1 immune responses ( 21 - 23 ) .
brown et al . suggested that low - dose naltrexone presents a safe and promising approach to prevention and/or treatment of many autoimmune diseases and cancer variants , as well as potentially various viral ( aids ) and neurological diseases ( multiple sclerosis ) that are exacerbated by compromised immunity ( 22 ) .
moreover , naltrexone was reported to stimulate ifn- production and induced effective immune responses against fibrosarcoma tumor and led to a significant inhibition of tumor growth in balb / c mice ( 23 ) .
the finding of the current study about adjuvant activity of naltrexone and previous studies about adjuvant activity of naloxone ( which are structurally similar pure opiate receptor antagonists ) and propranolol ( 24 - 28 ) , emphasizes that the local microenvironment at the time of up taking and processing an antigen by apcs has a key role in the fate of subsequent acquired immune response against infection ( 29 , 30 ) .
one important mechanism for adjuvant activity of naltrexone is blocking the opioid receptors which accelerates local inflammation via direct effect on monocytes , macrophages and dendritic cells ( 31 , 32 ) .
ifn- secreted by t cells , plays a key role in protection against intracellular infectious agents and is the cytokine primarily used as a marker for the existence of th1 immune responses .
cytokine analysis revealed that the administration of leishmania proteins with al ntx or ntx induced high levels of th1 cytokine ( ifn- ) .
furthermore , the spleens of mice immunized with al ntx vac showed significantly the highest ifn- /il-5 ratio compared with the other immunized groups which is an important correlate of immune protection against l. major .
these results are in agreement with other studies showing that ntx induce th1 type of immune response ( 23 , 32 ) .
ntx can shift the immune response toward a th1 pattern while alum is a th2-type adjuvant . however , despite the opposing effects of these adjuvants on skew the immune system toward a th1/th2 response , our results show that co - administration of naltrexone with alum more skewed the immune response in balb / c mice towards a th1-type than administration of naltrexone alone .
evaluation of lymphocyte proliferation response revealed that immunization of mice with al ntx vac stimulate the proliferation of spleen cells significantly moreover footpad swelling after infectious with l. major was measured and the results demonstrated that al ntx vac elicited strong dth responses .
unexpectedly , immunization of balb / c mice with different groups and challenging with l. major did not show any significant differences in the death rate until 10 weeks after infection .
immunization with the alum naltrexone mixture as an adjuvant , in combination with the autoclaved l. major promastigotes antigens , can enhance cellular immunity and shift the immune responses to a th1 pattern .
to our awareness , this study is the first evaluation for using an alum naltrexone mixture as an adjuvant for vaccination .
therefore , follow - up studies are needed to elucidate the effect of naltrexone on the mice immune system and to examine adjuvant activity of naltrexone when combined with vaccines against other microorganisms .
|
backgroundnaltrexone , an opioid receptor antagonist shifts the immune response toward a th1 profile . in the current study , we evaluated the efficacy of the mixture of ntx and alum , as a new adjuvant , to enhance immune response and induce protection against leishmania major in a mouse model.methodsbalb/c mice were immunized three times either autoclaved l. major promastigotes antigens alone or in combination with the adjuvant alum , naltrexone or the alum
naltrexone mixture . both humoral and cellular immune responses
were assessed two weeks after the last immunization and compared with control mice.resultsthe administration of alum- ntx in combination with the parasite antigen , significantly increased production of ifn- ifn- /il-5 ratio , lymphocyte proliferation and improved dth response against l. major .
there was no significant difference in survival following challenge among groups.conclusionimmunization with the alum naltrexone mixture as an adjuvant , in combination with the autoclaved l. major promastigotes antigens , can enhance cellular immunity and shift the immune responses to a th1 pattern .
|
Introduction
Materials and Methods
Parasites and sample preparation
Immunization of BALB/c mice
Lymphocyte proliferation test
Cytokines analysis
Delayed type hypersensitivity (DTH) assessment
Challenge with live promastigotes of L. major
Statistical analysis
Results
Lymphocyte proliferation
In vitro cytokine production by splenocytes
Delayed type hypersensitivity responses (DTH)
Challenge results
Discussion
Conclusion
|
psoroptid mites of the genus caparinia affect hedgehogs and a few other mammals causing skin disease .
the mite passes all stages on the host and feeds on sloughed skin cells and epidermal debris , similar to chorioptes species . among 5 species of the genus caparinia
that have previously been classified , 2 species are known to infest hedgehogs ; c. tripilis and c. erinacei . among them , c. tripilis shows higher pathogenicity than c. erinacei and may burrow into the skin of the head , ears , flanks , and inner sides of the legs where they form clusters .
it can cause irritation , dermatitis , and self - trauma due to pruritus which can lead to a secondary infection and the host may eventually die .
first reported in england in 1889 by michael who found very active mites on the surface of hedgehogs and described " running up and down the spines of hedgehogs with great rapidity " , the mite was introduced to new zealand but received little attention until brokie examined 100 hedgehogs from wellington between 1954 and 1957 . in relatively recent years , c. tripilis has also been introduced to new mexico , united states , through breeding colonies of african hedgehogs for sale as pets .
so far , the mite has not been reported in asia , possibly because the animal has neither been popular as pet nor has been considered as endangered species . although there has been a great increase in the population of hedgehogs as pets , limited studies on the ectoparasitic diseases of hedgehogs are available .
we report in this paper an outbreak of dermatitis in hedgehogs caused by c. tripilis with description of keys for the identification of the family psoroptidae and the genus caparinia .
in february 2010 , dermatitis characterized by scale and self - trauma due to pruritus was recognized in a group of 22 four - toed hedgehogs ( a. albiventris wagner , 1841 ) from a local pet shop in gwangju , korea .
three of the hedgehogs were males and the rest were females with an average weight of 339.3 g. the mantle of the skin was flaking , and scales and crusts were present .
two of the severely affected hedgehogs died of self - trauma and secondary bacterial infections . for parasitological examinations , skin scraping samples from severely affected areas of the skin were collected and preserved in 70% methanol . for morphological comparison , adult male mites of otodectes cynotis and of chorioptes texanus were obtained from naturally - infested dogs and a holstein cow , respectively .
the identification of the genus caparinia was based on the key provided by lawrence and description by fain .
body length and width of 5 mites per each developmental stage were measured ( table 1 ; fig .
tarsal caruncles were bell - shaped on all legs of male while they were absent on legs iii and iv of females .
there were 3 long setae on the third pair of legs in both sexes ( fig .
adult males had a posterior end of the abdomen with a trilobate projection on each side , each lobe with a long seta ( fig .
average length and width of adult males and females were 313.6240.5 m and 418.6287.1 m , respectively . based on these morphological features ,
4 ) , and the quadrilateral space ( fig . 3a , double - sided arrow ) of the abdomen of males was wider than its length .
a distinctive feature was that they had 3 long setae both on the third and fourth pairs of legs .
two pairs of long setae which were present at the posterior end of the body were well separated from the posterior margin ( fig .
long humeral seta of the dorsal surface was inserted in a small distinct oval scutum ( fig .
based on these features , the species of the mite was identified as c. tripilis .
this study reports the first outbreak of c. tripilis infestation in a colony of african pygmy hedgehogs in korea .
the genus caparinia belongs to the family psoroptidae and is closely related to chorioptes except for the presence of a caruncle on leg iv of female mites and to otodectes except for the presence of posterior lobate projections in male mites ( fig .
3b ) . caparinia spp . infest hedgehogs and some other mammals , including the cape polecat , hyena and european fox leading to skin diseases .
since this is the first report of caparinic mite infestation in korea , identification keys for the family psoroptidae and the genus caparinia that have been modified from lawrence , fain , and michael are provided at the end of the discussion .
the genus caparinia have been classified into 3 well - documented species ; c. tripilis , c. erinacei , and c. ictonyctis .
two additional species , c. setifera ( mgnin , 1880 ) and c. vulpis ( mgnin , 1880 ) , were mentioned by lawrence but his description was based only on females and were too brief to differentiate between these 2 species and c. tripilis . for this reason , the diagnostic key provided in this paper includes features of female mites of 5 species , while features of male mites of c. setifera and c. vulpis are not provided .
the taxonomic status of c. tripilis and c. ictonyctis is based on the mophological characteristics of adult male mites which differ from c. erinacei by the number of lobes on each laminate projection on the posterior end of the body and the number of the setae attached on each lobe .
male mites of c. tripilis and c. ictonyctis have 3 paired posterior lobes , each equipped with a long seta , whereas c. erinacei has 2 paired lobes with a long seta on each lobe .
the presence of posterior lobate projections is also an important feature when it is needed to be distinguished from the genus otodectes which does not have these lobate projections ( fig .
3b ) . the posterior dorsal scutum is wider than its length in c. tripilis , whereas that in c. ictonyctis is longer than its width ( fig .
4 ) . furthermore , while the 2 posterior projections of males enclose a quadrilateral space which is as long as its width in c. ictonyctis and wider than its length in c. tripilis ( fig .
features of adult female mites of c. tripilis are very similar to those of both c. ictonyctis and c. erinacei .
legs iii and iv do not have a caruncle , which is a characteristic feature to distinguish from chorioptes spp .
female mites have 3 long setae at the end of the third and fourth pairs of legs whereas male mites have those on only leg iii . on the other hand ,
two pairs of long setae were present at the posterior end of the body of adult females ( fig . 2i , black arrow ) .
furthermore , 1 of the distinctive features of c. tripilis females is that the insertion of posterior abdominal setae is well separated from the posterior margin .
by contrast , both c. ictonyctis and c. erinacei had posterior abdominal setae inserted on the edge of the posterior margin .
the life cycle of c. tripilis includes an egg , larva , protonymph , deutonymph ( including pubescent female ) , and either an adult male or an adult female .
two bosses are present on the surface of an egg and are on the same side of the cleavage ( fig .
the adult males and pubescent females form an attachment pair which is more or less permanent up until the time of emergence of the adult females from the deutonymphal exuviae ( fig .
2h ) . except for the presence of a couple of copulatory tubercles at the posterior end of a pubescent female ( female deutonymph , fig . 2f , arrow head ) ,
egg , larvae , protonymph , and deutonymph stages of caparinia do not show sexual dimorphism .
observations suggest that the complete life cycle encompass about 3 weeks . c. tripilis may burrow into the skin of hedgehogs , and symptoms may include pruritus , hair loss , spine loss , deformation of the ears , and scaly , encrusted skin lesions , leading to secondary infections . severely infected animals
become feeble , lose weight , scratch the affected skin , and may abandon their normal nocturnal behavior to become active in the daytime .
c. erinacei , on the other hand , has low pathogenicity and does not form clusters on its hosts . a sarcoptic mange mite , notoedres muris ,
one of the most common pet breed of hedgehogs is the african pygmy hedgehog or 4-toed hedgehog ( a. albiventris ) . it is smaller than the western european hedgehog , has a white abdomen , and is characterized by lacking the first toe of the hind leg .
the animal had previously been classified in the genus erinaceus ( macdonald , 1986 ) like western european hedgehogs , but recently it has been reclassified as part of the genus atelerix ( wilson , 1993 ) .
hedgehogs are becoming popular pet animals but relatively little research has been carried out on their ectoparasitic diseases . as previously stated , this is the first outbreak of c. tripilis in a colony of hedgehogs in korea .
some identification key aspects regarding the morphological features of the family psoroptidae and the genus caparinia are provided .
the identification keys in this article are adapted from lawrence , fain , and michael .
pedicel of tarsal caruncles segmented and long -- psoroptespedicle of tarsal caruncles not segmented , short ----------- 2only leg iii of the female without a caruncle ---- choriopteslegs iii and iv of the female without a caruncle ----------- 3posterior of abdomen of male with paired lobate projections ; tarsus iii in both sexes with 3 long setae - capariniaposterior of abdomen of male without lobate projections ; tarsus iii in both sexes with 2 long setae ----------- otodectes
pedicel of tarsal caruncles segmented and long -- psoroptes pedicle of tarsal caruncles not segmented , short ----------- 2 only leg iii of the female without a caruncle ---- chorioptes legs iii and iv of the female without a caruncle ----------- 3 posterior of abdomen of male with paired lobate projections ; tarsus iii in both sexes with 3 long setae - caparinia posterior of abdomen of male without lobate projections ; tarsus iii in both sexes with 2 long setae ----------- otodectes
posterior end of abdomen with bilobate laminate projection on either side , each lobe with a long seta , host hedgehog ----------------------------------------------------------- c. erinaceiposterior end of abdomen with two trilobate laminate projections , each lobe with a long seta ----------------------- 2posterior dorsal scutum is wider than it is long , quadrilateral space of abdomen is wider than it is long , side of the body are rounded , larger than c. ictonyctis , host hedgehog -------------------------------------------------------------------- c. tripilisposterior dorsal scutum is longer than it is
wide , quadrilateral space of abdomen is as long as it is wide , side of the body are parallel , smaller than c. tripilis , host cape polecat ----------------------------------------------------------- c. ictonyctis
posterior end of abdomen with bilobate laminate projection on either side , each lobe with a long seta , host hedgehog ----------------------------------------------------------- c. erinacei posterior end of abdomen with two trilobate laminate projections , each lobe with a long seta ----------------------- 2 posterior dorsal scutum is wider than it is long , quadrilateral space of abdomen is wider than it is long , side of the body are rounded , larger than c. ictonyctis , host hedgehog -------------------------------------------------------------------- c. tripilis posterior dorsal scutum is longer than it is wide , quadrilateral space of abdomen is as long as it is wide , side of the body are parallel , smaller than c. tripilis , host cape polecat ----------------------------------------------------------- c. ictonyctis
abdomen with 2 pairs of posterior setae --------------------- 2abdomen with 1 pair of posterior setae ---------------------- 4insertion of posterior abdominal setae well separated from posterior margin ; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilisinsertion of posterior abdominal setae on the edge of posterior margin ; humeral seta of dorsal surface not inserted in a scutum , or this scutum very indistinct ------------------ 3host : cape polecat -------------------------------------- c. ictonyctishost : hedgehog ------------------------------------------- c. erinacei
length and width of body 360280 m ; posterior abdominal setae as long as body ; host , hyena --------- c. setiferalength and width of body 450400 m ; posterior abdominal setae half as long as those of c. setifera ; host ,
european fox -------------------------------------------------------- c. vulpis
abdomen with 2 pairs of posterior setae --------------------- 2 abdomen with 1 pair of posterior setae ---------------------- 4 insertion of posterior abdominal setae well separated from posterior margin ; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilis insertion of posterior abdominal setae on the edge of posterior margin ; humeral seta of dorsal surface not inserted in a scutum , or this scutum very indistinct ------------------ 3 host : cape polecat -------------------------------------- c. ictonyctis host : hedgehog ------------------------------------------- c. erinacei
length and width of body 360280 m ; posterior abdominal setae as long as body ; host , hyena --------- c. setifera length and width of body 450400 m ; posterior abdominal setae half as long as those of c. setifera ; host , european fox -------------------------------------------------------- c. vulpis
pedicel of tarsal caruncles segmented and long -- psoroptespedicle of tarsal caruncles not segmented , short ----------- 2only leg iii of the female without a caruncle ---- choriopteslegs iii and iv of the female without a caruncle ----------- 3posterior of abdomen of male with paired lobate projections ; tarsus iii in both sexes with 3 long setae - capariniaposterior of abdomen of male without lobate projections ; tarsus iii in both sexes with 2 long setae ----------- otodectes
pedicel of tarsal caruncles segmented and long -- psoroptes pedicle of tarsal caruncles not segmented , short ----------- 2 only leg iii of the female without a caruncle ---- chorioptes legs iii and iv of the female without a caruncle ----------- 3 posterior of abdomen of male with paired lobate projections ; tarsus iii in both sexes with 3 long setae - caparinia posterior of abdomen of male without lobate projections ; tarsus iii in both sexes with 2 long setae ----------- otodectes
posterior end of abdomen with bilobate laminate projection on either side , each lobe with a long seta , host hedgehog ----------------------------------------------------------- c. erinaceiposterior end of abdomen with two trilobate laminate projections , each lobe with a long seta ----------------------- 2posterior dorsal scutum is wider than it is long , quadrilateral space of abdomen is wider than it is long , side of the body are rounded , larger than c. ictonyctis , host hedgehog -------------------------------------------------------------------- c. tripilisposterior dorsal scutum is longer than it is
wide , quadrilateral space of abdomen is as long as it is wide , side of the body are parallel , smaller than c. tripilis , host cape polecat ----------------------------------------------------------- c. ictonyctis
posterior end of abdomen with bilobate laminate projection on either side , each lobe with a long seta , host hedgehog ----------------------------------------------------------- c. erinacei posterior end of abdomen with two trilobate laminate projections , each lobe with a long seta ----------------------- 2 posterior dorsal scutum is wider than it is long , quadrilateral space of abdomen is wider than it is long , side of the body are rounded , larger than c. ictonyctis , host hedgehog -------------------------------------------------------------------- c. tripilis posterior dorsal scutum is longer than it is wide , quadrilateral space of abdomen is as long as it is wide , side of the body are parallel , smaller than c. tripilis , host cape polecat ----------------------------------------------------------- c. ictonyctis
abdomen with 2 pairs of posterior setae --------------------- 2abdomen with 1 pair of posterior setae ---------------------- 4insertion of posterior abdominal setae well separated from posterior margin ; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilisinsertion of posterior abdominal setae on the edge of posterior margin ; humeral seta of dorsal surface not inserted in a scutum , or this scutum very indistinct ------------------ 3host : cape polecat -------------------------------------- c. ictonyctishost : hedgehog ------------------------------------------- c. erinacei
length and width of body 360280 m ; posterior abdominal setae as long as body ; host , hyena --------- c. setiferalength and width of body 450400 m ; posterior abdominal setae half as long as those of c. setifera ; host , european fox -------------------------------------------------------- c. vulpis
abdomen with 2 pairs of posterior setae --------------------- 2 abdomen with 1 pair of posterior setae ---------------------- 4 insertion of posterior abdominal setae well separated from posterior margin ; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilis insertion of posterior abdominal setae on the edge of posterior margin ; humeral seta of dorsal surface not inserted in a scutum , or this scutum very indistinct ------------------ 3 host : cape polecat -------------------------------------- c. ictonyctis host : hedgehog ------------------------------------------- c. erinacei
length and width of body 360280 m ; posterior abdominal setae as long as body ; host , hyena --------- c. setifera length and width of body 450400 m ; posterior abdominal setae half as long as those of c. setifera ; host , european fox -------------------------------------------------------- c. vulpis
pedicel of tarsal caruncles segmented and long -- psoroptespedicle of tarsal caruncles not segmented , short ----------- 2only leg iii of the female without a caruncle ---- choriopteslegs iii and iv of the female without a caruncle ----------- 3posterior of abdomen of male with paired lobate projections ; tarsus iii in both sexes with 3 long setae - capariniaposterior of abdomen of male without lobate projections ; tarsus iii in both sexes with 2 long setae ----------- otodectes
pedicel of tarsal caruncles segmented and long -- psoroptes pedicle of tarsal caruncles not segmented , short ----------- 2 only leg iii of the female without a caruncle ---- chorioptes legs iii and iv of the female without a caruncle ----------- 3 posterior of abdomen of male with paired lobate projections ; tarsus iii in both sexes with 3 long setae - caparinia posterior of abdomen of male without lobate projections ; tarsus iii in both sexes with 2 long setae ----------- otodectes
posterior end of abdomen with bilobate laminate projection on either side , each lobe with a long seta , host hedgehog ----------------------------------------------------------- c. erinaceiposterior end of abdomen with two trilobate laminate projections , each lobe with a long seta -----------------------
2posterior dorsal scutum is wider than it is long , quadrilateral space of abdomen is wider than it is long , side of the body are rounded , larger than c. ictonyctis , host hedgehog -------------------------------------------------------------------- c. tripilisposterior dorsal scutum is longer than it is wide , quadrilateral space of abdomen is as long as it is wide , side of the body are parallel , smaller than c. tripilis , host cape polecat ----------------------------------------------------------- c. ictonyctis
posterior end of abdomen with bilobate laminate projection on either side , each lobe with a long seta , host hedgehog ----------------------------------------------------------- c. erinacei posterior end of abdomen with two trilobate laminate projections , each lobe with a long seta ----------------------- 2 posterior dorsal scutum is wider than it is long , quadrilateral space of abdomen is wider than it is long , side of the body are rounded , larger than c. ictonyctis , host hedgehog -------------------------------------------------------------------- c. tripilis posterior dorsal scutum is longer than it is wide , quadrilateral space of abdomen is as long as it is wide , side of the body are parallel , smaller than c. tripilis , host cape polecat ----------------------------------------------------------- c. ictonyctis
abdomen with 2 pairs of posterior setae --------------------- 2abdomen with 1 pair of posterior setae ---------------------- 4insertion of posterior abdominal setae well separated from posterior margin ; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilisinsertion of posterior abdominal setae on the edge of posterior margin ; humeral seta of dorsal surface not inserted in a scutum , or this scutum very indistinct ------------------ 3host : cape polecat -------------------------------------- c. ictonyctishost : hedgehog ------------------------------------------- c. erinacei
length and width of body 360280 m ; posterior abdominal setae as long as body ; host , hyena --------- c. setiferalength and width of body 450400 m ; posterior abdominal setae half as long as those of c. setifera ; host , european fox -------------------------------------------------------- c. vulpis
abdomen with 2 pairs of posterior setae --------------------- 2 abdomen with 1 pair of posterior setae ---------------------- 4 insertion of posterior abdominal setae well separated from posterior margin ; long humeral seta of dorsal surface inserted in a small distinct oval scutum --------------- c. tripilis insertion of posterior abdominal setae on the edge of posterior margin ; humeral seta of dorsal surface not inserted in a scutum , or this scutum very indistinct ------------------ 3 host : cape polecat -------------------------------------- c. ictonyctis host : hedgehog ------------------------------------------- c. erinacei
length and width of body 360280 m ; posterior abdominal setae as long as body ; host , hyena --------- c. setifera length and width of body 450400 m ; posterior abdominal setae half as long as those of c. setifera ; host , european fox -------------------------------------------------------- c. vulpis
|
in february 2010 , dermatitis characterized by scale and self - trauma due to puritis was recognized in a group of 22 four - toed hedgehogs ( atelerix albiventris wagner , 1841 ) from a local pet shop in gwangju , korea .
microscopic examinations of skin scraping samples showed numerous mites of all developmental stages .
morphologically , pedicels of adult mites were short and unjointed .
tarsal caruncles were bell - shaped on all legs of males while they were absent on legs iii and iv of females .
three long setae on the third pair of legs in both sexes were present .
adult males had posterior end of the abdomen with trilobate projection on each side , each lobe with a long seta .
based on these features , the mites were identified as caparinia tripilis .
this is the first report of caparinic mite infestation in hedgehogs from korea .
identification keys for the family psoroptidae and the genus caparinia are provided .
|
INTRODUCTION
CASE DESCRIPTION
DISCUSSION
Identification keys for the family Psoroptidae and the genus
Key to the genera of the Family Psoroptidae
Key to the species of
Key to the species of
|
under innate immune conditions , the inflammatory responses mediated by macrophages , mast cells , and neutrophils comprise an important barrier against infectious pathogens , such as viruses , fungi , and bacteria , as well as chemical toxins [ 1 , 2 ] . among the cellular components of innate immunity
, macrophages are regarded as central inflammatory cells , as they identify external pathogens using special surface receptors ( e.g. , toll - like receptors ( tlrs ) ) and are widely distributed in the human body .
the inflammatory responses mediated by macrophages and their role in pathophysiology have been previously studied .
activated macrophages induce various intracellular signaling cascades , including src , syk , phosphatidylinositide 3-kinase ( pi3k ) , akt , inhibitor of b ( ib ) kinase ( ikk ) , and ib [ 46 ] .
the signaling pathway also stimulates the nuclear translocation of nuclear factor- ( nf- ) b and activator protein ap-1 , triggering the expression of inflammatory genes that lead to secretion of inflammatory mediators ( e.g. , nitric oxide ( no ) , reactive oxygen species ( ros ) , prostaglandin e2 ( pge2 ) , chemokines , and cytokines ( e.g. , tumor necrosis factor- ( tnf- ) ) ) [ 79 ] .
recently , ample evidence has suggested that unchecked , prolonged inflammatory responses can cause serious immunological diseases , including diabetes , septic shock , cancer , arthritis , and cardiovascular disease .
the understanding of inflammatory responses and exploration of strategies for suppressing inflammation are thus considered appropriate approaches to reducing disease incidence [ 1013 ] .
the cordyceps genus including cordyceps sinensis , cordyceps militaris , cordyceps pruinosa , and cordyceps bassiana grow in korea , japan , china , and the congo .
the cordyceps genus can be administered through traditional routes and is known to ameliorate various inflammatory diseases , including chronicle bronchitis , asthma , and eczema .
the biological and pharmacological activities of cordyceps genus are antioxidative , antiviral , antifibrotic , anti - inflammatory , antinociceptive , antiangiogenic , antiplatelet aggregation , and antidiabetic [ 14 , 15 ] .
studies have also demonstrated the anti - inflammatory mechanisms of butanol ( bf ) and hexane ( hf ) fractions of cordyceps bassiana . however , the specific chemical compounds responsible for the plant 's anti - inflammatory properties have not yet been elucidated .
recently , we isolated a promising novel compound [ kth-13 : 4-isopropyl-2,6-bis(1-phenylethyl)phenol ] with anticancer activity from cordyceps bassiana . despite the novel chemical structure of this compound ,
we have established a method for its total synthesis and derivatization to develop more effective molecules .
so far , almost 60 compounds were newly synthesized and tested to check their activities by employing no assay and antiproliferative activity . of them , interestingly , kth-13-amine - diastereomer 1 [ 4-isopropyl-2,6-bis(1-phenylethyl)aniline 1 ( kth-13-ad1 ) ] has been reported to have stronger activity than that of the original compound in terms of anticancer activity ( data not shown ) . in this study , therefore , we further aimed to demonstrate the anti - inflammatory potential of kth-13-ad1 , a derivative of kth-13 , and to explore its mechanism of action using activated macrophages .
sodium nitroprusside ( snp ) , 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) , dihydrorhodamine 123 ( dhr123 ) , fluorescein isothiocyanate- ( fitc- ) dextran , ascorbic acid , and lps ( e. coli 0111:b4 ) were purchased from sigma chemical co. ( st . louis , mo , usa ) . fetal bovine serum and rpmi 1640
the murine macrophage cell line raw264.7 and human embryonic kidney ( hek ) 293 cells were purchased from the american type culture collection ( rockville , md , usa ) .
luciferase constructs containing binding promoters for nf-b and ap-1 were gifts from professors chung , hae young ( pusan national university , pusan , korea ) and rhee , man hee ( kyungpook national university , daegu , korea ) .
phospho- and total protein - specific antibodies to p65 , p50 , c - fos , c - jun , ib , ikk , akt , p85 , src , syk , lamin a / c , and -actin were obtained from cell signaling technology ( beverly , ma , usa ) .
primers ( table 1 ) designed in our laboratory were synthesized by bioneer ( daejeon , korea ) . to synthesize kth-13-ad1 ( figure 1(a ) ) , a solution of 4-isopropylaniline ( 4.00 g , 29.6 mmol ) in xylene ( 14 ml ) was mixed with styrene ( 9.48 g , 91.1 mmol ) and cf3so3h ( 1.0 ml , 11.4 mmol ) .
the reaction mixture was allowed to heat to 160c and stirred for 24 h. at that time , the reaction was allowed to cool to room temperature and the volatiles were removed under vacuo .
the resulting residue was purified by silica gel column chromatography ( hexanes : etoac = 9 : 1 ) to afford the desired kth-13-ad1 ( 2.60 g , 7.57 mmol , and 1 : 1 diastereomers ) in 26% yield as a brown oil . infrared ( ir ) spectra of kth-13-ad1 were recorded on a bruker vertex 70 spectrophotometer , max in cm .
bands are characterized as strong ( s ) , medium ( m ) , and weak ( w ) .
h nuclear magnetic resonance ( nmr ) spectra of this compound were recorded on a jeol jnm - al400 ( 400 mhz ) spectrometer .
chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard ( cdcl3 : 7.27 ppm ) .
data are reported as follows : chemical shift , multiplicity ( s = singlet , d = doublet , t = triplet , q = quartet , and m = multiplet ) , coupling constants ( hz ) , and integration .
c nmr spectra were recorded on a jeol jnm - al400 ( 100 mhz ) spectrometer with complete proton decoupling .
chemical shifts are reported in ppm from tetramethylsilane with the solvent resonance as the internal standard ( cdcl3 : 77.16 ppm ) .
low - resolution mass spectrometry was performed on an agilent 6890n gc ( hewlett - packard co. , palo alto , california , usa ) .
h nmr ( 400 mhz , cdcl3 , diastereomer 1 ) : 7.407.12 ( m , 12h ) , 4.07 ( q , j = 6.9 hz , 2h ) , 3.29 ( br s , 2h ) , 3.00 ( sept , j = 6.9 hz , 1h ) , 1.67 ( d , j = 7.1 hz , 6h ) , 1.37 ( d , j = 7.1 hz , 6h ) ; c nmr ( 100 mhz , cdcl3 ) : 145.8 , 139.4 , 138.9 , 130.2 , 128.7 , 127.5 , 126.3 , 123.5 , 40.2 , 33.7 , 24.4 , 22,2 ; ir ( neat ) : 3471 ( s ) , 3384 ( s ) , 3060 ( m ) , 3024 ( m ) , 2982 ( s ) , 2869 ( s ) , 2293 ( w ) , 1947 ( w ) , 1878 ( w ) , 1803 ( w ) , 1623 ( s ) , 1600 ( s ) , 1471 ( s ) , 1450 ( s ) , 1373 ( m ) , 1318 ( m ) , 1257 ( m ) , 1172 ( m ) , 1027 ( m ) , 881 ( s ) , 761 ( s ) , 700 ( s ) cm ; lr - ms ( esi ) : m / z calculated for c25h30n ( [ m + h ] ) 344.2 , and found 344.2 .
raw264.7 and hek293 cells were cultured with rpmi1640 medium supplemented with 10% heat - inactivated fbs , glutamine , and antibiotics ( penicillin and streptomycin ) at 37c in a 5% co2 atmosphere . in each experiment , cells were detached with a scraper .
examination of cell densities at 2 10 cells / ml revealed that the proportion of dead cells was consistently < 1% according to trypan blue dye exclusion as the criterion for viability .
raw264.7 macrophage cells ( 1 10 cells / ml ) were cultured for 18 h , pretreated with kth-13-ad1 ( 0 to 200 m ) for 30 min , and further incubated with lps ( 1 g / ml ) for 24 h. the inhibitory effect of kth-13-ad1 on lps - induced no production was determined by analyzing no level using griess reagent , as previously described [ 18 , 19 ] .
the od at 550 nm ( od550 ) was measured using a spectramax 250 microplate reader ( molecular devices , sunnyvale , ca , usa ) .
the level of intracellular ros was determined by recording the change in fluorescence resulting from the oxidation of the fluorescent probe dhr123 .
briefly , 5 10 raw264.7 cells were exposed to kth-13-ad1 ( 0 to 150 m ) for 30 min and then incubated with snp ( 0.25 mm ) at 37c for 20 min to induce ros production .
the cells were further incubated with 20 m of the fluorescent probe dhr123 for 30 min at 37c .
the degree of fluorescence , which corresponded to the level of intracellular ros , was determined using a facscan flow cytometer ( becton - dickinson ) , as reported previously . to measure the phagocytic activity of raw264.7 cells , we modified a previously reported method .
raw264.7 cells ( 5 10 ) were pretreated with kth-13-ad1 ( 0 to 150 m ) for 1 h , resuspended in 100 l phosphate buffered saline ( pbs ) containing 1% human ab serum , and then incubated with fitc - dextran ( 1 mg / ml ) at 37c for 20 min .
the reactions were stopped by adding 2 ml ice - cold pbs containing 1% human serum and 0.02% sodium azide .
the cells were then washed three times with cold pbs - azide and analyzed on a facscan flow cytometer ( becton - dickinson , san jose , ca , usa ) , as reported previously .
the level of fitc - dextran or ros in raw264.7 cells was determined through flow cytometric analysis [ 22 , 23 ] .
10 cells / ml ) treated with kth-13-ad1 in the presence or absence of fitc - dextran ( 1 mg / ml ) or dhr123 ( 20 m ) were washed with staining buffer containing 2% rabbit serum and 1% sodium azide in pbs and then incubated with direct - labeled antibodies for an additional 45 min on ice . after washing three times with staining buffer , stained cells were analyzed on a facscan flow cytometer ( becton - dickinson ) .
raw264.7 cells ( 1 10 cells / ml ) were cultured for 18 h , after which kth-13-ad1 ( 0 to 150 m ) was added for the final 24 or 8 h of culture .
the cytotoxic effects of ats - e3 kth-13-ad1 were then evaluated using a conventional mtt assay , as reported previously [ 24 , 25 ] . for the final 3 h of culture
, 10 l mtt solution ( 10 mg / ml in pbs , ph 7.4 ) was added to each well .
the incubation was stopped by the addition of 15% sodium dodecyl sulfate ( sds ) to each well , which solubilized the formazan .
the absorbance at 570 nm ( od570630 ) was measured using a spectramax 250 microplate reader ( biotek , bad friedrichshall , germany ) .
raw264.7 cells ( 1 10 cells / ml ) were cultured for 18 h , pretreated with kth-13-ad1 ( 0 to 150 m ) for 30 min , and further cultured with lps ( 1 g / ml ) for 6 h. the inhibitory effect of kth-13-ad1 on the expression of inos and tnf- was determined using semiquantitative rt - pcr and real - time quantitative reverse transcription - polymerase chain reaction ( qrt - pcr ) , as reported previously [ 18 , 27 ] .
the primers ( bioneer , daejeon , korea ) used in these reactions are listed in table 1 .
hek293 cells ( 1 10 cells / ml ) were transfected with 1 g plasmids to drive the expression of -galactosidase and either nf-b - luc or ap-1-luc in the presence or absence of an inducing molecule ( myd88 , trif , or ha - src ) .
transfections were performed using the pei method in 12-well plates , as previously outlined [ 28 , 29 ] .
luciferase assays were performed using the luciferase assay system ( promega , madison , wi , usa ) , as previously reported .
raw264.7 cells ( 5 10 cells / ml ) were washed three times in cold pbs supplemented with 1 mm sodium orthovanadate , resuspended in lysis buffer ( 20 mm tris - hcl , ph 7.4 , 2 mm edta , 2 mm ethyleneglycotetraacetic acid , 50 mm -glycerophosphate , 1 mm sodium orthovanadate , 1 mm dithiothreitol , 1% triton x-100 , 10% glycerol , 10 g / ml aprotinin , 10 g / ml pepstatin , 1 mm benzamide , and 2 mm pmsf ) , lysed by sonication , and rotated for 30 min at 4c .
the lysates were clarified by centrifugation at 16,000 g for 10 min at 4c and stored at 20c until use .
the soluble fractions of the cell lysates were immunoblotted , and total and phosphoprotein levels of c - fos , c - jun , p50 , p65 , ib , ikk , akt , p85 , src , syk , lamin a / c , and -actin were visualized , as previously reported .
data are expressed as the mean standard deviation ( sd ) , as calculated from one ( n = 6 ) of two independent experiments .
other data are representative of three different experiments with similar results . for statistical comparisons ,
the results were analyzed using analysis of variance / scheffe 's post hoc test and the kruskal - wallis / mann - whitney test . a p value < 0.05 was considered to be statistically significant .
all statistical tests were conducted using spss ( spss inc . , chicago , il , usa ) .
in previous studies , we and other investigators have reported on the anti - inflammatory , anticancer , and immunomodulatory activities of cordyceps bassiana [ 3235 ] . unlike other cordyceps species , such as cordyceps militaris and cordyceps sinensis ,
recently , we identified a promising new compound called 4-isopropyl-2,6-bis(1-phenylethyl)phenol with anticancer activity against several cancer cell lines , such as c6 glioma and mda - mb-231 .
we further established a method for total synthesis of this compound in order to facilitate its mass production and derivatization . in this study , we tested one ( kth-13-ad1 ( figure 1(a ) ) ) of its derivatives , specifically to see whether this compound is able to suppress macrophage - mediated inflammatory responses .
as shown in figure 1(b ) , left panel , the production of no was reduced by up to 70% in a dose - dependent manner by kth-13-ad1 in lps - stimulated macrophage - like raw264.6 cells at 150 m , though it did not downregulate no released from snp , a drug that directly releases no in vitro ( figure 1(b ) , right panel ) .
these results indicate that kth-13-ad1 does not act as a chemically directed neutralizing agent but rather as a modulator of no biosynthesis during tlr4 stimulation following lps treatment . in similar fashion ,
the radical scavenging activity of kth-13-ad1 was observed only at 100 and 150 m when applied to snp - treated cells ( figure 1(c ) , left panel ) but not in the dpph assay ( figure 1(c ) , right panel ) . since kth-13-ad1 did not suppress the viability of raw264.7 cells ( figure 1(d ) ) , the results demonstrate that though this compound does not have direct antioxidative activity , it displays anti - inflammatory properties that indirectly suppress the production of inflammatory mediators , such as no , similarly to ginsenosides that have only anti - inflammatory properties [ 37 , 38 ] .
however , unlike these compounds , flavonoids ( e.g. , quercetin , luteolin , and kaempferol ) and anthraquinones ( e.g. , rhodoptilometrin ) , are active components of numerous medicinal plants and have been reported to demonstrate both antioxidative and anti - inflammatory activities [ 3942 ] . to determine the mechanism by which kth-13-ad1 suppresses no production , we investigated tlr4-mediated transcriptional activation levels in lps - treated raw264.7 cells pretreated with kth-13-ad1 .
first , we evaluated mrna levels in proinflammatory genes , including inos and tnf- , using quantitative real - time and semiquantitative pcr .
as shown in figures 2(a ) and 2(b ) , kth-13-ad1 ( 150 m ) significantly decreased the mrna levels of inos and tnf- , suggesting that kth-13-ad1-driven suppression of inflammatory responses occur at the transcriptional level .
since nf-b and ap-1 are representative of transcription factors regulating inflammatory responses [ 43 , 44 ] , we determined the ability of this compound to suppress the activation of nf-b and ap-1 using a luciferase reporter gene assay with promoter sites for nf-b and ap-1 . to determine how kth-13-ad1 regulates promoter activity in inflammatory genes
, we pretreated the kth-13-ad1 for 30 min before activating hek293 cells with pma , an activator of pkc that induces the inflammatory signaling pathway .
as shown in figure 2(c ) , luciferase activity mediated by nf-b and ap-1 was greatly enhanced by pma , up to 500- and 40-fold , respectively .
interestingly , kth-13-ad1 ( 100 and 150 m ) strongly suppressed nf-b - mediated luciferase activity ( figure 2(c ) , left panel ) but not ap-1 ( figure 2(c ) , right panel ) .
it has also been reported that myd88 and trif , adaptor molecules regulating the tlr4-mediated intracellular signaling pathway , are good inducers of luciferase activity in hek293 cells [ 47 , 48 ] .
the results in figure 2(d ) support this conclusion , as cotransfection of these molecules increased both nf-b- and ap-1-mediated luciferase activity up to 4,000- and 250-fold , respectively , as seen in previous studies . comparing these two conditions ,
myd88-induced nf-b activation was strongly suppressed by kth-13-ad1 , with 100 m of the compound inhibiting activation by 45% ( figure 2(d ) ) . in addition
, kth-13-ad1 reduced the nuclear translocation of the nf-b subunits p65 and p50 at 30 and 60 min , although there was no inhibition at 15 min ( figure 2(e ) ) .
these results suggest that the nf-b signaling pathway affecting the activation of nf-b dimerization at 30 to 60 min might be a potential target for kth-13-ad1 .
these inhibitory patterns observed by kth-13-ad1 treatment seem to also imply that nf-b activation signals might be timely regulated by some upstream signaling units . to prove such raised possibility , we subsequently focused on identifying the nf-b regulatory pathways that are recognized by this compound .
first , we determined the phosphorylation level of ib , an essential step in the nuclear translocation of nf-b .
the phosphorylation of ib was blocked by kth-13-ad1 at 15 , 30 , and 60 min , while the total form of ib was greatly increased at 30 and 60 min , compared to lps treatment alone ( figure 3(a ) ) , implying that this compound is able to suppress both phosphorylation and degradation of ib between 30 min and 1 h. in addition , kth-13-ad1 was found to reduce the phosphorylation of ikk/ , upstream of ib , at 5 min ( figure 3(a ) ) .
similarly , the phosphorylation of akt and p85/pi3k , upstream enzymes for ikk activation , was blocked by kth-13-ad1 at 3 to 5 min ( figure 3(b ) ) .
additionally , src phosphorylation occurring at 1 and 3 min was also suppressed by this compound ( figure 3(b ) ) . to confirm our results
, we evaluated the dose - responsive activity of kth-13-ad1 on the phosphorylation levels of putative target proteins , p85/pi3k and src .
as seen in figure 3(c ) , the phosphorylation of p85 and src was continuously diminished by kth-13-ad1 ( 100 and 150 m ) under the same conditions . as these results strongly suggest that the putative target of kth-13-ad1 is src , we next analyzed whether this compound is able to regulate the autophosphorylation of src , using an overexpression strategy , as reported previously [ 50 , 51 ] .
as shown in figure 3(d ) , kth-13-ad1 ( 100 and 150 m ) clearly decreased the level of src phosphorylation , implying that src is directly modulated by this compound .
this is further supported by the fact that the promoter activity of nf-b , as stimulated by src overexpression , was also blocked by kth-13-ad1 ( figure 3(e ) ) . based on these results
, we propose that src serves as a potential target protein in kth-13-ad1-mediated anti - inflammatory actions .
many anti - inflammatory compounds , both naturally occurring and chemically synthesized , have been found to possess direct inhibitory activity against src kinase [ 5052 ] .
additionally , previous studies have reported on src activity under nf-b activation and inflammatory gene expression [ 51 , 53 , 54 ] , suggesting an important role of src in inflammatory responses and in oncogenic activity .
finally , to demonstrate whether kth-13-ad1 modulates macrophage phagocytic uptake , fitc - dextran was applied to raw264.7 cells during exposure to this compound . as shown in figure 4
, there was no inhibition of phagocytosis by kth-13-ad1 , as measured by fitc - derived fluorescence [ 55 , 56 ] ; however , significant enhancement was observed at 100 and 150 m concentrations , implying that the compound stimulates macrophage phagocytosis , an important step in the innate immune response .
this result suggests that innate immune responses might be regulated by kth-13-ad1 via different cellular mechanisms .
in summary , kth-13-ad1 demonstrated impressive suppression of inflammatory mediator production , including ros and no , without inducing cytotoxicity , while this compound strongly enhanced the phagocytotic uptake , suggesting that kth-13-ad1 is able to regulate cellular responses related to innate immunity .
in addition , the transcriptional activation of nf-b and its upstream signaling pathway , composed of src , p85/pi3k , and akt , was blocked by kth-13-ad1 , as summarized in figure 5 .
thus , the present study proposes that kth-13-ad1 , a chemical analog of kth-13 extracted from cordyceps bassiana , has clinical utility as an anti - inflammatory drug .
|
the cordyceps species has been a good source of compounds with anticancer and anti - inflammatory activities .
recently , we reported a novel compound ( 4-isopropyl-2,6-bis(1-phenylethyl)phenol , kth-13 ) with anticancer activity isolated from cordyceps bassiana and created several derivatives to increase its pharmacological activity . in this study , we tested one of the kth-013 derivatives , 4-isopropyl-2,6-bis(1-phenylethyl)aniline 1 ( kth-13-ad1 ) , with regard to anti - inflammatory activity under macrophage - mediated inflammatory conditions . kth-13-ad1
clearly suppressed the production of nitric oxide ( no ) and reactive oxygen species ( ros ) in lipopolysaccharide ( lps ) and sodium nitroprusside- ( snp- ) treated macrophage - like cells ( raw264.7 cells ) .
similarly , this compound also reduced mrna expression of inducible no synthase ( inos ) and tumor necrosis factor- ( tnf- ) , as analyzed by rt - pcr and real - time pcr .
interestingly , kth-13-ad1 strongly diminished nf-b - mediated luciferase activities and nuclear translocation of nf-b family proteins . in accordance
, kth-13-ad1 suppressed the upstream signaling pathway of nf-b activation , including ib , ikk/ , akt , p85/pi3k , and src in a time- and dose - dependent manner .
the autophosphorylation of src and nf-b observed during the overexpression of src was also suppressed by kth-13-ad1 .
these results strongly suggest that kth-13-ad1 has strong anti - inflammatory features mediated by suppression of the src / nf-b regulatory loop .
|
1. Introduction
2. Materials and Methods
3. Results and Discussion
|
a 52-year - old female patient visited the hospital complaining of pain in both breasts .
the pain started immediately after breast reduction surgery she underwent 3 years previously at a private plastic surgery hospital , and it had continued despite a subsequent surgery .
the pattern of the pain included burning , shooting and itching sensations at the operation site as well as unbearable burning sensations when the patient was in the places where the temperature was higher than normal .
the pain as measured by the visual analogue scale ( vas ) was approximately 70/100 mm .
in particular , the degree of pain was dependent on the season , and it was unbearable under hot weather , with the patient complaining of pain at vas 80 - 90/100 mm . before coming to the hospital , the patient had taken aceclofenac ( airtal , daewoong pharmaceutical ) 200 mg , pregabalin ( lyrica , pfizer ) 300 mg , acetaminophen 1,300 mg and tramadol hcl 150 mg ( ultracet , janssen korea ) per day , but the pain was not relieved .
thus , the patient tried pregabalin 600 mg , acetaminophen 1,300 mg and tramadol hcl 150 mg per day , but there was no effect .
complaining of dizziness due to ultracet , the patient started to take 20 mg of oxycodone hcl ( oxycontin , mundipharma korea ) each day , but she eventually stopped taking it because of dizziness , nausea , and vomiting .
subsequently , the patient underwent drug treatment with pregabalin 300 mg a day , but she visited the hospital because the pain did not subside .
there was no specific finding in a simple plane chest x - ray spectrograph , while digital infrared thermographic imaging ( diti ) showed a slight temperature drop in both breasts .
because the previous treatments were not effective in improving the symptoms and because the patient wanted another method of treatment due to the continued pain , it was decided that a thoracic epidural block would be performed , and informed consent was obtained from the patient .
the patient was laid on her side and the epidural space was checked using the loss - of - resistance method with a 22-gauge epidural needle ( hakko , japan ) in between the t4 and t5 , applying an aseptic technique in the interlaminar approach .
0.5% mepivacaine hcl ( emcaine 2% inj. reyon pharm ) 6 ml was then injected .
the pain was relieved into the vas range of 40 - 50/100 mm after the procedure , and the patient was discharged .
the vas was decreased to 40 - 50/100 mm after an additional procedure a week later , but the patient complained of continued pain .
a decision was made to carry out prf to the spinal nerve and its root and . to measure the level and determine the effect
, informed consent was received from the patient after she was provided with an explanation of the procedure and the side effects that may occur after the procedure . for the diagnosis ,
selective nerve root block ( snb ) was performed at t3 , t4 and t5 , one level each time in order with an interval of 2 days , by injecting 1% lidocaine 0.5 ml with a 25-gauge quincke needle ( spinocan , bbraun ) .
prf was carried out at the fourth thoracic spinal nerve and its root after giving an explanation to the patient about the procedure and the side effects that may occur after the procedure and receiving informed consent once again . after supporting the patient 's lower abdomen with a pillow and placing her in a prone position ,
the site of the procedure was disinfected by a common sterilizing method and a standard monitor was attached .
the area between t4 and t5 was identified in the anteroposterior view using a c - arm device and the 4 cm point right lateral from the t4 spinous process was chosen as the needle implantation point .
local anesthesia was carried out with 1% lidocaine , and a 20-gauge 10 cm long rf cannula ( owl sharp curved rf insulated cannula , diros technology inc . ,
usa ) with a 10 mm - active tip was then introduced into the anesthetized part . by continuously monitoring the position of the cannula with the c - arm
, it continued to the inferior of end plate of fourth thoracic vertebral body ( fig .
1 ) , and the tip of the cannula was located at the posterior border of the vertebral space in the lateral view ( fig .
3 ) , and an rf generator ( rfg-3c plus , radionics , usa ) was then connected and an electric stimulus of 50 hz at 0.1 volt was applied .
electric stimulus of 2 hz at 0.1 volt was applied , and it was verified that the muscle did not contract up to 0.8 volt .
one minute after injecting 1% mepivacaine 1 ml , prf was performed two times for 120 seconds at 42. the same procedure was performed on the opposite side after one week . during the procedure , the vital signs of the patient were normal and no side effect took place . after the procedure , the pain was reduced to vas 20 - 30/100 mm .
the patient currently undergoes oral administration of pregabalin 300 mg / day and nortriptyline ( sensival , ilsung pharm ) 10 mg / day and this was followed up at the hospital for 9 months without particular exacerbation of the pain or any other inconvenience in her daily tasks .
pain after breast surgery , which is commonly complaint by patients , is known to be related with the injury , ischemia and inflammation of the soft tissue .
if the pain in the affected part and arm continues for one year after the surgery , it is considered as chronic pain in general .
the mechanism is not yet accurately known , but the chronic pain may be caused by injuries in various peripheral nerves that arise during the surgery and can develop into neuropathic pain such as stabbing , pricking , burning , shooting and sharp pains .
wallace et al . investigated 282 women who underwent different types of breast surgery , and their result showed that 31% of women who underwent mastectomy , 49% of those who underwent mastectomy with reconstruction , 38% of those who underwent cosmetic augmentation and 22% of those who underwent breast reduction complained of pain up to one year after the surgery .
particularly , as the survival rate of breast cancer patients has increased , the number of patients who complain of chronic pain after breast cancer survey has also increased .
the patient in this case study was also a chronic neuropathic pain patient whose pain had continued for 3 years following breast reduction surgery .
she complained of pricking , shooting and burning sensations at the site of the operation .
although the patient complained of pain immediately after the surgery , she continued to suffer neuropathic pain for 3 years without being given appropriate pain treatment because she was not able to take medicine due to the side effects of the drugs .
macrae stated that severe acute pain after the surgery may be a risk factor for chronic pain .
this can be one possible cause of the chronic pain of the patient in this case report , because she complained severe pain at vas 70 - 80/100 mm immediately after the surgery but the pain was not properly controlled .
in general , it is thought that the intercostal nerve ( icn ) from t1 to t6 and the nerves that originate from those braches can be damaged by breast surgery . for the patient in this case report , because she complained of the pain at the dermatome of the t4 spinal nerve , neuropathic pain caused by damage at the 4th icn and at the branch was suspected .
the effect of a nerve block in controlling pain after breast surgery is well known . in particular , it was reported that a paravertebral block ( pvb ) reduced pain , nausea and vomiting after breast surgery .
in addition , kairaluoma et al . reported that preoperative pvb reduced chronic pain after breast surgery .
these results show that a nerve block can be used in controlling pain following breast surgery effectively , preventing it from becoming chronic .
a nerve block using a local anesthetic is easy to perform in the treatment of pain and is effective in reducing pain .
however , because the duration of desensibilization may not be sufficient depending on the disease causing the pain and because many repeated procedures may be required , radiofrequency thermocoagulation is sometimes employed to maintain the effect of the nerve block .
radiofrequency thermocoagulation was first introduced in 1930 's by kirschner , who applied it treating trigeminal neuralgia at the gasserian ganglion .
recently , the application of radiofrequency thermocoagulation in the case of various diseases has gradually increased as quality of the radiofrequency generator , the associated equipment , and the catheter needle has been improved .
this treatment is highly advantageous , in that a more accurate lesion can be generated compared to other nerve - destructive procedures . moreover ,
the mechanism of radiofrequency thermocoagulation is known to change the nerve tissue due to the heat around the electrode , blocking and the inflow of the nerve stimulus . on the other hand ,
prf was introduced as a new method because another mechanism of radiofrequency - aided treatment arises in that a clinical effect is noted by the heat applied to the nerve and the electromagnetic field that forms around the electrode .
prf is a radiofrequency - aided treatment in which pulsed radiofrequency stimulus of 20 msec is generated two times every 0.5 second at a temperature of 42 , which is low enough for the nerve tissue to remain undamaged .
prf is a novel treatment method that has corrected the problems of previous radiofrequency - aided treatments based on heat .
it does not cause nerve destruction , side effects such as neuritis , or the complications of other types of radiofrequency - aided treatments based on heat .
it can be performed to treat neuropathic pain even for body parts where side effects take place frequently due to radiofrequency thermocoagulation .
it can be also performed on parts near bone or scar tissue , where the risk of complications is high when radiofrequency thermocoagulation is performed .
rohof applied prf to the suprascapular nerve in the treatment of chronic shoulder pain and reported it to be a novel treatment method that is free from side effects or complications , in contrast to the conventional thermocoagulation .
moreover , positive effects were reported when prf was applied to peripheral nerves such as the facial nerve , superior laryngeal nerve , suprascapular nerve , intercostals nerve , obturator nerve and the joint - dominating peripheral nerve articular branch , whose main nerve is sensory . in this case report as well , although the pain was controlled by an epidural block with a local anesthetic , repeated procedures were required due to the short duration of the first treatment .
long - lasting pain reduction was achieved without any specific side effect , as the prf was carried out on the spinal nerve and its root under monitoring by a c - arm device . in this article
, we report a case in which prf was applied to control the pain of a patient who came to the hospital complaining of chronic severe neuropathic pain at the operation site that arose after breast reduction surgery .
appropriate pain control after breast surgery can reduce the suffering of the patient due to early pain and greatly affect the quality of life of the patient by preventing the pain from being chronic .
if an appropriate pain treatment had been given after breast surgery to the patient in this case report , she would have not suffered from chronic pain . in the case of breast surgery , considerable attention should be paid to postoperative pain as well as surgical pain , and pain treatment should be carried out positively .
it is thought that a nerve block as well as general analgesics can be helpful as a pain control strategy .
in addition , prf may be helpful in reducing the pain of the patients who are not responsive to a nerve block using analgesics or local anesthetics .
furthermore , additional studies are needed regarding the reduction of chronic neuropathic pain by prf after breast surgery .
|
breast surgery is a common procedure performed in women .
many women who undergo breast surgery suffer from ill - defined pain syndromes .
a nerve block is used in the treatment of the acute and chronic pain , but the effectiveness of the treatment has been limited because of its short duration .
recently , the advent of pulsed radiofrequency lesioning ( prf ) has proved a successful treatment for chronic refractory pain involving the peripheral nerves .
we experienced a case of a 52-year - old female patient complaining of chronic breast neuropathic pain after breast reduction , which was relieved after prf lesioning of the 4th thoracic spinal nerve and its root .
|
Case Report
Discussion
|
hepatitis c virus ( hcv ) infection is a leading cause of end - stage liver disease and hepatocellular carcinoma [ 1 , 2 ] .
hcv - related liver disease is the most common indication for orthotopic liver transplantation in the usa and northern europe and for living donor liver transplantation ( ldlt ) in japan [ 3 , 4 , 5 ] .
unfortunately , liver transplantation is not a cure for hcv infection , and the occurrence of graft reinfection with hcv is universal , leading to progression of liver fibrosis and occasionally to graft loss at rates higher than in transplant patients not infected with hcv [ 5 , 6 ] . in japan , because of long - standing legal difficulties associated with cadaveric donation , ldlt is the main type of liver transplantation for end - stage liver disease .
the sustained virologic response ( svr ) rate was 40% in donors of ldlt treated with pegylated interferon ( peginterferon ) plus ribavirin , although adverse events were commonly observed even in nontransplanted patients receiving this treatment .
reported that the dose reduction rate and the discontinuation rate of peginterferon plus ribavirin treatment was 40 and 42% , respectively .
recent progress in the development of direct - acting antivirals ( daas ) against hcv has made it possible to eradicate hcv effectively and to shorten the treatment duration compared to the previous standard of care , i.e. peginterferon plus ribavirin .
treatment with the hcv ns5b polymerase inhibitor sofosbuvir plus ribavirin for 12 weeks was shown to lead to high rates of svr ( 97% ) in japanese patients infected with chronic hcv genotype 2 .
asian - pacific countries , including japan , account for 50% of all chronic hepatitis b virus ( hbv ) infection globally . given the prevalence of hbv , ldlt from hepatitis b core antibody - positive donors to recipients
was occasionally performed with passive immunization with hyperimmune hepatitis b immunoglobulin ( hbig ) plus nucleos(t)ide analogs . here , we report on an ldlt recipient who was reinfected with hcv genotype 2a after receiving a graft from a hepatitis b core antibody - positive donor .
hcv genotype 2a was eradicated by a 12-week treatment with sofosbuvir plus ribavirin with hbig plus entecavir , one of the nucleos(t)ide analogs , for the successful prevention of hbv reappearance .
a 66-year - old japanese woman developed liver failure due to cirrhosis and hcv genotype 2a infection .
she was a treatment - nave patient , but her il28b rs8099917 was a favorable genotype ( tt ) .
two years prior to ldlt , she has been diagnosed with liver cirrhosis due to hcv infection without liver biopsy .
she had been infected with hcv after having received a blood transfusion during childbirth at the age of 30 years . at ldlt , she had peripheral edema with a meld ( model for end - stage liver disease ) score of 12 .
ldlt with a right liver graft from a hepatitis b core antibody - positive donor was performed in february 2015 .
five months after ldlt , the hcv rna level was 5.8 log iu / ml , and she was diagnosed with graft reinfection with hcv genotype 2a .
combination treatment with 400 mg daily of sofosbuvir and 400 mg daily of ribavirin was commenced .
her height , body weight , and body mass index were 1.56 m , 50 kg , and 20.5 , respectively .
her laboratory data before treatment are shown in table 1 . the hcv rna level before treatment was 5.8 log iu / ml .
after ldlt , she received triple immunosuppressive therapy consisting of tacrolimus ( 3 mg daily ) , mycophenolate mofetil , and basiliximab with passive immunization with hbig and 0.5 mg daily of entecavir .
four weeks after initiating the combination treatment with sofosbuvir and ribavirin , hcv rna levels were undetectable .
she completed this treatment for 12 weeks and achieved svr at 24 weeks following the termination of this treatment ( svr24 ) ( fig .
1 ) . there was no evidence of hbv reactivation . because her hemoglobin level was 12.2 g / dl before the commencement of this treatment , an oral iron preparation was also started .
after 2 months of treatment , her hemoglobin level fell to 10.8 mg / dl .
then , the dose of ribavirin was decreased to 200 mg daily , and her hemoglobin level improved to 12.8 g / dl ( fig .
, she did not develop evidence of bone marrow suppression , such as observed in peginterferon - plus - ribavirin treatment .
no serious adverse events were observed . during treatment , the trough level of tacrolimus remained stable .
we presented a female patient with living donor - related graft reinfection with hcv genotype 2a who was treated with a combination of sofosbuvir and ribavirin for 12 weeks .
although several daas have drug - drug interactions , no changes in this patient 's drug protocol , which included immunosuppressants and combination treatment of sofosbuvir plus ribavirin , were necessary in this case .
peginterferon plus ribavirin with or without daas may be attempted , but the use of peginterferon is restricted by severe side effects and inadequate efficacy . in addition , interferon has an immune - mediated cytotoxicity , occasionally causing allograft dysfunction .
interferon - free therapy is a viable treatment option and improves treatment efficacy [ 15 , 16 , 17 , 18 , 19 , 20 ] .
this case suggests that daas are well tolerated and effective for the eradication of hcv from post - liver transplantation patients . in the present case of ldlt , graft reinfection with hcv genotype 2
the patient achieved svr24 , although she had anemia as an adverse event , and anti - hcv treatment was continued without blood transfusion .
curry et al . reported that common adverse events of combination treatment with sofosbuvir and ribavirin were fatigue ( in 38% of patients ) , headache ( 23% ) , and anemia ( 21% ) after liver transplantation .
charlton et al . reported that the most common adverse events were fatigue ( 30% ) , diarrhea ( 28% ) , headache ( 25% ) , and anemia ( 20% ) during a 24-week combination treatment course of sofosbuvir plus ribavirin after liver transplantation . in the present study ,
the patient received a liver graft from a hepatitis b core antibody - positive donor , and , in general , hbv from these donors is transmitted to recipients at a high rate .
therefore , passive immunization with hbig plus treatment with entecavir were provided to prevent hbv reactivation .
curing hcv infection with daas in hbv / hcv coinfection and monitoring for hbv reactivation should be performed , because daas against hcv do not have any effect on hbv replication , unlike interferon . in the present case ,
the patient was treated with hbig and entecavir , and there was no evidence of hbv reactivation .
recently , more effective regimens with daas against hcv have been reported [ 22 , 23 ] .
these regimens may make it possible to shorten the duration of treatment and to make it easier to achieve svr .
the use of interferon - free regimens is possible for the eradication of hcv in post - ldlt patients with grafts from hepatitis b core antibody - positive donors , as was demonstrated by our patient , who was successfully treated with hbig and entecavir to prevent hbv reactivation . in conclusion ,
12-week treatment with sofosbuvir plus ribavirin is relatively safe and highly effective for the eradication of hcv genotype 2 in ldlt patients .
the other authors declare that there is no conflict of interest regarding the publication of this manuscript .
|
direct - acting antivirals ( daas ) are relatively safe and highly effective for the eradication of hepatitis c virus ( hcv ) in liver transplant recipients . in this case study
, we present a female with a graft reinfected with hcv genotype 2 who was treated with a combination of sofosbuvir and ribavirin after living donor liver transplantation ( ldlt ) . because the graft was from a hepatitis b core antibody - positive donor , passive immunization with hyperimmune hepatitis b immunoglobulin ( hbig ) and entecavir
were also provided to prevent hepatitis b virus ( hbv ) reactivation .
it became clear that the combination of sofosbuvir and ribavirin promptly led to a sustained virologic response and that this combination was safe to treat graft reinfection with hcv genotype 2 after ldlt .
adverse events caused by daas were not observed , except for slight anemia .
hbig and entecavir were useful in the prevention of hbv reactivation . in conclusion
, the present case indicated that daa treatment for graft reinfection with hcv is safe and effective in ldlt from hepatitis b core antibody - positive donors .
|
Introduction
Case Report
Discussion
Statement of Ethics
Disclosure Statement
|
spontaneous ovarian hyperstimulation syndrome ( sohss ) is now a recognized entity , defined as ohss appearing in patients with no history of stimulatory infertility treatment .
ohss , which otherwise may occur in normal pregnancies ( type i ohss ) , is now reported to be associated with mutated fsh receptor ( fshr ) gene , which may lead to recurrent sohss in patients presenting the mutations .
type ii is secondary to high levels of human chorionic gonadotropin ( hcg ) , while type iii coexists with hypothyroidism .
a 29-year - old female married since 1 year , primigravida with 3 months amenorrhea and ultrasonographic gestational age of 8 weeks and 5 days , presented with acute onset of abdominal distension , vulval edema and breathlessness .
there was no vomiting , trauma , fever , bleeding , or spotting per vaginum .
she had regular past menstrual cycles and no symptoms suggestive of renal , cardiac , or liver disorders .
ultrasonography done a few weeks prior to presentation revealed a normal intrauterine pregnancy and adnexae [ figure 1 ] .
ultrasonography images showing normal endometrial cavity ( intrauterine pregnancy ) and bilateral ovaries the patient was conscious , cooperative , afebrile , with no tachycardia and normotensive .
abdominal examination revealed shiny skin , tense ascites with no guarding , rigidity , or tenderness .
history was reconfirmed ; patient had not sought any medical advice or treatment for fertility enhancement .
blood investigations revealed hyponatremia ( 125 meq / l ) and hypoalbuminemia ( 3.4 gm / dl ) .
hemoglobin , white blood cell count , platelet count , coagulation profile , liver enzymes , blood urea nitrogen , and serum creatinine were normal .
estradiol was elevated at 12927 pg / dl ( normal range 188 - 2497 pg / dl ) .
ultrasonography revealed a multiloculated cystic lesion of size 17 cm 11 cm with vascularity within the wall of cystic lesion .
features of ohss were confirmed on magnetic resonance imaging ( mri ) [ figure 3 ] .
ultrasonography b - mode images show intrauterine fetus with multiple cysts involving both the right and left ovary .
there is mild free fluid in the intra - peritoneal cavity with left pleural effusion t2-weighted magnetic resonance imaging pelvic coronal images show t2-hypo intense lesion in the intrauterine cavity suggestive of fetus .
there are hyper intense multiple cysts seen without any mural nodule arising from both right ( in right iliac fossa ) and left ovary ( in pouch of douglas ) vital parameters , intake - output , and abdominal girth were monitored .
additional intravenous fluids were omitted , oral fluids were encouraged , and diuretics were initially required .
intravenous albumin infusion was instituted ( 25 g in 0.1 l solution per day for 7 days ) .
no interventional procedure was done for ascites and pleural effusion , both of which reduced , along with the adnexal mass .
unfractionated heparin 5000 u subcutaneous injection twice daily was instituted and continued for 9 days .
regular ultrasonographic monitoring showed further reduction in size of adnexal mass . at 40 weeks gestation ,
patient underwent cesarean section in view of nonprogress of labor and delivered a healthy male of 2.85 kg .
intraoperatively , an left ovarian simple cyst of 5 cm 5 cm was observed , for which no intervention was done .
postoperative mri revealed simple left ovarian cyst of 2 cm 2 cm , right side normal and involuting uterus [ figure 4 ] . t2-weighted magnetic resonance imaging pelvis images
6 weeks postpartum show complete resolution of bilateral cystic lesions with a single follicular cyst in the left ovary and multiple small follicles in the right ovary to investigate further , if ovarian hyperstimulation observed in this patient was due to any mutation in fshr gene , the patient was called back after delivery . the entire exonic region of fshr gene was screened for known / novel mutations as described earlier . to our interest
, we identified a novel heterozygous mutation in fshr gene leading to a change at amino acid position 449 [ figure 5 ] .
this was observed to be a transversion at nucleotide position 1346 ( act to aat ) in exon 10 yielding a threonine to asparagine ( thrasn ) substitution in the transmembrane domain helix 3 of the fshr .
( a ) electropherogram of 5-3 strand of the exon 10 of fshr gene , showing a wild type sequences ( upper panel ) of a control subject and heterozygous mutant sequence ( lower panel ) resulting in change of nucleotide c to a / c ( 1346 position ) leading to thrasn substitution identified in patient who developed spontaneous ovarian hyperstimulation syndrome during pregnancy .
the majority of ohss cases are usually iatrogenic and are induced by exogenous hormonal therapy .
the pathophysiology is one of the fluid extravasations , and the mediator appears to vascular endothelial growth factor . with respect to the severity of clinical picture
, it can be classified into :
grade 1 : abdominal distension and discomfortgrade 2 : grade 1 + nausea , vomiting , and/or diarrhea , ovaries 512 cmgrade 3 : grade 2 and ultrasonic evidence of ascitesgrade 4 : grade 3 and/or hydrothorax or dyspneagrade 5 : all above with hemoconcentration , coagulation abnormalities , and diminished renal perfusion ( critical ohss ) .
grade 1 : abdominal distension and discomfort grade 2 : grade 1 + nausea , vomiting , and/or diarrhea , ovaries 512 cm grade 3 : grade 2 and ultrasonic evidence of ascites grade 4 : grade 3 and/or hydrothorax or dyspnea grade 5 : all above with hemoconcentration , coagulation abnormalities , and diminished renal perfusion ( critical ohss ) .
however , it is now being recognized and slowly coming to light , in the form of individual case reports that a similar clinical picture can occur in patients , unrelated to exogenous hormone administration . after a sufficient number of cases of sohss
were reported based on the underlying primary abnormality and the triggers leading to the sohss , de leener et al . ,
have proposed a 4-group classification for this abnormality , which is shown in table 1 .
classification of fsh receptor mutation our patient was classified as type 1 critical sohss in pregnancy and managed as per existing guidelines .
though the reports of successful completion of pregnancies complicated by sohss have been few , the conservative management recommended by the existing guidelines is validated by these few successful outcomes .
the understanding of the molecular basis of sohss is very recent , evident by the fact that the first recognition of a mutation in the fshr gene resulting in ohss was described very recently .
interestingly , all the activating mutations of fshr gene reported in women till date are associated with sohss leading to the activation of the receptor .
these mutations have been reported to cause loss of ligand binding specificity and increased response to hcg ( especially in first trimester when the levels of hcg are high ) resulting in hyperstimulation .
this mutation leads to the substitution of threonine to asparagine at amino acid position 449 in fshr .
however , the functional analysis of this mutation needs to be investigated to understand the pathophysiology at the molecular level . in the wake of this finding
at this point in time , much is yet to be understood about sohss and the molecular mechanisms behind it , and hence the reason of presenting this case report : a novel mutation in a condition , not fully understood .
the general gynecologist / obstetrician should have a high index of suspicion for sohss , as this entity can have several implications .
genetic analysis should be considered in all patients with sohss to improve our understanding of this entity , paving way for an earlier recognition and improved management of this condition .
|
the objective is to study the fsh receptor ( fshr ) for mutations in a case of spontaneous ovarian hyperstimulation syndrome ( sohss ) .
this is a single case study and it examined patient who presented with spontaneous critical ohss in early pregnancy and had successful good obstetric outcome .
intervention of this study was analysis of blood for genetic analysis of fshr postdelivery . the main outcome measure noted was fshr mutation . the study resulted in a novel , here though unreported , heterozygous mutation in fshr gene at nucleotide position 1346 ( ac1346 t to aat ) in exon 10 yielding a threonine to asparagine ( thr449asn ) substitution in the transmembrane domain helix 3 of the fshr . to conclude fshr gene analysis can add to our understanding of sohss .
|
INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest
|
a 63-year - old man with diabetes and end - stage kidney failure had been receiving maintenance hemodialysis in the outpatient clinic at odense university hospital in odense , denmark , since 1997 .
a femoral femoral bridge graft was used for vascular access . in november 2013 , he was admitted to the hospital because of a fever he experienced during dialysis and inflammation around his bridge graft .
the organism was also cultured from the patient s bridge graft and from a sample of joint fluid from his right shoulder .
a transesophageal echocardiography revealed mitral valve endocarditis . despite relevant treatment with vancomycin and rifampin and surgical debridement of his shoulder joint
on inquiry by the staff , the patient had reported no previous history of mrsa infection or colonization and no direct or household - related contact with pigs .
four months before patient 1 s illness , mrsa cc398 spa type t011 had been cultivated from an infected decubitus ulcer of another patient who was receiving hemodialysis in the same outpatient clinic as patient 1 .
subsequent mrsa screening revealed that patient 2 was a nasal and pharyngeal carrier . on inquiry by the staff , the patient reported no direct or household - related contact with pigs .
a 74-year - old nursing home resident had hemiparesis and recurrent aspiration pneumonia after an apoplectic insult .
in april 2014 , he was admitted to the hospital with severe pneumonia . on admission
blood cultures grew mrsa cc398 spa type t034 , and the organism was found in a tracheal aspirate and from the area around a percutaneous gastrostoma tube . despite relevant treatment with piperacillin / tazobactam , metronidazole , and vancomycin , the patient died from respiratory failure after 1 week . on inquiry by the staff ,
the patient and his attending daughter reported no direct or household - related contact with pigs . in july 2010 , mrsa cc398 spa type t034 had been isolated from a bedsore of another patient living in the same nursing home as patient 3 . in november 2013 ,
the organism was isolated once more from the patient , this time from an indwelling urine catheter .
patients 3 and 4 lived in the same wing of the nursing home and shared common facilities .
after hospital staff recognized the transmission chain , all residents in this wing and all attached staff were screened for mrsa , but none tested positive .
the mrsa cc398 isolates identified in the 2 transmission chains were spa types t011 and t034 , which are common among livestock - associated mrsa strains ( 12 ) .
draft whole - genome sequencing was performed on 7 isolates from the 4 patients in the 2 transmission chains , and results were compared with similar data for cc398-related mrsa and methicillin - sensitive s. aureus isolates from denmark obtained during a previous study of the global dissemination of isolates belonging to this clonal complex ( 13 ) .
sequence files for the individual isolates from patients are available at the european nucleotide archive ( http://www.ebi.ac.uk/ena ; accession no .
a phylogenetic tree was constructed from single - nucleotide polymorphism differences in genome sequences of isolates from the 4 patients and from the isolates obtained during the previous study by using the s0385 complete genome sequence ( genbank accession no .
am990992.1 ) as reference ( figure ) . this analysis showed that the patient isolates within each transmission chain were closely related but that no close relation between the 2 chains existed .
in addition , the isolates from both chains clustered among isolates previously found to be animal associated ( clade iia ; 13 ) .
consistent with an animal origin , all isolates carried the tet(m ) and czrc determinants ( similar to most mrsa strains from clade iia ) and lacked the sa3 phage ( typical of the human clade i ) ( 13 ) .
phylogeny of methicillin - resistant staphylococcus aureus ( mrsa ) clonal complex ( cc ) 398 isolates linked to fatal septicemia in a hospital patient and a nursing home resident in denmark .
draft whole - genome sequencing was performed on 7 isolates from the 4 patients identified in the 2 transmission chains , and results were compared with similar genomic data for cc398-related mrsa and methicillin - sensitive s. aureus isolates obtained in denmark during a previous study of isolates belonging to cc398 ( 13 ) .
single - nucleotide polymorphism differences were identified , and a maximum - likelihood phylogeny was inferred from raw data by using the web tool csi phylogeny ( https://cge.cbs.dtu.dk//services/csiphylogeny ) .
the region of bp 12252135180 was excluded from analysis because it contains the spa region and disrupts the phylogenic signal ( 13 ) .
p , pig ; h , human ; met , methicillin susceptibility ; r , resistant ; s , susceptible .
a 63-year - old man with diabetes and end - stage kidney failure had been receiving maintenance hemodialysis in the outpatient clinic at odense university hospital in odense , denmark , since 1997 .
a femoral femoral bridge graft was used for vascular access . in november 2013 , he was admitted to the hospital because of a fever he experienced during dialysis and inflammation around his bridge graft .
the organism was also cultured from the patient s bridge graft and from a sample of joint fluid from his right shoulder .
a transesophageal echocardiography revealed mitral valve endocarditis . despite relevant treatment with vancomycin and rifampin and surgical debridement of his shoulder joint
on inquiry by the staff , the patient had reported no previous history of mrsa infection or colonization and no direct or household - related contact with pigs .
four months before patient 1 s illness , mrsa cc398 spa type t011 had been cultivated from an infected decubitus ulcer of another patient who was receiving hemodialysis in the same outpatient clinic as patient 1 .
subsequent mrsa screening revealed that patient 2 was a nasal and pharyngeal carrier . on inquiry by the staff , the patient reported no direct or household - related contact with pigs .
a 63-year - old man with diabetes and end - stage kidney failure had been receiving maintenance hemodialysis in the outpatient clinic at odense university hospital in odense , denmark , since 1997 .
a femoral femoral bridge graft was used for vascular access . in november 2013 , he was admitted to the hospital because of a fever he experienced during dialysis and inflammation around his bridge graft .
the organism was also cultured from the patient s bridge graft and from a sample of joint fluid from his right shoulder .
a transesophageal echocardiography revealed mitral valve endocarditis . despite relevant treatment with vancomycin and rifampin and surgical debridement of his shoulder joint
on inquiry by the staff , the patient had reported no previous history of mrsa infection or colonization and no direct or household - related contact with pigs .
four months before patient 1 s illness , mrsa cc398 spa type t011 had been cultivated from an infected decubitus ulcer of another patient who was receiving hemodialysis in the same outpatient clinic as patient 1 .
subsequent mrsa screening revealed that patient 2 was a nasal and pharyngeal carrier . on inquiry by the staff , the patient reported no direct or household - related contact with pigs .
a 74-year - old nursing home resident had hemiparesis and recurrent aspiration pneumonia after an apoplectic insult . in april 2014 , he was admitted to the hospital with severe pneumonia . on admission , he had sepsis .
blood cultures grew mrsa cc398 spa type t034 , and the organism was found in a tracheal aspirate and from the area around a percutaneous gastrostoma tube . despite relevant treatment with piperacillin / tazobactam , metronidazole , and vancomycin , the patient died from respiratory failure after 1 week . on inquiry by the staff ,
the patient and his attending daughter reported no direct or household - related contact with pigs . in july 2010 , mrsa cc398 spa type t034 had been isolated from a bedsore of another patient living in the same nursing home as patient 3 . in november 2013 ,
the organism was isolated once more from the patient , this time from an indwelling urine catheter .
patients 3 and 4 lived in the same wing of the nursing home and shared common facilities .
after hospital staff recognized the transmission chain , all residents in this wing and all attached staff were screened for mrsa , but none tested positive .
the mrsa cc398 isolates identified in the 2 transmission chains were spa types t011 and t034 , which are common among livestock - associated mrsa strains ( 12 ) .
draft whole - genome sequencing was performed on 7 isolates from the 4 patients in the 2 transmission chains , and results were compared with similar data for cc398-related mrsa and methicillin - sensitive s. aureus isolates from denmark obtained during a previous study of the global dissemination of isolates belonging to this clonal complex ( 13 ) .
sequence files for the individual isolates from patients are available at the european nucleotide archive ( http://www.ebi.ac.uk/ena ; accession no .
a phylogenetic tree was constructed from single - nucleotide polymorphism differences in genome sequences of isolates from the 4 patients and from the isolates obtained during the previous study by using the s0385 complete genome sequence ( genbank accession no .
this analysis showed that the patient isolates within each transmission chain were closely related but that no close relation between the 2 chains existed .
in addition , the isolates from both chains clustered among isolates previously found to be animal associated ( clade iia ; 13 ) .
consistent with an animal origin , all isolates carried the tet(m ) and czrc determinants ( similar to most mrsa strains from clade iia ) and lacked the sa3 phage ( typical of the human clade i ) ( 13 ) .
phylogeny of methicillin - resistant staphylococcus aureus ( mrsa ) clonal complex ( cc ) 398 isolates linked to fatal septicemia in a hospital patient and a nursing home resident in denmark .
draft whole - genome sequencing was performed on 7 isolates from the 4 patients identified in the 2 transmission chains , and results were compared with similar genomic data for cc398-related mrsa and methicillin - sensitive s. aureus isolates obtained in denmark during a previous study of isolates belonging to cc398 ( 13 ) .
single - nucleotide polymorphism differences were identified , and a maximum - likelihood phylogeny was inferred from raw data by using the web tool csi phylogeny ( https://cge.cbs.dtu.dk//services/csiphylogeny ) .
the region of bp 12252135180 was excluded from analysis because it contains the spa region and disrupts the phylogenic signal ( 13 ) .
p , pig ; h , human ; met , methicillin susceptibility ; r , resistant ; s , susceptible .
a 74-year - old nursing home resident had hemiparesis and recurrent aspiration pneumonia after an apoplectic insult . in april 2014 , he was admitted to the hospital with severe pneumonia . on admission , he had sepsis .
blood cultures grew mrsa cc398 spa type t034 , and the organism was found in a tracheal aspirate and from the area around a percutaneous gastrostoma tube . despite relevant treatment with piperacillin / tazobactam , metronidazole , and vancomycin , the patient died from respiratory failure after 1 week . on inquiry by the staff ,
the patient and his attending daughter reported no direct or household - related contact with pigs .
in july 2010 , mrsa cc398 spa type t034 had been isolated from a bedsore of another patient living in the same nursing home as patient 3 . in november 2013 ,
the organism was isolated once more from the patient , this time from an indwelling urine catheter .
patients 3 and 4 lived in the same wing of the nursing home and shared common facilities .
after hospital staff recognized the transmission chain , all residents in this wing and all attached staff were screened for mrsa , but none tested positive .
the mrsa cc398 isolates identified in the 2 transmission chains were spa types t011 and t034 , which are common among livestock - associated mrsa strains ( 12 ) .
draft whole - genome sequencing was performed on 7 isolates from the 4 patients in the 2 transmission chains , and results were compared with similar data for cc398-related mrsa and methicillin - sensitive s. aureus isolates from denmark obtained during a previous study of the global dissemination of isolates belonging to this clonal complex ( 13 ) .
sequence files for the individual isolates from patients are available at the european nucleotide archive ( http://www.ebi.ac.uk/ena ; accession no .
a phylogenetic tree was constructed from single - nucleotide polymorphism differences in genome sequences of isolates from the 4 patients and from the isolates obtained during the previous study by using the s0385 complete genome sequence ( genbank accession no .
this analysis showed that the patient isolates within each transmission chain were closely related but that no close relation between the 2 chains existed .
in addition , the isolates from both chains clustered among isolates previously found to be animal associated ( clade iia ; 13 ) .
consistent with an animal origin , all isolates carried the tet(m ) and czrc determinants ( similar to most mrsa strains from clade iia ) and lacked the sa3 phage ( typical of the human clade i ) ( 13 ) .
phylogeny of methicillin - resistant staphylococcus aureus ( mrsa ) clonal complex ( cc ) 398 isolates linked to fatal septicemia in a hospital patient and a nursing home resident in denmark .
draft whole - genome sequencing was performed on 7 isolates from the 4 patients identified in the 2 transmission chains , and results were compared with similar genomic data for cc398-related mrsa and methicillin - sensitive s. aureus isolates obtained in denmark during a previous study of isolates belonging to cc398 ( 13 ) .
single - nucleotide polymorphism differences were identified , and a maximum - likelihood phylogeny was inferred from raw data by using the web tool csi phylogeny ( https://cge.cbs.dtu.dk//services/csiphylogeny ) .
the region of bp 12252135180 was excluded from analysis because it contains the spa region and disrupts the phylogenic signal ( 13 ) .
p , pig ; h , human ; met , methicillin susceptibility ; r , resistant ; s , susceptible .
we report 2 fatal mrsa cc398 infections after human - to - human transmission in institutional settings .
both patients had debilitating underlying diseases but were in a stable condition until the time of their infections .
the sequence of events leaves no doubt that septicemia attributable to mrsa cc398 was the cause of death in both cases .
the cc and spa types of the isolates causing fatal infections were typical for mrsa isolates from pigs .
phylogenetic analyses of whole - genome sequences indicated that the human isolates from the 2 transmission chains were located in different clusters that intermingled with isolates from pigs .
the different spa types and the clustering of the mrsa isolates from the 2 chains clearly indicate 2 separate chains of infection .
however , epidemiologic investigations and typing analyses strongly suggested that the 2 mrsa - infected patients could have acquired their infections from an asymptomatic carrier in the outpatient hemodialysis unit ( transmission chain 1 , patient 2 ) and the nursing home ( transmission chain 2 , patient 4 ) .
transmission of mrsa cc398 in hospitals and institutions has been reported elsewhere , which underscores its potential to spread through person - to - person contact ( 911 ) . in conclusion ,
the organism implicated in these 2 fatal cases was by all accounts spread from person to person .
these findings suggest that this clonal complex can be of high pathogenicity and is readily transmissible among humans .
|
we describe 2 fatal cases of methicillin - resistant staphylococcus aureus ( mrsa ) clonal complex 398 septicemia in persons who had no contact with livestock .
whole - genome sequencing of the isolated mrsa strains strongly suggest that both were of animal origin and that the patients had been infected through 2 independent person - to - person transmission chains .
|
The Study
Transmission Chain 1
Patient 1
Patient 2
Transmission Chain 2
Patient 3
Patient 4
Conclusions
|
celiac disease ( cd ) is an immune - based enteropathy triggered by dietary wheat gluten and similar proteins in barley and rye in genetically susceptible individuals .
recently , the espghn ( european society of paediatric gastroenterology , hepatology and nutrition ) proposed that cd may be defined as an immune - mediated systemic disorder elicited by gluten and related prolamins in genetically susceptible individuals and characterized by a variable combination of gluten - dependent manifestations , cd - specific antibodies , and hla - dq2 or hla - dq8 haplotypes , causing duodenal chronic inflammation .
cd is a t - cell mediated disease , in which gliadin - derived peptides activate lamina propria infiltrating t lymphocytes .
this leads to the release of proinflammatory cytokines , such as ifn- and il-15 which are responsible for the activation of the cytotoxic activity of intraepithelial lymphocytes ( iels ) that leads to a profound tissue remodeling .
this is a complex disorder , with environmental and immune - genetic factors contributing to its etiology .
the main genetic influence on cd is the hla locus , specifically mhc class ii genes that encode hla - dq2 ( hla - dq2.5 and hla - dq2.2 ) and hla - dq8 heterodimers .
the prevalence of disease is usually reported to be about 1% in the general us population but there are emerging data suggesting an increase in some countries . although there is a strong genetic predisposition , resulting from the presence of the hla gene dq2/dq8 in the development of cd , and gluten is the main environmental factor responsible for the signs and symptoms of this disorder , neither genetic nor environmental factors show 100% correlation .
thus other immune , genetic , and environmental factors must be involved in cd onset .
evidence for a genetic component is best exemplified by the strong dependence on the presence of the hla - dq2 ( encoded by the alleles dqa105 and dqb102 ) and hla - dq8 ( dqa103 and dqb1 - 0302 ) haplotypes . more than 95% of those with cd express hladq2 while the rest express hla - dq8 . however , about 3040% of the general population expresses hla - dq2 , so while these hla genes are necessary , they are not sufficient for developing disease and clearly non - hla genes are also involved .
at least 39 non - hla genes have been identified through genome - wide association studies as strongly associated with cd .
most of these genes are involved in control of the innate and adaptive immune response .
however , the role of these genes in the development of cd is not completely clear .
the primary environmental trigger is gluten , the protein fractions of wheat , barley , and rye .
the incomplete digestion of this protein by humans , due to high concentrations of glutamine and proline , results in the formation of residual partially digested peptides .
these peptides are responsible for innate and adaptive immune responses underlying the disease in genetically predisposed subjects .
currently , some studies are evaluating the role of the modifications of gut microbiota as a factor contributing to the onset of disease .
although the precise immune mechanisms that are involved in the progressive destruction of the small intestinal mucosa remain to be elucidated , the hallmark of cd is an immune - mediated enteropathy that involves both of the innate and adaptive immune system . in relation to innate immune system response
, we should consider that some gluten peptides can induce tissue damage by directly activating components of innate immunity .
the peptide ( -gliadin 31 - 43 ) p31 - 43/49 has been shown to activate the production of il-15 and the nk receptor - mediated cytotoxicity by iels [ 11 , 12 ] , independent of tcr specificity , and to induce apoptosis of enterocytes , upregulate mhc class i molecules , activate map kinase pathway , and upregulate the expression of cd83 , a maturation marker of dendritic cells .
the presence of a receptor for p31 - 43/49 in intestinal epithelial cells has not been found yet and , thus , the molecular mechanism underlying the biological effects observed as a consequence of the interaction of this peptide with the gut mucosa remains still unclear . the dq2 and dq8 molecules confer susceptibility to cd by presenting disease - related peptides to t - cells in the small intestine or by shaping the t - cell repertoire during t - cell development in the thymus .
initially , paracellular passage of gliadin peptides is due to an increase of gut permeability which , in turn , is due to an upregulation of zonulin , an intestinal peptide involved in epithelial tight junction control .
also , il-15 contributes to promoting the cd4 + t - cell adaptive immune response [ 3 , 16 ] .
il-15 up - regulates both cd94/nkg2c and nkg2d nk receptors boosting their ability to lyse enterocytes .
the adaptive immune response in cd is characterized predominantly by production of the proinflammatory cytokine interferon- ( ifn- ) from gluten - specific cd4 + t - helper cells triggered by gluten - derived peptides recognized by hla - dq2 or hla - dq8 heterodimers of antigen - presenting cells ( apcs ) .
tissue transglutaminase-2 ( ttg2 ) converts noncharged glutamine into negatively charged glutamic acid through a process called deamidation .
in fact , the peptide - binding motifs of dq2 and dq8 predict a preference for negative charges at anchor positions of the bound peptides .
dq2 has a preference for negatively charged residues at the p4 , p6 , and p7 anchor positions , whereas dq8 has a preference for negatively charged residues at anchor positions p1 , p4 , and p9 .
generally , gluten proteins contain few negatively charged residues but in active cd the level of expression of enzyme tg2 is increased and the ratio of deamidation to transamidation is markedly increased . in active cd tg2 is expressed at the epithelial brush border , as well as being expressed extracellularly in the subepithelial region .
the ph in the proximal small intestine is ~6.6 which should allow marked tg2-mediated deamidation of peptides in the brush border .
this process increases the negative charge on the peptide molecule and enhances binding of the peptide within the peptide binding groove of the hla - dq2 molecule on the surface of the apcs .
this is a prerequisite for a gluten - specific t - cell response as well as a b - cell response that results in production of the anti - ttg antibodies that represents an epiphenomenon of the cd pathogenesis and may be used in the diagnostic pathway [ 15 , 21 ] .
furthermore , deamidated gluten peptides are presented to cd4 + t - cells that release proinflammatory cytokines activating cytotoxic cd8 iels ( cd8 + tcr+ and tcr+ t - cells ) contributing to a profound tissue remodeling and damage [ 13 , 22 ] . while iga antibodies against either gluten or the autoantigen tg2 serve as a highly useful means of testing for cd , their precise role in the immunopathogenesis of the condition remains yet unknown .
whether they are byproducts of the intestinal adaptive immune response or they play a direct role in cd pathogenesis remains unclear .
there is evidence that il-21 plays a role in the pathogenesis of cd as high levels of this cytokine can be demonstrated in biopsies from those with active disease that are not on treatment .
however , the mechanism whereby il-21 is produced and the precise role it plays in the disease process remains unexplained .
more recently , it has been proposed that , in the intestine of cd subjects , normal gluten peptides may be complexed to intraluminal secretory iga , bound to an iga receptor and transported , and protected from lysosomal degradation by a specific transcytosis pathway involving the transferrin receptor cd71 .
compared to nonceliac subjects or cd subjects on a gluten - free diet , cd71 expression is increased in cd sufferers and colocalizes with iga at the apical enterocyte membrane . in summary ,
the disease develops as a result of an abnormal cd4 + t - cell - initiated immune response to gluten . generally , gluten gains access to peyer 's patches physiologically via m cells or supraphysiologically during periods of increased epithelial permeability
; thus , it is processed by peyer 's patches dendritic cells and presented to cd4 + t - cell . in nonceliac subjects
the presentation of gluten peptides on hla - dq2/dq8 induces a th2 response ; in celiac subjects in the presence of ttg2 activity and deamidation of gluten peptides in the apcs the response is biased towards th1 response .
presentation of gluten to t - cells could be carried out not only by dendritic cells but also by macrophages , b - cells , and even enterocytes that express hla class ii .
enterocytes can present antigens to lipoprotein lipase ( lpl ) via evaginations through the basement membrane and can express costimulatory molecules under inflammatory conditions .
the primed cd4 + t - cells would then recirculate to the lamina propria [ 32 , 33 ] , and subsequent contact with gluten would induce their activation and proliferation , with production of proinflammatory cytokines , such as tnf- , il-1 , il-6 , il-15 , il-17a , il-17f , il-21 , il-22 , il-23 , and il-26 ( figure 1 ) [ 34 , 35 ] .
this would result in synthesis and release of metalloproteases mmp-1 , mmp-3 , and keratinocyte growth factor ( kgf ) by stromal cells , which would induce cryptal hyperplasia .
the next stage , villous atrophy , would be due to enterocyte death induced by iels [ 39 , 40 ] . in fact cd8
+ iels from cd patients have been shown to respond to gluten peptides presented by hla - a2 .
additionally , there is an overexpression of membrane bound il-15 on enterocytes in active cd which induces the expression of the nk receptors cd94 and nkg2d by cd3 + iels .
the ligand for nkg2d and mic - a is overexpressed on enterocytes in active cd , and this supports the involvement of the mic - a / nkg2d pathway in the epithelial atrophy of cd .
it is characterized by an interplay between different cells and their defense systems , food particles , molecules derived from digestion , and the vast array of residing microbial species with their secretory products .
these microorganisms present in the gut lumen form the microbiota that performs several physiological functions , that is , the absorption and digestion processes , tolerance to non - self - food antigens , and defense from pathogens .
the composition of main bacterial populations does not stabilize until after the first few years of life . in this period ,
the microbiota gradually colonizes the mucosal and skin surfaces of the neonate and exerts the greatest effect on the development of the immune system .
components of the intestinal microbiota play a crucial role in the postnatal development of the immune system . during the early postnatal period ,
the intestinal microbiota stimulates the development of both local and systemic immunity , while later on these components evoke inhibitory regulatory mechanisms intended to keep both mucosal and systemic immunity in check . in this postnatal period ,
components of the normal microbiota induce a transient physiological inflammatory response in the gut associated with enlargement of the mucosal - associated lymphatic tissue and increases in its cellularity [ 43 , 44 ] .
many studies have shown that microbial colonization of animals living in germ - free conditions results in an increase in immunoglobulin levels , the production of specific antibodies , and substantial changes in mucosal - associated lymphocyte tissues and cell populations [ 45 , 46 ] .
interestingly , the microbial colonization of germ - free mice also speeds up the biochemical maturation of enterocytes , resulting in a shift in the specific activities of brush - border enzymes nearly to the extent found in conventional mice .
moreover , a similar introduction of microorganisms alters the synthesis of sugar chains in membrane - associated glycoproteins , which could influence the gut barrier function [ 4749 ] . in some cases , impaired function of the intestinal barrier leads to an increase in antibodies directed against antigens present in the intestinal lumen .
it was recently shown that the appearance of these antibodies or / and autoantibodies in individuals lacking clinical symptoms may have important predictive value for the development of inflammatory and autoimmune diseases [ 50 , 51 ] . in the case of autoimmune diseases
, considerable effort has been made to understand mechanisms leading to the loss of self - tolerance .
a principal function of the microbiota is to protect the intestine against colonization by exogenous pathogens and potentially harmful indigenous microorganisms .
however , in certain conditions , some species of bacteria are thought to be capable of causing disease by producing inflammation , infection , or increasing cancer risk for the host .
particular bacterial populations that are typically found in very low abundance can acquire pathogenic properties .
these conditions include inherent immune defects as well as changes in diet and/or acute inflammation and can result in the disruption of the normal balanced state of the gut microbiota , which is referred to as dysbiosis .
dysbiosis involves the abnormal accumulation or increased virulence of certain commensal populations of bacteria , thereby transforming former symbionts into pathobionts .
the breakdown of the normal microbial community contributes to increase in the risk of pathogen infection and the overgrowth of harmful pathobionts and inflammatory disease .
the intestinal dysbiosis of cd patients is characterized by increases in numbers or proportions of bacteroides spp . and reductions in those of bifidobacterium spp . and b. longum , which were not completely normalized after patient adherence to a gluten - free diet ( gfd ) [ 5456 ] .
e. coli and staphylococcus numbers were also higher in feces and biopsies of untreated cd patients than in those of controls , but the differences were normalized after gluten withdrawal .
the analyses of the prevalence of bacterial species associated with duodenal biopsies also revealed a reduction in bacteroides species diversity in patients , untreated and treated with the gfd , in comparison with controls .
in addition to dysbiosis , infections may also disrupt the intestinal homeostasis leading to chronic inflammation and tissue damage , which could eventually contribute to reduced gluten tolerance .
in fact , infections have often been considered to initiate the process in genetically predisposed individuals .
these include polyclonal lymphocyte activation , increased immunogenicity of organ autoantigens secondary to infection - mediated inflammation , or antigen mimicry molecular mechanisms .
one major hypothesis explaining how infectious components can cause autoimmune reactions is based on the concept of cross - reactivity , also known as molecular mimicry , that is the similarity between the epitopes of autoantigens and epitopes of harmless environmental antigens .
the adjuvant activity of microbial components may participate in the stimulation of apcs , such as dendritic cells , that leads to the abnormal processing and presentation of self - antigens .
homeostasis of the intestinal mucosa may be disturbed by pathogenic microorganisms and toxins attacking the intestine or by inadequately functioning components of the immune system , as observed in immunodeficiency or in cases of dysregulated mechanisms of the mucosal immune system .
the intestinal mucosa can be affected as a consequence of either insufficient activity or exaggerated activation of the immune system .
various complex diseases may occur as a consequence of disturbances of mucosal barrier function or of changes in mechanisms regulating mucosal immunity to food or component of the microbiota [ 62 , 63 ] .
the complexity and interindividual variation of the gut microbiota composition in humans represent a confounding factor in the efforts to determine the possible significance of individual commensal microbial organisms in disease pathogenesis .
recent data have described a possible link between cd onset in susceptible patients and diverse infectious agents , which may have occurred as early as during the perinatal period .
. demonstrated a possible protective role that infections with ebv , cmv , and rubella may have on susceptible individuals ; it seems that encountering certain infections may establish a particular immunological background that disfavours the evolution of autoimmune conditions .
moreover , several intestinal viral triggers including adenovirus , hepatitis c virus ( hcv ) , and rotavirus and bacterial infections capable of initiating or augmenting gut mucosal responses to gluten were suggested to play a role in the pathogenic mechanism of cd .
an environmental factor , such as an infectious agent , is thought to precipitate the disease , via various pathogenic mechanisms , such as molecular mimicry , resulting in modulation of the host 's immune tolerance .
several other gastrointestinal pathogens have been associated with the development of cd , with varying outcomes ; most are isolated case reports .
other pathogens such as campylobacter jejuni , giardia lamblia , rotavirus infection , and enterovirus infection have also been associated with development of cd .
abnormal components found among the microbial inhabitants adhering to the diseased jejunal mucosa have been described and recently analyzed using new microbiological methods .
profound changes in the fecal and duodenal microbiota composition of patients with active disease who are on a gluten - free diet have also been demonstrated .
bacteroides and clostridium leptum groups were more abundant in faeces and biopsies of cd patients than in controls regardless of the stage of the disease .
escherichia coli and staphylococcus counts were also higher in faces and biopsies of nontreated cd patients than in those of controls .
interestingly , some commensal bacteria , such as escherichia coli , promoted the activation of innate immune cells by gliadin , whereas others such as bifidobacteria exerted inhibitory effects .
dietary factors seem particularly relevant at early stages when the immature neonate 's gut is acquiring and shaping its own microbiota and undergoing major physiological and immunological developments up to the point when the immune system acquires full competence and tolerance to nonharmful antigens .
the period in which the human host is most acutely influenced by the microbiota is the postnatal period , during which the germ - free neonate moves from the sterile environment of its mother 's uterus into a world full of microorganisms and during which the neonate 's mucosal and skin surfaces become gradually colonized . particularly relevant
as early as the seventies , it had become clear that the introduction of gluten in the diet after the fourth month of life would reduce the incidence of cd onset .
infants who carry either the hla - dr3 or dr4 alleles or who have a first - degree relative affected by cd have a fivefold increased risk of developing cd autoimmunity with the presentation of positive tissue transglutaminase ( ttg ) autoantibody if they are exposed to gluten in the first three months of life .
infants introduced to gluten at 7 months or later also had an increased risk of cd compared with those exposed between 4 and 6 months .
for the increased risk of cd when first gluten exposure occurs in younger and older children instead of at the age of 46 months . on the one hand , in younger children , early introduction of solid foods ( i.e. , before the intestinal immune system reaches a certain level of maturation ) may lead to intolerance .
the increased risk of cd in children introduced to gluten at 7 months or older might be due to the larger amounts of gluten intake at the first exposure .
the espghan committee on nutrition has outlined possible practical suggestions on the introduction of complementary feeding to avoid both early ( < 4 months ) and late ( 7 months ) introduction of gluten and to gradually introduce small amounts of gluten whilst the infant is still breast - fed in order to reduce the predisposition to cd later in life .
however , the time of first exposure to potentially allergenic foods in infants differs significantly between countries and occurs much earlier than recommended in some countries , as reported in .
a recent meta - analysis of observational retrospective studies was used to analyze the protective role of breast - feeding against cd onset and concluded that increased duration of breastfeeding is associated with a reduced risk of cd .
a study of 627 cases with confirmed cd revealed that the risk of cd was reduced in a group of children aged < 2 years if they were still being breast - fed when dietary gluten was introduced and the risk increased when the gluten was introduced in the diet in large amounts .
it is also important to examine further whether favorable infant dietary patterns postpone cd onset or in fact reduce the overall lifetime risk of the disease .
other recent studies have pointed to the role of breast - feeding in delaying cd in infancy .
d'amico et al . showed that children with cd who had been exclusively breast - fed had a delayed onset and less severe disease symptoms than those who had not been exclusively breast - fed . in spite of all the evidence reported
, it remains unclear whether those children breast - fed during the introduction of gluten are more likely to develop an extraintestinal ( atypical ) cd .
a series of 162 celiac children registered by the university of chicago revealed that children breast - fed at the time of gluten introduction were just as likely to develop both intestinal and extraintestinal symptoms , whereas children who were not breast - fed when weaned with gluten had a much higher chance of developing intestinal symptoms .
human milk provides many bioactive factors , including antimicrobial and anti - inflammatory agents , enzymes , hormones , and growth factors , many of which are involved in gut maturation and development of the infant 's innate and acquired immunity .
breast - feeding might affect tolerance induction in infants because of the possible transfer of small amounts of gluten and gluten - specific iga antibodies through breast milk and the presence of factors in breast milk that affect immune system maturation and responses .
also , it seems that the lymphocyte subset profile of breast - fed infants is associated with a better response to gliadin after gluten introduction .
other reasons that could explain a protective effect of breast - feeding against cd development could be related to the role human milk plays in defining microbiota composition and in the incidence of infections .
in particular , innate immunity cells , located in the lamina propria , promote immunological unresponsiveness to commensal bacteria , which is important for maintaining gut homeostasis .
specifically , gut - resident phagocytes are hyporesponsive to microbial ligands and commensal bacteria , and they do not produce biologically significant levels of proinflammatory molecules upon stimulation . however , the microbiota is essential for upregulating the production of pro - il-1 , the precursor to il-1 , in resident innate immunity cells .
this role is , at least partly , mediated by its ability to induce the expression of endothelial adhesion molecules that contribute to neutrophil recruitment and pathogen clearance in the intestine .
initiation of the innate immune response in the intestine is triggered by pathogen - recognition receptors ( prrs ) .
these prrs serve as sensors of pathogen - associated molecular patterns ( pamps ) from the intestinal lumen .
tlrs are transmembrane proteins that are typically expressed by intestinal epithelial cells either on the cell surface or in endosomes .
tlr signaling in the intestine is involved in epithelial cell proliferation , immunoglobulin a ( iga ) production , and antimicrobial peptide expression , functions that are crucial for maintaining a healthy epithelial barrier .
prrs are also expressed by other immune cells in the lamina propria and they can activate an inflammatory response involving both innate and adaptive immune system .
unfortunately , in some cases the innate immune system 's attempts to protect the host fail and chronic inflammation and intestinal autoimmunity occur , such as in the case of celiac disease and ibd .
recent experimental data demonstrated the existence of a strong and direct interaction between tlrs and intestinal microbiota .
for example , cheng et al . demonstrated that pediatric cd patients have lower duodenal expression of tlr2 and higher expression of tlr9 as compared to healthy controls confirming that microbiota may have a role in cd .
in fact , cheng and colleagues concluded that the overall composition , diversity , and the estimated microbe associated molecular pattern ( mamp ) content of microbiota were comparable between cd and healthy subjects , but a subpopulation profile comprising eight genus - like bacterial groups was found to differ significantly between healthy subjects and cd . in healthy subjects , increased tlr2 expression was positively correlated with the expression of tight junction protein zo-1 . in cd , the expression of il-10 , ifn - gamma , and cxcr6 was higher as compared to healthy subjects .
other recent data comparing the composition of microbiota between cd patients and healthy controls demonstrated that tlrs activation by intestinal microbiota may determine different effects on the human gut of cd patients and healthy controls .
these data suggest that microbiota and altered expression of mucosal receptors have a role in cd . in cd subjects ,
the increased expression of il-10 and ifn - gamma may have partly resulted from the increased tlr9 expression and signaling .
studied the expression of tlrs and cytokines and revealed that they occur in duodenal mucosa and are elevated in both children and adult celiac patients .
cd patients with high levels of tlr4 also showed high levels of proinflammatory cytokines ( il-1 , il-6 , il-8 , and il-17 ) as well as transcription factors ( irak4 , myd88 , and nf-b ) .
these data support the hypothesis that a unique pattern of tlr expression is associated with cd independently of age at diagnosis .
the evidence that pediatric and adult patients have a similar inflammatory profile will encourage treatment of both with the same immunological therapy in the future .
the only approved treatment for cd is the lifelong complete exclusion of the gluten from the diet .
indeed , we need novel strategies , targeting various factors at different levels in cd pathogenesis . for example
, the reduction of gluten antigenicity and/or elimination of toxic peptides from gluten could be walkable ; in this manner , the food gluten would not have the negative effect on activation of host immune system and the consequential gut inflammation .
these modifications of gluten antigenicity may be achieved in several manners , from the selection of natural cereal cultivars to the genetic modification of gluten peptide sequences or more likely producing genetically modified organisms , in which toxic sequences are deleted or silenced .
the other translational strategy of cd therapy derives from the knowledge of immunomodulatory mechanisms of disease .
in fact , in cd , food gluten peptides activate lamina propria t - cells to produce inflammatory cytokines leading to tissue destruction .
thus , a way to block this pathogenetic event in cd could be a specific modulation of gluten reactive pathogenetic target t - cells .
this modulation in gluten related to t - cell reactivity may be borrowed from the actual evidences on other immune hypersensitivity related diseases , such as allergy and autoimmune diseases .
in fact , in these diseases , such as in cd , there is a pivotal role of cd4 + t - helper cells in driving and orchestrating gut inflammation and tissue destruction .
the goal of pathogenetic translational therapy may be to develop vaccines consisting of synthetic peptides representing t - cells epitopes that may have the function to restore the balance between inflammatory and regulatory responses to the causative antigens [ 91 , 92 ] . in cd , this could be achieved using a therapeutic vaccination constructed with the panel of most immunodominant gluten epitopes .
the efficacy of vaccine therapy has been just demonstrated in allergy in which it is able to reduce allergen sensitivity and improve regulatory t - cell function .
furthermore , there are recent findings in the alterations of the microbiota in various medical conditions , such as cd , inflammatory bowel disease , allergy , and colorectal cancer .
even though changes of the microbiota could be linked to the etiopathogenesis of these diseases , further studies are needed to understand how to modulate microbiota to ameliorate the morbidity of cd .
chronic inflammatory diseases have multifactorial etiologies that involve environmental components and many immune and genetic factors .
an environmental factor that precipitates disease is known ( gluten ) ; the hla molecules that confer predisposition to the disease have been identified ( hla - dq2/hla - dq8 ) ; and access to the small intestine is simple .
thus , understanding the interaction of the microbiota with pathogens and host might provide new insights into the pathogenesis of disease , as well as novel avenues for preventing and treating intestinal and systemic disorders . the high increase in incidence of autoimmune disorders
can not be explained only by genetic drift and is thought to be the result of changes in the environmental factors and in their complex interaction with innate and adaptive immunity .
although there is strong support for the role of microorganisms as triggers of innate immune activation , there is no evidence of a role for molecular mimicry in cd . in other words
, we do not have any evidence so far that cd pathogenesis is the result of a t - cell response against a microbial peptide cross - reacting with gluten .
the gut microbiota has been studied for more than a century ; however , we have only recently begun to understand the ever - expanding roles for these microscopic organisms in health and disease . despite the complexity of the gut microbiota
, there is a delicate balance in bacterial populations such that any disruption in this balance leads to dysbiosis and , consequently , to decreased resistance to pathogen colonization , to the favoured growth of pathobionts , and to pathological both innate and adaptive immune responses .
so , in genetically predisposed individuals , gluten in association with microbial antigens can stimulate and modulate innate and adaptive immune response , sustaining a chronic mucosal inflammation , underlining this chronic disease . in summary
, cd may be considered as a model to explain the pathogenesis of several other autoimmune diseases . in particular , how in the steady state condition homeostasis is maintained due to the complete balance between pro- and anti - inflammatory factors whereas , during disease , this balance is altered .
the breakdown of the interaction among microbiota , innate immunity , and genetic and dietary factors leads to the disruption of homeostasis leading to inflammation and tissue damage .
thus , focusing the attention on this interaction and its breakdown may allow a better understanding of the cd pathogenesis and finally get novel translational avenues for preventing and treating this widespread disease .
|
celiac disease ( cd ) is an immune - mediated enteropathy , triggered by dietary wheat gluten and similar proteins of barley and rye in genetically susceptible individuals .
this is a complex disorder involving both environmental and immune - genetic factors .
the major genetic risk factor for cd is determined by hla - dq genes .
dysfunction of the innate and adaptive immune systems can conceivably cause impairment of mucosal barrier function and development of localized or systemic inflammatory and autoimmune processes .
exposure to gluten is the main environmental trigger responsible for the signs and symptoms of the disease , but exposure to gluten does not fully explain the manifestation of cd .
thus , both genetic determination and environmental exposure to gluten are necessary for the full manifestation of cd ; neither of them is sufficient alone .
epidemiological and clinical data suggest that other environmental factors , including infections , alterations in the intestinal microbiota composition , and early feeding practices , might also play a role in disease development .
thus , this interaction is the condicio sine qua non celiac disease can develop .
the breakdown of the interaction among microbiota , innate immunity , and genetic and dietary factors leads to disruption of homeostasis and inflammation ; and tissue damage occurs .
focusing attention on this interaction and its breakdown may allow a better understanding of the cd pathogenesis and lead to novel translational avenues for preventing and treating this widespread disease .
|
1. Introduction
2. Pathogenesis and Pathogenic Model of Celiac Disease
3. Immunopathogenesis
4. Association between Microbiota and Dietary Factors in Development of Innate and Adaptive Immunity
5. Dietary and Microbiota Changes and Immunomodulatory Approaches in Celiac Disease: Translational Applications
6. Conclusion
|
the demand for food of animal origin increases each year . to satisfy this demand , livestock production within the european union ( eu ) in 2011 was approximately 10 million heads of goats , 80 million heads of sheep , 80 million heads of cattle , and 150 million heads of pigs . to produce safe and nutritional food products , the animals need to be in good health .
even though veterinary medicines contribute to improving and maintaining animal health , administration of these medicines by the farmer is carried out under licence via a veterinarian .
the amount of drugs employed in food production estimated by kools et al . was 6051 t , with antibiotics the most frequently used class of drug ( 5393 t ) .
the groups of tetracyclines and -lactams were used in high amounts and antiparasitic agents ( 194 t ) were the second most frequently used class of drug .
intensively produced animals are often fed with concentrated feed , a mixture of various materials ( oats , wheat , barley , rye , cottonseed , and crambe ) and additives .
antibiotic sulphonamides , tetracyclines , and -lactam are the most frequently used antibiotics , and coccidiostats and ivermectin are the most frequently used antiparasitic agents .
coccidiostats and histomonostats are a group of antiparasitic agents that have been shown to be persistent during the manufacture of feed and carry - over of this type of drugs has been demonstrated .
the eu introduced maximum levels for these substances in nontarget feed in 2009 ; this regulation was later modified for some coccidiostats by the regulation ec/574/2011 .
cross contamination between medicated and nonmedicated feed could occur with any type of drug added to the feed , not only to coccidiostats , particularly when the cleaning process between batches is inefficient .
recently , a study conducted by stolker et al . in the netherlands confirmed that nonmedicated feed batches were contaminated with antibiotic residues such as tetracyclines , penicillins , and sulfonamides . from 140 samples
the fact that antibiotics could be present as contaminants in feed without the farmers ' knowledge implies that withdrawal times will not be considered and antibiotic residues could remain in animal products ( meat , eggs , milk , honey , seafood , and fish ) , in addition to the development of antibiotic resistant bacteria . due to the problems related to food safety , the authorities involved regularly monitor the presence of veterinary drugs in food of animal origin ( eggs , milk , muscle , and liver ) .
controls on the water and food consumed by the animals have also been implemented , but the analysis conducted only evaluated the presence of substances such as pesticides [ 7 , 8 ] , nitrofurans , and mycotoxins [ 10 , 11 ] .
methods based on hplc - ms / ms detection are considered confirmatory methods because these types of methods provide full or complementary information , enabling substances to be unequivocally identified and if necessary quantified at the level of interest , according to the european commission decision 2002/657/ec .
therefore , it is recommended to use confirmatory methods to detect the presence of antibiotics in nontarget feed .
van poucke et al . reported a method for the analysis of zinc bacitracin , spiramycin , tylosin , and virginiamycin with quantification limits below 500 g / kg .
published a multiclass method for the analysis of 33 analytes for 14 groups of antibiotics ( including tetracyclines , quinolones , penicillins , ionophore coccidiostats , macrolides , and sulphonamides ) with quantification limits of 3.865.0 g / kg .
of the eight forms of commercially available tetracyclines , four are frequently used in food animal production ( chlortetracycline , doxycycline , oxytetracycline , and tetracycline ) .
maximum residue limits ( mrl ) for these four tetracyclines and their three epimers have been introduced for various foods of animal origin , including eggs , muscle , and milk . as maximum levels ( ml ) for these substances in feed samples
concentrations of tetracyclines in medicated feed are variable and depend on the target animal , with dosage rates between 25 and 700 mg / kg .
therefore , carry - over should be expected in the batch of feed manufactured after a medicated feed .
reported that 100% of samples from the first batch of feed produced after the manufacture of medicated feed were contaminated with tetracyclines , with concentrations of 0.5154 mg / kg .
based on these results , carry - over contamination is also expected for the other two commonly used tetracyclines ( chlortetracycline and tetracycline ) as they have similar chemical properties [ 16 , 17 ] .
based on the common use of tetracyclines in food animal production and the absence of confirmatory methods for the presence of the four tetracyclines in animal feed , the aim of this work is to present an hplc - ms / ms method for the analysis of tetracyclines in nonmedicated feed samples at levels of g / kg .
disodium hydrogen phosphate dehydrate , anhydrous citric acid , ethylenediaminetetraacetic acid disodium salt ( edta ) , trichloroacetic acid ( tca ) , and formic acid ( purity > 99% by analysis ) were purchased from sigma - aldrich ( mo , usa ) .
tetracycline , chlortetracycline , doxycycline , and oxytetracycline ( purity > 98% ) and demeclocycline , used as the internal standard ( is ) , were supplied by sigma - aldrich ( mo , usa ) .
organic solvents , methanol and ethyl acetate , hplc or analytical grade , were purchased from scharlau chemie ( barcelona , spain ) and demineralised water ( resistivity 18 mu cm ) was prepared in - house with a milli - q water system ( millipore , bedford , ma , usa ) . mobile phase a consisted of milli - q water acidified to 0.04% with formic acid and mobile phase b consisted of methanol , acidified to 0.1% with formic acid . to prepare the individual stock solution of tetracycline ,
20 mg of tetracycline was dissolved in 20 ml of methanol and stored at 20c for up to six months .
the intermediate solution , a mixture of tetracyclines , was prepared by diluting the stock solution of each tetracycline to a final concentration of 50 g / ml and stored at 20c for up to one month .
a standard working solution of tetracycline was prepared freshly each day by diluting the intermediate stock solution to a final concentration of 1 g / ml . for the internal standard stock solution ,
mcilvaine buffer was prepared with 10.8 g of citric acid , 10.93 g of disodium hydrogen phosphate , and 33.62 g of ethylenediaminetetraacetic acid disodium salt dihydrate ( c10h14n2na2o8 ) .
once the three reagents were completely dissolved they were mixed and the volume was made up to 1 l and the ph adjusted to ph 4 .
hplc - ms / ms determination of tetracyclines was performed according to a previously reported method .
the hplc - ms / ms consisted of an hplc alliance 2795 and a ms quattro premier xe triple quadrupole ( waters , manchester , uk ) controlled by the software masslynx 4.1 ( waters , manchester , uk ) .
the chromatographic analyses were performed by injecting 25 l of extract into a sunfire c18 column ( 150 2.1 mm i.d .
, 5.0 mm ) ( waters , manchester , uk ) . mobile phases a and b were mixed on a gradient mode and with a flow rate of 0.25 ml / min .
an electrospray ionisation ( esi ) probe was set up on the triple quadrupole ms to evaporate the mobile phase coming from the hplc and to ionise the tetracyclines .
analytes were detected in positive - ion mode and under the following conditions : capillary voltage 3 kv , source temperature 120c , desolvation temperature 350c , cone gas flow 49 l h , and desolvation gas flow 650 l h. as indicated in the decision 2002/657/ec , tetracyclines were identified on the basis of their selected reaction monitoring ( srm ) transition and their retention time ( rt ) .
different volumes of the tetracycline working standard solution were added to 2 g feed samples ( exempt of tetracycline ) and shaken in the dark for 30 min .
concentrations of tetracycline in the matrix matched feed samples were 0 , 400 , 800 , 1200 , 1600 , and 1600 g / kg .
to extract the tetracyclines from the feed samples , 2 g of grounded feed , 8 ml of mcilvaine buffer , 300 l of tca , and 0,1 ml of is working solution were added to a 50 ml polypropylene tube . after shaking the sample in the dark for 10 min ,
6 ml of ethyl acetate was added and the samples were shaken in an orbital shaker at 200 rpm for 20 min .
after 15 of centrifugation at 4500 rpm , in a model 5415d centrifuge ( eppendorf , hamburg , germany ) , 2 ml of the supernatant was transferred to a 10 ml amber conical tube and evaporated to dryness , in a turboevaporator model turbo vap ii de zyrmark ( hopkinton , ma , usa ) .
the final residue was dissolved in 0.5 ml of a mixture of mobile phase components ( 90a:10b ) and vortexed . to filtrate the final extract ultrafree - mc centrifugal filter ( millipore , ma , us ) was employed and centrifuged at 9000 rpm for 10 min .
the filtrate was transferred into an hplc vial which contained a 0.3 ml microinsert and stored at 20c ; analyses were conducted within 3 days .
the guidelines used to validate the method and to interpret the results were those established in the commission decision 2002/657/ec .
the decision establishes criteria and procedures for the validation of analytical methods to ensure the quality and comparability of analytical results generated by official laboratories .
aspects such us trueness / recovery , precision ( under repeatability and reproducibility conditions ) , specificity , and applicability / ruggedness / stability of the method were investigated .
trueness and the other validation parameters were assessed through recovery of additions of known amounts of tetracyclines in blank feed samples ( except tetracyclines ) as no certified reference material exists for this type of analysis and following the recommendation included in the decision 2002/657/ec . for validation one batch of matrix - matched samples
was prepared with 21 samples fortified with tetracyclines at six concentrations ( 0 , 400 , 800 , 1200 , 1600 , and 4000 g / kg ) .
for concentrations 400 , 800 , and 1200 g / kg six replicated samples were prepared with only one sample for the remaining concentrations ( 0 , 1600 , and 4000 g / kg ) .
additionally , two reagent samples were prepared for control , a blank reagent ( containing only the reagents ) and fortified reagent ( containing 1200 g / kg of tetracycline and the reagents ) . to conduct the validation three batches of matrix - matched samples were prepared ; each batch consisted of 21 samples fortified with tetracyclines at 0 , 400 , 800 , 1200 , 1600 , and 4000 g / kg .
while for levels of 400 , 800 , and 1200 g / kg six replicated samples were prepared , only one sample was used for levels of 0 , 1600 , and 4000 g / kg .
after fortification , and prior to extraction , samples were shaken on an orbital shaker at 200 rpm for 10 min .
after the samples extraction procedure explained above was applied . as well as the 21 matrix - matched samples , with each batch of samples two additional samples
were prepared with reagents ( no feed ) ; one was spiked with tetracycline at 1200 g / kg ( fortified reagents ) and the other was not spiked with tetracyclines ( blank reagent ) . additionally , 20 feed samples were analysed to determine selectivity / specificity .
ten were spiked with tetracyclines at a validation level ( 800 g / kg ) and with 400 g / kg of three antimicrobial drugs commonly used in food animal production ( sulfadiazine , sulfamethoxazol , and trimethoprim ) .
the other ten samples were analysed without adding any veterinary drugs ( decision 2002/657/ec ) .
the decision limit ( cc ) and detection capability ( cc ) were determined as described by freitas et al .
, following the decision 2002/657/ec requirements and applying the validation level of 800 g / kg .
nonmedicated feed samples were collected from 50 milk farms located in galicia , spain , to investigate the presence of tetracycline residues in feed that are being consumed by cows that are producing milk daily .
additionally , samples were supplied by feed manufacturers to investigate carry - over levels of tetracyclines after making medicated feed .
the sampling procedures in both cases were conducted following the requirement of the regulation 691/2013 .
feed samples were stored at room temperature and in the dark with the objective of using similar store conditions compared to that in the industry and farms .
the main raw materials of the feed samples according to the labels were corn genetically modified ( between 36 and 15% ) , soy flour produced from soya been genetically modified ( present in some samples , between 4 and 38% ) , colza flour ( present in some samples , between 3 and 47% ) , and barley ( present in some samples , between 6 and 18% ) ; each feed sample had a particular composition which normally depends on the manufacture .
on the other proximate composition the feed samples were crude protein ( between 18 and 26% ) , crude fibre ( between 4 and 10% ) , oil and fat crude ( between 2.5 and 6% ) , crude ash ( between 6 and 10% ) , and sodium ( between 0.7 and 4% ) .
tetracyclines have more than three hydroxyl groups that are easily ionisable by esi to enhance their detection .
therefore , esi is commonly used for the ionisation of tetracyclines when analysis is conducted by hplc - ms / ms , independently of the matrix type [ 14 , 2024 ] . to optimise the ms parameters for a high ms signal response , standard solutions of 1
g / ml of individual tetracyclines were infused directly into the ms . during this procedure ,
one precursor ion and two product ions were selected for each tetracycline to conduct srm analysis ( table 1 ) . with the hplc - ms / ms operating on srm mode , two transitions and the retention time were used to achieve four identification points for each of the analytes , as required in the decision 2002/657/ec .
it should be highlighted that with other detection methods , such as diode array or a fluorescence detector , only one identification point is achieved and more steps are required .
the use of precursor and product ions identified in this research for tetracyclines was also reported by boscher et al .
2010 for the analysis of these analytes in feed , royal jelly , and muscle [ 14 , 20 ] .
the separation gradient could be run at ambient temperature ; however , it was observed that an increase in mobile pressure can cause the system to collapse ; therefore a temperature of 35c is recommended .
based on previously reported methods for tetracycline analysis in food and feed matrices [ 14 , 2527 ] different extraction protocols were tested .
firstly , simple extractions with ethyl acetate , hexane , acetonitrile , methanol , and dichloromethane were tested .
however , recoveries were low , due to the tendency of tetracyclines to form chelation complexes with different cations .
therefore , initial extraction with mcilvaine / edta buffer was employed and gave satisfactory results , as in previously reported methods [ 7 , 21 , 28 , 29 ] .
tetracyclines dissolved in the mcilvaine / edta solution were then extracted with ethyl acetate , as these two solutions are immiscible , and separation could be easily conducted as the organic phase stays on the top layer .
other authors employ spe cartridges such as oasis for dispersive spe instead of ethyl acetate in order to purify the extract .
the use of spe was avoided to reduce time and cost of the analysis as satisfactory results were achieved with the presented method .
animal feed is derived from a multitude of raw materials from plant and animal origin , as well as pharmaceutical and industrial sources .
as feed ingredients vary depending upon the animal , that is , poultry , swine , and cattle , analysis of tetracyclines in different feed types could be more complicated , particularly as the fat content will vary .
the method used in this paper has been tested in feed for cattle , laying birds and chickens , rabbits , and dairy cows , and satisfactory results were obtained in all cases .
these matrices were tested by preparing matrix - matched calibration curves with each type of feed , depending on the animal that was going to consume the feed .
calibration curves to quantify concentrations of tetracyclines were obtained by spiking feed samples with the analytes at different concentrations . if linear regression coefficients ( r ) were below 0.98 the extraction procedure was repeated . even if a signal to noise ratio ( s / n ) higher than 10 was achieved at 300 g / kg , validation was conducted at 800 g / kg to provide acceptable results at 0.5 , 1 , and 1.5 times the validation level ( 800 g / kg ) recommended by the decision 2002/657/ec .
figure 1 shows chromatograms of a blank sample , figure 2 shows a blank sample spiked with all the tetracyclines at 400 g / kg , and figure 3 shows the srm transition employed for each tetracycline in one of the samples spiked at 400 g / kg .
reference materials were not available ; therefore trueness of the method was calculated in terms of recoveries .
however , the advantage of the presented extraction protocol is that it does not require solid phase extraction and the four main tetracyclines can be identified and quantified simultaneously .
results for repeatability , calculated as the mean rds of the rsd ( n = 6 ) for each concentration on each day of the validation , were below 17% for chlortetracycline , doxycycline , oxytetracycline , and tetracycline ( table 2 ) .
results for reproducibility , calculated as the rds of 21 samples at the same concentration , were below 23% for all tetracyclines
( table 2 ) . to determine selectivity / specificity , 10 blank samples and the same samples spiked with the four tetracyclines at 800 g / kg were analysed .
the successful quantification of tetracyclines and the absence of interfering peaks at the retention times of each analyte demonstrated the selectivity / specificity of the method .
the limit of detection ( lod ) and limit of quantification ( loq ) of the method were calculated and verified with feed samples spiked with the tetracyclines at different concentrations .
based on s / n above 3 for lod and above 10 for loq in matrix - matched samples , the lod and loq of the method were set at 35 and 47 g / kg for chlortetracycline , 40 and 60 g / kg for oxytetracycline , 24 and 40 g / kg for tetracycline , and 100 and 150 g / kg for doxycycline .
cc and cc were determined using the conditions for substances for which no permitted limit has been established .
cc and cc were higher than lod and loq for all the compounds , meaning that tetracyclines detected at a higher level than the cc will be positive and levels of tetracyclines were quantifiable , without doubt .
cc and cc for chlortetracycline , doxycycline , oxytetracycline , and tetracycline were below 400 g / kg ( table 2 ) .
validation results already published have shown , in some cases , higher repeatability , reproducibility , and lower loq , such as the work conducted by boscher et al . who reported rsd lower than 12% and loq of 20 g / kg .
similarly , the method reported by stolker et al . achieved a loq of 0.1 g / kg for doxycycline and oxytetracycline .
however , it should be highlighted that none of the methods reported , based on the authors knowledge , have been validated according to the decision 2002/657/ec . from 75 feed samples investigated
oxytetracycline was the tetracycline more frequently detected present in 8% of the samples ( n = 6 ) ; its concentration range was between 90 and 400 mg / kg .
each tetracycline was detected in individual samples and their concentrations were 150 and 110 mg / kg .
chlortetracycline was the pharmaceutical detected at the highest concentration in this study , 15.14 mg / kg in feed samples for calves .
it is important to highlight that the consumption of contaminated feed at level such as 15.14 mg / kg could cause food safety problems giving positive food samples .
carry - over during feed manufacture has been proved for veterinary drugs such as coccidiostats and a similar case can be considered for tetracyclines , a group of antimicrobial agents commonly used in food animal production , due to its low cost .
the research work presents a simple and fast method for the analysis of the four tetracyclines regulated in the production of food of animal origin ( chlortetracycline , doxycycline , oxytetracycline , and tetracycline ) .
the method was validated according to the european guideline and successfully applied to 75 nonmedicated feed samples .
results showed the presence of tetracyclines in 15% of the samples , indicating that cross contamination occurs and maximum levels for tetracyclines may be required in the future .
|
the commission regulation 574/2011/ec set up maximum levels of coccidiostats and histomonostats in nonmedicated feed as a consequence of carry - over during manufacturing .
carry - over takes place from medicated to nonmedicated feed during feed production .
similar contamination could also occur for other pharmaceuticals such as tetracyclines , a group of antibiotics commonly employed in food production animal .
the objective of this work is to present a simple and fast method for the simultaneous detection of four tetracyclines ( chlortetracycline , doxycycline , oxytetracycline , and tetracycline ) in nontarget feed at a g / kg level .
validation of the method was performed according to the guideline included in the commission decision 2002/657/ec for official method .
the validated method was successfully applied to 50 feed samples collected from different milk farms and 25 samples obtained from feed manufacturers .
while oxytetracycline was the tetracycline most frequently detected , chlortetracycline was the analyte measured at the highest concentration 15.14 mg / kg . from 75 nonmedicated feed analysed 15% resulted to be positive for the presence of one tetracycline .
|
1. Introduction
2. Experimental
3. Results and Discussion
4. Conclusion
|
de ridder , vanneste , and focquaert address concerns relating to the definition of a ced and the distinction between treatment and enhancement .
they raise a number of problems with the treatment - enhancement distinction and suggest that we need to ask whether we are prepared to change the definition of health used by the medical devices directive. there are in fact three questions raised here .
the first is whether our proposal requires a robust characterization of the treatment - enhancement distinction , the second is whether our suggestions , as we intended them , actually involve changing the definition of heath implicit in the mdd ( there is no explicit definition ) , and the third is whether the mere inclusion of ceds within the mdd will have the de facto effect of changing the implicit regulatory concept of health or
treatment. in response to the first question , we would maintain that it is in fact an advantage of our approach that it minimizes the importance given to the treatment - enhancement distinction and thus diminishes the need to characterize it in a way that is immune to criticism .
on our approach , devices intended for enhancement are regulated in a similar manner to therapeutic medical devices .
thus , if some enhancement devices are misclassified as therapeutic devices , or vice versa , this will not have major implications for their regulation .
what matters , from our perspective , are risks and benefits . by contrast , some alternative approaches , including the one recommended by de ridder and collaborators , would regulate ceds and therapeutic medical devices quite differently , with the result that misclassification would have more significant regulatory effects , since devices regulated under the general product safety directive alone are held to less specific and less stringent standards . in short ,
we agree that the treatment - enhancement distinction is problematic and some of us have elsewhere rejected it but we believe a desire to mitigate the problems it raises counts in favor of our approach , not against it
. we would also dispute the suggestion that our approach requires any modification to the concept of health .
our proposal was not to change the definition of health ( nor treatment ) but , rather , to bring some non - therapeutic devices , which do not aim to improve health problems , within the remit of the mdd .
our proposals involved leaving the definition of a medical device used by the mdd unaltered .
ceds would not be medical devices on the mdd definition . instead , an ancillary positive list is proposed to bring specific devices with a non - medical purpose within the remit of the mdd alongside those devices defined as medical devices according to the criteria employed in the directive . by proposing an ancillary list for specific cognition - affecting devices without a medical purpose ,
we in fact reinforce the concepts ( ie medical and non - medical ) underpinning the european commission 's proposed creation of annex xv for implantable and other invasive devices for which the manufacturer claims only a non - medical purpose .
we proposed that this list should be extended to include non - invasive neuromodulation for non - medical purposes .
however , the third implicit question indeed requires consideration : will there be a de facto change in what is meant by health and
treatment if ceds are regulated under the mdd ? arguably , the mere association of ceds with therapeutic medical devices could result in an expansion of the concepts of treatment and health .
the possible implications of a shift in what is seen as health - related or
there , they suggest that medical device regulation might underscore the illusion that devices are beneficial .
however , we suggest that the idea that tdcs and tms techniques can yield cognitive enhancement in healthy adults is not a mere illusion .
further , we suggested that regulatory approval of moderately risky brain stimulation devices should be dependent on evidence of at least some degree of
performance. in this respect , it would be important to notify consumers explicitly on which population the ced has shown to be beneficial while reminding that it can be ineffective or detrimental to other populations .
regulation would therefore go some way towards ensuring that effects are not illusory for those devices that are approved ( although individuals could believe effects to be bigger than they are ) .
whilst we concede de ridder , vanneste , and focquaert 's point that there is currently no substantive evidence that ceds produce lasting effects outside of research and clinical settings ( p. 320 ) , there is a wealth of scientific research that provides proof of concept for the cognitive enhancing effects of tdcs and tms techniques . that the ceds on the market have not been subject to objective assessment is part of the reason why regulation is needed and counts in its favor .
they argue that contemporary scholarship shows no serious harms associated with the use of neurofeedback or tdcs . in response to this
, we wish to point out particularly in relation to active devices used for tdcs and tms that , whilst devices have indeed been use safely in the laboratory , and the regulated devices that researchers use in such contexts are indeed conducive to safe use , these facts do nothing to ensure that the unregulated devices on sale for enhancement exhibit the same safety profile .
first , the exclusion criteria for brain stimulation research are much more conservative than marketed exclusion criteria .
second , while brain stimulation experiments lasts usually between a single session to a dozen ( the latter is a relatively rare scenario ) , there is not safety data on the usage of brain stimulation over a long period of time such as months or years .
essentially , regulation is needed to ensure that devices sold for enhancement are as similar as possible to those devices about which kuersten and hamilton make their safety claims . indeed and to illustrate the point from another perspective
fitz and reiner raised concerns about the foc.us device in their commentary :
the internal electrodes exceed the general safety guidelines for current density at all stimulation levels , the voltage limits do not behave as specified in the manual , the device behaves unpredictably when its connection to the head is lost , and under some circumstances the foc.us can generate small voltage current spikes .
the internal electrodes exceed the general safety guidelines for current density at all stimulation levels , the voltage limits do not behave as specified in the manual , the device behaves unpredictably when its connection to the head is lost , and under some circumstances the foc.us can generate small voltage current spikes .
accordingly , we emphasize : just because tdcs can be safe does not mean that all of the particular devices on sale are meeting the same level of safety . further , at the end of their commentary , and perhaps in tension with their assertion that the sorts of ceds we discuss present no significant risks , kuersten and hamilton say that low - risk devices should not be excluded from on - going oversight , since so much concerning the brain , what affects it , and how remains unknown ( p. 347 ) .
we wish here to emphasize that low - risk devices , as we envisioned them , would typically be non - active devices such as neurofeedback equipment , which they correctly claim pose no risks qua devices ( see below ) . given their comments on the safety of neurofeeback , especially considered simply as a device , we would be surprised if they would think that such equipment required on - going oversight .
thus , even if there is room to disagree about whether ced is the right label for non - active devices ( as we discuss below ) , we in fact seem to agree on the lack of need for the continual regulation of low - risk devices , such as those that do not transfer energy through the skull . kuersten and hamilton object to our inclusion of neurofeedback as a ced .
however , in offering our definition of ceds , we were not aiming to provide the definitive view on what does and what does not fall within this category .
our interest in offering a definition was only to facilitate identification of a class of non - therapeutic devices that should receive regulatory attention and , on that definition , neurofeedback plausibly does qualify as a ced .
note also that doubts about whether neurofeedback equipment qualifies as a ced on a precise understanding of that term could also be raised regarding tdcs equipment , which kuersten and hamilton believe does qualify .
they argue that what actually affects the brain is therapy used with [ neurofeeback equipment] ( p. 341 ) .
but tdcs also only enhances cognitive performance when combined with cognitive training exercises , so could be excluded from the category of ceds on similar grounds .
nevertheless , kuersten and hamilton are right to highlight that tdcs directly modulates brain activity whereas neurofeedback modulates it through psychological mechanisms .
we have no problem with the suggestion that this is a significant difference , and indeed it informs our suggestion that tdcs and tms should be classed as iia or iib devices , due to their active nature , and neurofeedback should be in class i , if regulated at all .
kuersten and hamilton next argue that including ceds under the definition of a medical device would not result in our suggested problem of overbroadness ( which we suggested would occur if the definition of a medical device were to be altered ) , since devices , they claim , would be limited by the definition to those that
investigate , replace or modify the anatomy or a physiological process. their claim is based on the assumption that ceds could fall under the definition as it is currently articulated .
however , it was the possibility that an altered definition would be overbroad that we thought counted in favor of a supplementary positive list . in setting out our proposals , we worked on the assumption that the current definition did not in fact capture ceds ( due to the principal criterion that a medical device be intended to treat , prevent or diagnose ) and that changing the definition to remove this principal criterion across the board would have the result that it would then be overbroad . if the principal criterion were simply that a medical device investigate , replace or modify the anatomy or a physiological process then all sorts of devices would fall under the remit of the mdd from earrings to nail extensions .
indeed , the european commission used precisely this argument to support the creation of a positive list of implantable or other invasive devices without a medical purpose .
according to their impact assessment on the revision of the regulatory framework for medical devices , the definition of a medical device stipulates that it be intended for treatment , prevention or diagnosis , and the removal of this criterion would result in overbroadness , making a positive list the preferred solution in the case of certain cosmetic devices .
our argument for an analogous list for ceds is based on the assumption that the european commission is correct in its understanding of the mdd and the implications of the current and possible definitions employed therein .
fitz and reiner broadly support our proposals and endorse our central claim , viz . that the regulatory framework for medical devices should be extended to include ceds .
nevertheless , they raise a concern about the ability of ced users to evaluate the risks of ceds , saying that evidence that consumers are in a strong position to evaluate the risks associated with ced use is lacking ( p. 323 ) . whilst we agree that consumers will not be perfect rational calculators
, we emphasize that our proposals would eliminate devices from the market that were manifestly dangerous or far more dangerous than needed to serve their intended function .
in addition , our liberal view is committed to the contention that allowing some room for error in consumers weighing of benefits vs. risks in relation to their individual well - being can be a justifiable cost of allowing individuals greater autonomy and freedom of action .
we stress also that what the risks of devices are their nature and likelihood is something that would , in line with the procedure for medical devices , be assessed by a group of experts prior to approval for the market .
so , although we agree that leaving some room for consumer valuation of risk leaves open the possibility that individuals will not factor this carefully into their decision whether to use a product , we contend that this is an acceptable cost of a liberal approach to regulation .
further , we would like to emphasize that the current lack of regulation implicitly signals to home users that there are no real concerns with ceds , as otherwise they would have been regulated
ie the perception is likely to be that , given the lack of regulation , there are no risks for consumers to evaluate at all .
thus , even if consumers are not perfect risk calculators , the current situation it likely to be more misleading than one in which risks have been identified by regulators .
king , gavaghan , and mcmillan provide an interesting critique of our proposals relating to consumer assessment of ceds , and in particular their risks and benefits .
they agree that the concept of medical benefit is not always appropriate for ceds ( especially where designed and sold only for enhancement ) and that well - being would be the theoretically appropriate construct when assessing such ceds .
however , they suggest that well - being , harm and risk are difficult to assess in the pre - market approval process and raise a particular concern about the phenomenon of risk compensation. the central feature of this phenomenon is that some individuals have a propensity for taking a certain level of risk and will increase the riskiness of what they are doing until this propensity is met .
they therefore challenge the assumption that pre - market approval will make the use of ceds safer overall , especially in the context of experimental home use .
they suggest that , where users are determined to use devices in a risky way , they will do so despite safety standards . in response to this
, we raise a point of empirical uncertainty , and emphasize what can still be achieved through regulation , despite the phenomenon of risk compensation instantiated in some individuals .
it might be true that people will tend to fulfill their propensity for a particular total amount of risk in their lives , but it is unclear whether they do this by assuming superfluous risk to achieve a particular goal or seeking further risky goals . perhaps , as king and colleagues suggest , achieving one goal more safely allows one to assume additional risk in relation to other pursuits .
however , a risk quota might also be met by increasing or maintaining the level of risk one exposes oneself to in pursuit of one 's current primary goal . which is the case is likely to depend on the number of goals one believes to be valuable to pursue .
if ceds are made safer , would users with high risk thresholds seek to obtain riskier devices , or would their risk quota be freed up to pursue additional risky activities ?
the question is essentially whether individuals tend to increase riskiness across all activities until their risk propensity is met , or whether they increase the riskiness of every activity to some threshold level for each activity , regardless of how superfluous this risk may be .
if the former , risk reducing regulation could still have benefits in the form of facilitating pursuit of other risky , but perhaps all - things - considered valuable , activities .
even if individuals with a high risk threshold are inclined to practice riskier use as the device itself becomes safer , we assume that king and colleagues would agree that it is still better that the devices available pose as low a risk as possible to achieve their effects , and that unjustifiably dangerous devices are prohibited from placement on the market .
eg by using failsafe mechanisms , audible warnings , and limits on stimulation duration and strength .
an individual 's propensity to take risks with a product does not render consideration of that product 's safety redundant .
by analogy , just because some individuals routinely drive dangerously , this does not negate the need to try to make cars as safe as possible .
even if those engaging in risk compensation reap no overall benefit from making ceds safer , failing to minimize risks would unfairly jeopardize those who do not so compensate .
johnson , gillett , and snelling suggest that the most convincing argument in favor of our position is based on the regulatory assessment of risk , which they believe should be the sole consideration in pre - market assessment .
we suggest here that our respective proposals are not as different as they may have seemed , as we proposed that risk should be the primary regulatory concern . however , we diverge from the position advocated by them in our contention that objective improvements to cognitive capacities are amenable and relevant to assessment .
baseline ( or , we infer , upper limit ) level of risk that society feels an individual should be able to take ( eg skydiving or climbing ) , and then require manufacturers to quantify risks only .
they argue for this position by pointing out that when technologies are new , and particularly when new technologies are used for an innovative application , variation in the effects on individuals and useful endpoints my not be obvious ab initio .
our first response is to note that the same applies for risks novel applications may present novel and variable risks that are as difficult to predict as the cognitive improvements one might obtain from such applications .
our second response is to emphasize that the model we proposed is actually quite close to a risk - only model ; at least , it is a risk - first model .
we suggested using risk as the primary way of categorizing a device and that benefits should only be considered in terms of objective improvements eg demonstrated improvements in a cognitive capacity , such working memory which are precisely the sorts of effects about which manufacturers make their claims .
importantly , this consideration of objective benefit is not intended to downplay the broader benefits that an individual might attain from using a device .
whilst objective benefits can be considered , they must not be thought to constitute the only determinants of well - being .
nonetheless , regulators should still ensure that consumers are provided with information on objective benefits .
indeed , the distinction between thick and thin considerations of well - being drawn by king , gavaghan , and mcmillan is illuminating in this regard .
thin well - being , they say , consists only in all - purpose goods , whereas a thick well - being incorporates the individual agent 's conception of value and his particular circumstances .
an objective impairment in , say , verbal working memory ( that occurs as a trade - off of an objective improvement in , say , visuospatial working memory ) , will present the very same challenges for quantification , despite such impairment constituting a risk rather than a benefit . to the extent , as the risk only model assumes , that objective risks can be quantified ( and impairment in verbal working memory indeed permits such quantification ) objective benefits along the same sorts of dimensions can also be quantified ) .
further , we suggested that room should be made to account for the difficulty of measuring effects ( positive or negative ) on thick well - being .
our proposal was that , once the manufacturer had made it clear what the objective benefits and risks are for moderately risky devices , the consumer should have the freedom to decide whether that benefit risk ratio is acceptable for them ( for example , because they expect it to result in a net gain in thick well - being ) .
on the other hand , we proposed that high - risk devices , presenting risks that might be classed all - purpose bads ( eg a high risk of seizure ) , should be prohibited for sale on the consumer market .
whatever the individual 's life plans , in the preponderance of cases of healthy adult use , a seizure is unlikely to promote these plans . where there is scope for disagreement about the value of an effect for example as to whether a small impairment in verbal working memory is a reasonable price to pay for a similarly sized improvement in visuospatial working memory
level of acceptable risk therefore plays a greater role than quantification of benefits in our overall framework . as an objection to our model , king , gavaghan , and mcmillan
present the example of the surgical insertion of a ventriculoperitoneal shunt to treat normal pressure hydrocephalus .
they note that this would be a high - risk procedure on our proposals , as it comes with the risk of seizures , stroke , paralysis , and death .
moreover , there has never been a comprehensive multicenter randomized controlled trial to demonstrate the performance of this product .
this is thus a high - risk procedure for which there is no good evidence of benefit .
nevertheless , they seem to suggest , the use of this procedure should be permitted .
it might seem , then , that consistency requires permitting the use of some high - risk ceds as well , calling into doubt our claim that high - risk ceds should not be allowed on the market . in response
, we would note that patients with normal pressure hydrocephalus typically have much more to lose from non - intervention than do healthy individuals considering whether to employ ceds .
indeed , one might think that advancing dementia is amongst the worst fates that might befall a person .
there is thus a case for tolerating much greater risk in devices intended to treat this condition than in ceds .
king and collaborators also use the ventriculoperitoneal shunt example to suggest that it may not be practical to enforce the provision of evidence - based measures of efficacy to consumers for ceds . in response to this
, we emphasize that we do not have in mind a requirement such that each manufacturer must perform a randomized controlled trial using their device .
research investigating tdcs and tms in healthy adults is demonstrating efficacy , and we envisage that manufacturers should be required to show how their device complies with design parameters equivalent to those that have been shown in the scientific literature to produce enhancement effects . for example , devices with electrodes positioned in locations on the scalp that have no evidence of producing the particular effect claimed by the manufacturer should not be approved for sale alongside such claims .
manufacturers must be able to identify a credible body of scientific literature that supports the claimed efficacy and safety , given the product 's characteristics electrode location , stimulation intensity , duration , and so on .
further , we agree with king , gavaghan , and mcmillan that manufacturers should be required to state clearly the lack of evidence for any unsubstantiated claims , if they are to be allowed to make them at all .
kuersten and hamilton also comment on the risk benefit assessment of ceds , and the standard to which we proposed they should be subjected .
they argue that there is a lot of room for maneuver in the mdd to assess acceptable risks when weighed against the benefits ( p. 346 ) .
this is true , but we nonetheless think a framework for assessment is helpful , especially when the devices in question purport to offer non - medical benefits , a concept for which medical device regulators have little precedent . indeed , we argued against adopting the approach originally suggested by the mhra for cosmetic devices , which is very risk - averse : according to their approach such devices must present no or the minimum acceptable risk , as they confer no clinical benefit. conceding that some framework could be helpful , kuersten and hamilton suggest that , because the concept of benefit is nebulous , in the case of ceds , only risk should matter for pre - market approval .
in fact , as discussed above , we are broadly in agreement with such a position
benefits become increasingly harder to quantify and assess as they move beyond the sorts of benefits that enable individuals to pursue the standard range of activities most people wish to pursue . for example , not being in great pain would count as a benefit for most people , regardless of what they valued , but a small improvement to mathematical ability , for example , would not permit such evaluative consensus .
as noted above , our inclusion of regulatory consideration of benefits for moderately risky devices was intended only to refer to objective improvements about which consumers should not be misled . for example , if manufacturers claim that their device improves , say , the user 's linguistic fluency , this is a claim that can be tested and measured objectively .
beneficial such an objective improvement is to any particular person will vary depending on their goals , but there will be a fact of the matter about whether the device is able to confer such an improvement aside from the subjective question of how valuable this improvement might be .
certainly , we would advocate regulating the claims that manufacturers make in relation to what their devices can achieve .
however , far from our proposals being overly paternalistic , we argued that where there was room for disagreement about how to quantify a benefit in relation to risks , regulation should err on the side of consumer freedom .
further to their concerns regarding individuals abilities to weigh risks , fitz and reiner emphasize the remaining problem of do - it - yourself ( diy ) users who , in constructing devices from scratch , will be afforded no protection by the regulation of direct - to - consumer devices .
one of their proposals , with which we agree , is to bolster our recommendations with additional attempts at active harm reduction. they suggest that members of the professional community could join together to create an inclusive online community , where information could be gathered and disseminated with professional oversight .
we are broadly sympathetic with much of what they propose and would support the creation of an inclusive online community , were there to be sufficient expert interest in creating and maintaining it .
indeed , we are currently exploring how expert advice could be dispensed via existing fora , such as reddit .
de ridder , vanneste , and focquaert also raise the problem of unsupervised use and suggest potential solutions in the form of age limits , safety - by - design , and requiring user licenses . we agree that the unsupervised use of ceds needs consideration , but do not agree that this should lead to their apparent conclusion that we should regulate the use of ceds rather than their placement on the market .
the first task must be to ensure that the devices that people use ( unsupervised or otherwise ) are not unnecessarily dangerous or simply defective by design .
this can be achieved for example through european medical device law , which focuses on premarket requirements and reporting on post - market experience , even though it does not regulate use ( apart from requirements related to the instructions the manufacturers must provide ) .
there is no point carefully regulating use if the devices being used are not safe to begin with .
further , the authors suggestion that ceds should be made safe by design requires enforcement beyond that granted by the general product safety directive , and this is precisely the sort of thing that could be achieved by a model of the sort we propose .
de ridder , vanneste , and focquaert raise the concern that the societal benefits of enhancement might be lost if devices are in fact effective but regulated . in relation to this
, we emphasize that the regulation we advocate is not akin to prohibition . to the extent that our proposals would prevent devices from being placed on the market
, this would be limited to very dangerous devices or devices making implausible claims hardly the sorts of devices that would confer net societal benefits .
the important point that many other commentators on our model appear to overlook is that the effectiveness of brain stimulation techniques as a type of intervention does not guarantee the effectiveness of particular token devices claiming to be of this effective type .
moreover , as low risk , effective devices are developed , these would be available under our proposal and a vigorous market will emerge .
de ridder , vanneste , and focquaert raise concerns about the misuse of resources , which assumes a position markedly different from that which we set out in our paper .
they argue , persuasively , that resources should be directed to the most important causes and that enhancements might not be of a high enough priority to warrant use of our limited resources .
we agree that limited resources need to be allocated carefully , but our proposal does not threaten such allocation : the devices that would be prohibited under our proposal are ones that would certainly not warrant significant investment of public resources , and our proposal leaves open how resources should be allocated among those that would be permitted .
perhaps de ridder and collaborators assumed us to be advocating the inclusion of ceds under the healthcare funding arrangements , but this is not what we envisaged .
the extension to the scope of the mdd , as we recommend it , has no direct implications for the distribution of healthcare resources .
relatedly , the authors ask whether the regulation of ceds requires new regulations regarding who can use them . again
, the answer is for the most part no : our proposals merely affect what can be placed on the market for consumers to purchase .
the only exception should be restrictions governing the use of ceds on children or vulnerable adults through contraindication labeling and the criminal law .
in contrast , adult consumers will not need a prescription to purchase and use ceds on themselves . indeed , king , gavaghan , and mcmillan provide a compelling argument in their commentary for why medical practitioners should not serve as gatekeepers to cognitive enhancement . also of relevance to the question of the correct place of enhancement in healthcare , king ,
gavaghan , and mcmillan suggest that our reference to formally trained practitioners was not developed .
we used this phrase in relation to the use of ceds in children and vulnerable adults , since our proposals envisaged that healthy adults would be able to directly purchase and use ceds , with no medical intermediary .
they ask whether we envisage a further tier of regulation and raise a number of interesting points that would need to be addressed if medical professionals or those offering alternative therapies were to serve as gatekeepers to the use of ceds in such populations .
they are correct that the regulation of such practice would indeed introduce a further tier of regulation , and we acknowledge that this issue was not addressed in our original paper .
however , we believe that the scope for the permissible use of ceds on children may be limited .
further , neuromodulation offered to vulnerable adults overseen by medical professionals will primarily be governed by existing medical ethical guidelines , since such use is likely to be therapeutic and hence occurs within the clinical domain . in relation to children ,
king , gavaghan , and mcmillan are correct that further thought is needed to establish how use in adolescents should be controlled , and with whose oversight .
however , ensuring the safety of devices that might be used for enhancement in children and placing controls on such use are separate issues and both are important . whilst our proposals were concerned with controlling which devices are sold directly to consumers , we agree that the regulation of services , especially those offered to children needs close attention .
kuersten and hamilton object that we fail to cite much law on the remit of the mdd .
the suggestion seems to be that this has left us underinformed . attempting to clarify the current regulatory situation
, they say that instead of the definition of a medical device being the significant factor in the non - application of the mdd to ceds , ceds are instead unlikely to fall under the mdd because manufacturers targeting the general market are discouraged from intending them for a medical purpose ( which they argue manufacturers have considerable latitude to do ) ( p. 344 ) .
manufacturers are deterred , they say , because intending that a device be used for a medical purpose mandates costlier and more time - consuming requirements . we suggest that this is precisely the problem : manufacturers should not be able to evade regulation just because they deem it to be too burdensome .
we are fully aware that it is the manufacturers intentions identifiable from the claims they make in relation to their products that are instrumental in bringing a device within the definition of a medical device .
we argue that , particularly in the case of brain stimulation devices , opting not to fall under the definition of a medical device should not be a possibility .
however , kuersten and hamilton suggest that manufacturers intentions will in fact not matter once a new definition comes in to force , which will specifically define a medical purpose. they cite the mhra 's overview of the current proposals for the current revision of medical devices legislation , proposals with which we are very familiar .
they quote the document as follows , saying that the definition of medical purpose will remove this decision from manufacturers :
[ m]edical device means any instrument , apparatus , appliance , software , implant , reagent or other article , intended by the manufacturer to be used alone or in combination , for human beings for one or more of the specific medical purposes of : investigation , replacement or modification of the anatomy or of a physiological process or state ( pp .
[ m]edical device means any instrument , apparatus , appliance , software , implant , reagent or other article , intended by the manufacturer to be used alone or in combination , for human beings for one or more of the specific medical purposes of : investigation , replacement or modification of the anatomy or of a physiological process or state ( pp .
, we suggest that it can not be seen how the manufacturer 's intentions become irrelevant ; on the contrary , they are referred to explicitly .
further , rather than the indents defining medical purpose , the scope of medical purpose is constrained to the specific instances given in the indents . indeed , the consultation documents from the european commission and the uk 's medical and healthcare products agency underscore the continued relevance of the medical / non - medical purpose distinction in relation to devices that replace or modify anatomy : their proposal of a positive list ( in annex xv ) of implantable or other invasive devices without a medical purpose would be redundant if any replacement or modification of anatomy were to constitute a medical purpose .
again , the indents in the definition of a medical device serve to constrain , rather than define , medical purpose . thus , we rely not only on the court of justice decision , but also on the interpretation of the mdd offered by the mhra and other european commission documents .
kuersten and hamilton perhaps think that there should not be a gap , or may think that the definitions should be interpreted differently .
but the fact is , given the way the definitions are currently being understood by the relevant parties , it will take more than the cited revised definition of a medical device to bring ceds within the scope of the mdd .
we were gratified that our paper prompted so much discussion . in reading the commentaries and articulating our responses
, it became apparent that the comments and critiques served foremost to highlight the modest scope of our original proposals . whilst we still believe that pre - market approval is highly important for ceds especially for those that transfer energy across the skull
the further proposals made in these commentaries serve to set an agenda for continued discussion of the optimum integrated policy response to ceds and , indeed , other new and emerging technologies designed for enhancement .
|
our ( 2014 ) model for the regulation of cognitive enhancement devices ( ceds ) received a great deal of interest from those involved in european device regulation and from academic commentators .
further , since the publication of our recommendations , the number of manufacturers of brain stimulation devices for non - medical purposes has increased , underscoring the need for a regulatory response . in this paper , we clarify aspects of our original proposal and address additional regulatory issues beyond our original focus on the sale of devices .
we begin with theoretical points pertaining to the definition of a ced and the distinction between treatment and enhancement .
we then respond to practical challenges raised by the prospect of implementing our regulatory framework .
next , we address some wider societal considerations relating to users and other stakeholders . finally , we revisit the broader regulatory context within which the various discussions are situated .
|
THEORETICAL ISSUES
PRACTICALITIES AND STRUCTURE OF THE REGULATORY FRAMEWORK
WIDER SOCIETAL CONSIDERATIONS
THE CURRENT EUROPEAN REGULATORY CONTEXT
CONCLUSION
|
oral squamous cell carcinoma ( oscc ) is one of the most aggressive malignancies worldwide and accounts for more than 90% of all oral cancers .
it is ranked as the sixth leading cause of cancer mortality worldwide and the second leading cause of cancer mortality in india .
the most common sites of oscc are the lateral ventral surface of the tongue , the floor of the mouth and buccal mucosa .
a less frequent site to be affected is the gingiva which comprises of about 10% of all osccs and affects 91% of patients with gingival carcinoma aged above 66 years .
of all the intraoral carcinomas , gingival oscc is least associated with tobacco abuse and has the greatest predilection for females .
after these contradictory reports , it was suggested to analyze the cause of the male dominant tendency of gingival squamous cell carcinoma ( scc ) in asian patients .
these tumors commonly arise in the edentulous areas , although they may also develop at dentate areas .
it is generally agreed that carcinomas of the mandibular gingiva are more common than those of the maxillary gingiva and 60% of those are located posterior to the premolars .
although generally classified as a subset of oscc , gingival scc is a unique malignancy and can mimic a multitude of other lesions , especially those of inflammatory origin .
clinical presentations of sccs of the gingiva can be quite variable and hence are misdiagnosed as benign tumors or other inflammatory responses .
the 5-year survival rate of gingival scc is considerably less as compared to scc developing at other sites , suggesting a poor prognosis .
hence , scc of the gingiva should be considered in the differential diagnosis while dealing with gingival lesions particularly in elderly individuals and is of paramount importance that the lesion be diagnosed early to initiate treatment and thereby improve prognosis .
a 62-year - old female patient reported to a private dental clinic with pain in the right lower back tooth region for the past 2 weeks .
intraoral examination revealed the presence of reddish buccal gingival growth in relation to mesial aspect of tooth no .
extraoral examination revealed a single palpable , nontender , mobile and firm submandibular lymph node on the right side . on the basis of above findings
, the buccal growth was provisionally diagnosed as an inflammatory / reactive gingival growth and apical periodontitis in relation to 47 .
since the patient insisted only on symptomatic medical management , she was prescribed antibiotics , analgesics and chlorhexidine mouthwash for 3 days .
a complete hemogram and biochemical assay for blood sugar was requested and the patient was asked to report after a week .
one week recall visit revealed unsatisfactory healing and blood investigation reports were all within normal limits , excepting a slightly elevated erythrocyte sedimentation rate .
due to the persistence of the lesion and poor response to medical therapy a likelihood of noninfectious and noninflammatory pathology was strongly suspected .
since the patient did not want any further conservative management and insisted on an extraction , the dentist decided to extract the tooth . considering the innocuous appearance of the lesion , perceived lack of risk factors and the patients insistence of symptomatic management and unwillingness of the patient to undergo any radiographic examination , the dentist requested for an expert opinion from the speciality services . considering the age of the patient , ambiguous clinical presentation and the refractory nature of the lesion , a differential diagnosis of oscc and metastatic carcinoma to the gingiva
, did the patient agree for immediate biopsy along with extraction and the radiograph was taken only on follow - up .
on the 2 week recall , the patient reported with the panoramic radiograph and presented with a rapidly growing soft tissue mass in the extracted site .
clinical intraoral examination revealed an ovoid reddish , spongy mass measuring about 1 cm 1 cm from the extracted site [ figure 1 ] .
two weeks postextraction shows reddish , ovoid growth on the posterior alveolar ridge orthopantomogram showing the extracted site ( right mandible ) with no osseous changes histopathological examination revealed islands and sheets of dysplastic epithelium invading into the underlying connective tissue stroma with keratin pearl formation .
the overlying epithelium showed hyperkeratinized stratified squamous epithelium with dysplastic features suggesting a diagnosis of well - differentiated scc [ figures 35 ] .
photomicrograph showing squamous epithelium with islands of dysplastic cells infiltrating the connective tissue ( h&e stain , x40 ) photomicrograph showing well - differentiated squamous epithelial islands within the connective tissue ( h&e stain , x100 ) high power view showing island and nests of squamous epithelial cells within the connective tissue ( h&e , x400 ) the patient was referred to cancer speciality hospital for further management .
the patient was administered radiotherapy fractionated at 60 gy each session for a period of 5 weeks . the patient is continuously under follow - up 6 months postradiation and does not show any signs of recurrence [ figure 6 ] .
oral cancer is a major global public health problem with 5,00,000 new cases diagnosed annually . according to the international classification of diseases
, oral cancer refers to a subgroup of head and neck malignancies that develop on the lips , tongue , salivary glands , gingiva , floor of the mouth , oropharynx , buccal surfaces and other intraoral locations .
nevertheless , the term is synonymous to oscc of oral mucosal origin . despite rapid advances in treatment modalities ,
oral cancer still remains a life - threatening disease with no remarkable improvement in prognosis and survival .
the oral cavity is amenable to routine screening and clinical examination for malignant changes and therefore , in theory , these changes should be more easily detected and diagnosed at early stages leading to more effective treatment .
however , because of its varied site - related clinical presentation malignant oral disease is often difficult to distinguish from benign oral lesions .
carcinoma of the gingiva is an insidious disease , does not have the clinical appearance of a malignant neoplasm and is often misdiagnosed as one of the many inflammatory lesions of the periodontium .
gingival carcinoma typically arises from keratinized mucosa , most often in the posterior mandible , where it destroys the underlying bone structure causing tooth mobility .
the most common etiologic factors associated with oscc are smoking and smokeless tobacco , which increases the general risk fourfold .
of all the intraoral carcinomas , gingival oscc is least associated with tobacco abuse and has the greatest predilection for females .
other causes include phenol use , exposure to ultraviolet radiation , iron deficiency , candidal infections , oncogenic viruses and immunosuppression which may play much smaller roles .
gingival scc more frequently involves mandible than maxilla and is predominantly observed in older females over 50 years .
gingival oscc is more aggressive and in its early stage bears a resemblance to common mucosal infection and , therefore , has frequently led to a delay in diagnosis or misdiagnosis , leading to delay in treatment and making the prognosis grave .
gingival sccs carry a higher risk of metastases owing to its proximity to the underlying periosteum and bone which invites early invasion of these structures . as such
, gingival scc is diagnosed late , due its similarity with other common inflammatory lesions of the gingiva , invasive procedures such as curettage and extraction of the tooth worsens the prognosis , a hypothesis first suggested by peterson ( 1993 ) and it has been suggested that dissemination of cancer cells into the circulation during invasive procedures could increase the risks of distant metastases . eun
joo choi et al . studied the prognosis of gingival osccs in dentate patients diagnosed after invasive procedures such as curettage and extraction and found that bone invasion was seen in 75.8% patients who underwent invasive procedures and in the remaining 24.2% patients bone invasion was not demonstrated probably because the procedure included removal of floating teeth without curettage .
this result suggests that removal of floating teeth without curettage may not disseminate the cancer cells into the bone marrow . in this reported case ,
which was also initially misdiagnosed as an inflammatory mass , subsequently an atraumatic extraction was done for the mobile tooth based on the expert opinion .
such a cautious approach has to be exercised in the management of such suspicious lesions with an eye on prevention of untoward events .
however , even in the current case the outcome of the treatment has to be viewed with caution , as the patient is only in the 6 month follow - up period .
the prognosis with gingival carcinomas depends on the histological subtype and clinical extent of the tumor . a well - differentiated type such as in our case is generally considered to have a favorable prognosis . however , the most important indicator of the prognosis is the clinical stage of the disease .
if the neoplasm is small and localized , the 5-year cure rate is around 60%~70% ; however if cervical nodal metastasis occurs , the survival rate drops to about 25% suggesting that early diagnosis is imperative .
early detection of scc is vital as the prognosis is directly related to the size of the lesion .
lesions measuring < 1 cm are amenable to treatment and have a long - term prognosis .
thus , it is prudent to biopsy any unexplained lesion which remains after 2 weeks following removal of any suspected etiologic agent to avoid unnecessary delay in diagnosing such conditions .
the general dentist is frequently presented with oral lesions that are ambiguous in clinical presentation and behavior .
patients with oscc have a varied etiology , some of which are established while a few of the cases do not elicit classical risk factors . very often , the dentist is faced with the challenge of making a decision to commence treatment as desired by the patient or pursue further investigation to rule out more potentially morbid diagnosis .
such a cautious stance by the dentist can be possible only if the suspicion index for potentially life - threatening lesions is high on the differential list .
alternatively , these clinical situations necessitate the services of expert opinion that would obviate the chance of missing a diagnosis .
a missed diagnosis is a lost opportunity in instituting timely and definitive care for such life - threatening lesions .
gingival oscc is more aggressive in behavior and in its early stage bears a resemblance to common mucosal infections .
|
oral squamous cell carcinoma ( oscc ) is the most common epithelial malignant neoplasm affecting the oral cavity ; early detection is an important criterion for achieving high cure rate .
occasionally , it may be misdiagnosed because of its variable and innocuous clinical appearance .
carcinomas of the gingiva are a unique subset of oscc , constituting approximately 10% of osccs and can mimic a multitude of oral lesions especially those of inflammatory origin with benign features , often leading to delay in the diagnosis and hence delayed treatment .
this article reports a rare case of gingival oscc in a 62-year - old female patient mimicking an inflammatory gingival mass .
|
INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION
Financial support and sponsorship
Conflicts of interest
|
a significant number of these admissions are attributable to a small number of complex patients with other comorbidities who do not engage well with mainstream services .
assertive outreach teams have been used in the field of psychiatry to engage patients who are poorly compliant .
this study examines whether an alcohol assertive outreach team ( aaot ) can engage with this group and reduce hospital admissions .
the aaot is a multidisciplinary team with medical , psychiatric , substance misuse , psychology , nursing and social work specialists .
the team worked with patients with the highest number of alcohol - related admissions and case managed in a community setting for 6 months .
the admission and emergency department attendances of the cohort were compared for the 3-month period before and after the intervention .
christo inventory for substance misuse services ( ciss ) scores were determined pre and post the intervention period .
the total number of admissions in 3 months fell from 151 prior to the intervention period to 50 following the intervention .
emergency department attendances also fell from 360 in 3 months to 146 following the intervention period .
an aaot model appears to reduce hospital admissions and emergency department attendances in a complex group of patients that display high alcohol - related admissions .
in the uk nhs , a continued rise in alcohol - related admissions is clearly seen .
the current costs for alcohol - related admissions in england are estimated at 1.2 billion.1 within this general rise , there is a cohort of patients who frequently attend the emergency department and have high levels of repeated hospital admissions .
this group have complex physical health , psychiatric and social needs , but the most common factor is high levels of alcohol use.2 within this group , there are a high proportion of dual diagnosis patients : those with both mental health and alcohol problems .
unfortunately , they do not interact well with mainstream services and higher death rates are seen.3 previous studies have looked at strategies to impact patients who are frequent attenders at emergency departments .
patient care plans have been used to reduce emergency department attendances with mixed results.46 these plans involve a specific management plan that is implemented when the patient attends the emergency department .
case management strategies have also been used.7
8 this involves an ongoing clinical interaction which extends beyond the hospital into the community .
case management involves multidisciplinary teams usually comprising psychiatric , social work , substance misuse , nursing and medical specialists . in mental health services ,
these are multidisciplinary teams which work with people with complex health needs , drug , alcohol , mental health and housing issues .
they have been shown to be effective in patients with high levels of inpatient bed use.9 these teams are commonly targeted at people who engage poorly with services .
assertive outreach teams are typically defined by workers with a low caseload , usually 10 patients per worker , assertive engagement mechanisms and a high proportion of contact within the community.10 recent work has demonstrated the use of assertive treatment methods to improve engagement in alcohol services and to improve rates of patients entering recovery.11 in view of the high levels of alcohol - related admissions seen in the north west of england , the north west chief executives challenge , led by david dalton and other experts , developed strategies to improve the quality of alcohol care .
the two initiatives modelled were the development of an alcohol assertive outreach team ( aaot ) and a 7-day alcohol specialist nurse service.12 an aaot was developed to work with two separate cohorts of patients .
the first group are patients with alcohol - related liver disease who are increasingly using hospital services and exceed the threshold of two admissions wholly attributable to alcohol .
the aim of this study is to establish whether an aaot is an effective model for reducing hospital admissions and emergency department attendances in the top 30 most frequently admitted patients .
the study was carried out between january 2011 and july 2012 at the salford royal nhs foundation trust .
the study was a retrospective cohort analysis of the effects of assertive outreach methods on the admission rates pre and post the intervention period .
the aaot comprised a consultant in emergency medicine , a consultant psychiatrist specialising in addiction disorders , an emergency department nurse , a social worker , a psychologist , an alcohol worker , a support worker and an administrator . in the uk ,
a national audit system exists to record the number of alcohol - related admissions to each hospital .
a database was created to identify the adult patients with the highest number of alcohol - related unscheduled care admissions .
patients were case managed for a period of 6 months ; after this time the database was refreshed .
a new cohort of patients was then case managed for the subsequent 6 months .
to case manage the patients , case profiles were initially formed by information gathered from the patient , the acute trust , mental health trust , community alcohol team , primary care services , council and criminal justice services .
the face - to - face interaction as part of the case management was organised in the community away from the hospital .
common strategies were fast access to alcohol detoxification ; appropriate referral to outpatient specialities and support in getting there ; alcohol and psychological support ; facilitating housing solutions ; and robust responses to violence and aggression .
a part of the case management involved the production of individual care plans that would be implemented when a patient attended the emergency department .
the plans were immediately available to the emergency department staff at the point of triage and helped produce a consistent coordinated response whenever patients interacted with the hospital .
the primary outcome measure was the number of admissions per month in the 3-month period immediately before and after the 6 months of case management intervention .
emergency department attendances were also recorded during these periods . at the start and end of the 6-month case management period , christo inventory for substance misuse services ( ciss ) scores were calculated.13
this is a subjective score that reflects a patients social functioning , psychological and general health , compliance , drug and alcohol use and criminality .
the score is a 020 score , with lower scores reflecting better outcomes ( above 11 reflects high problem severity ) .
the score has been previously validated.13 ethics approval was not required as this study fulfils the criteria for service evaluation .
statistical analysis was performed with non - parametric statistical analysis as the ciss scores , admissions and attendance rates were not normally distributed .
medians are quoted and the wilcoxon rank sum test was used for comparing effects between groups .
the team was established and began to case manage patients in april 2011 . due to delays in fully establishing the team , only 24 patients were case managed in the initial cohort .
a further 30 patients were case managed in the 6-month period starting in october 2011 .
a significant reduction in hospital admissions was seen in the 3 months following case management as compared with the 3-month period immediately prior to the start of case management .
the total number of hospital admissions were 151 prior to case management , reduced to 50 following the case management period ( w=2070 , p<0.001 ) .
a similar significant reduction was also seen in the total number of emergency department attendances in the 3-month periods before and after intervention ( 360 reduced to 146 , w=2215 , p<0.001 ) .
the white bar represents the 6-month period in which the team actively case managed 54 patients .
there was a significant reduction in the ciss scores at the start as compared with the end of the case management period ( median 11 reduced to 8 , p<0.001 ) .
there were no deaths in the study group during the period of case management and up to 3 months after . however , one patient left the local area and contact was not able to be maintained .
there was loss of engagement with a second patient who did remain within the local area .
this study demonstrated that case management using the alcohol assertive outreach model can reduce emergency department attendances and hospital admissions .
this study is unique in that the focus is on patients with high level of alcohol admissions rather than frequent emergency department attendances alone .
while these groups share similar complexity , this is a slightly different cohort than the traditional group of emergency department regular attenders .
our pilot work demonstrated a lower level of homelessness in this group in contrast to our most frequent emergency department attenders .
our high admissions group had more stable accommodation and we were able to remain in contact and engage in the outreach aspect of the team 's work .
however , we had no exclusion criteria and homeless patients were included in the study .
a second reason for addressing a high admissions group rather than frequent emergency department attenders was the perceived cost benefit of reducing admissions rather than emergency department attendances alone .
based on national indicators and length of stay costs , an average alcohol - related admission costs a primary care organisation 1824 and an alcohol - related emergency department attendance costs 80.14 in the 3-month period after the end of case management alone , we demonstrated a reduction in admissions from 151 to 50 .
taking into account the reductions in admissions and attendances seen during the first year of the team alone , the cost reductions cover the 300 000 required to establish the service .
these initial cost reductions do not take into account any ongoing reduction in admissions and subsequent savings .
previous studies using care plans and case management strategies have shown mixed outcomes in emergency department frequent attenders .
studies from the uk , australia and the usa have all demonstrated reduction in emergency department attendances,2
4
5
8
15 but other teams have reported no benefit6
16 or even an increase in emergency department use.7 there is less evidence on the effects on admission rates .
a uk study reported a good impact on admission rates,4 but this was not repeated in other healthcare systems . a us randomised control trial reported no impact on admissions with case management;8 however , increased admissions were seen in an australian trial.7 traditional clinical approaches to medical , alcohol and psychiatric disorders are effective when a single disorder is identified .
unfortunately , when complex comorbidity is present , patients tend to fall between the gaps between services and commonly present and are admitted to inpatient hospital beds . we found that through effective multidisciplinary working , the aaot could more effectively support these complex individuals .
the most important factor that increased the effectiveness of the team was the creation of an extensive case profile for each patient from a wide ranging variety of information sources .
this permitted the team to plan effective interventions which supported the patient 's medical , psychiatric , substance misuse and social difficulties in a coordinated fashion .
as with all projects that impact the frequent users of emergency departments , there has to be concern that patients are not simply being diverted to other emergency departments .
this is true especially with homeless emergency department regular attenders who commonly move between areas .
one undoubtedly had moved out of the area and was likely to have attended other emergency departments .
the second patient remained within the local area but the team was not able to engage with him . for the rest of the patients , the ongoing community engagement allowed us to be confident that they remained within the locality of the hospital .
indeed , the reduction in the ciss scores that are demonstrated point to improved functioning and a reduction in problem severity .
the postintervention ciss score remains high in comparison with other settings such as abstinence based treatment centres or outpatient alcohol services , but this reflects the severity of the cohort that this group comprises .
assertive outreach is a uk term to refer to the north american assertive community treatment for psychiatric care .
it is more successful when hospital use is high.9 assertive approaches to engagement in uk community alcohol work have demonstrated to be beneficial .
passetti et al11 set up a clinic which targeted patients with a history of disengagement .
they used an assertive approach and saw more people completing withdrawal treatment and entering an aftercare placement .
we needed to modify the original model to adapt to our local application , but maintained the fidelity of the assertive approach.10 this was defined by a small caseload with 10 patients per worker , backed by a multidisciplinary team with psychiatry , nursing and substance misuse workers , and uses assertive engagement mechanisms and focuses on community engagement .
we used a preintervention and postintervention comparison whereby all patients were used as their own control .
some of the effects could be attributed to a natural reduction in emergency department attendances and admissions over time .
this effect has been previously noted in two us studies,6
8 but it is not a consistent effect with studies demonstrating no reduction16 or even an increase in emergency department attendance and admissions over time.7 a multicentre randomised controlled trial would be needed to adequately power a study to address this issue .
we took a pragmatic approach in using a prestudy and poststudy to describe the effects of alcohol assertive outreach work in reducing admissions in this cohort .
the main clinical findings of this study describe an aaot approach to the support and management of a cohort of patients who demonstrate high numbers of alcohol - related admissions to a uk inner city hospital .
this approach appears to reduce admissions , emergency department attendances and improves social functioning .
what is already known on this topica complex group of patients with high levels of repeat alcohol - related admissions are well recognised , but difficult to impact
what this study addsan alcohol assertive outreach team can engage with this complex patient group and reduce hospital admissions
how might it impact on clinical practice in the foreseeable futurealcohol assertive outreach teams provide a solution for reducing alcohol - related admissions in the highest users of inpatient services a complex group of patients with high levels of repeat alcohol - related admissions are well recognised , but difficult to impact an alcohol assertive outreach team can engage with this complex patient group and reduce hospital admissions alcohol assertive outreach teams provide a solution for reducing alcohol - related admissions in the highest users of inpatient services
|
objectivealcohol - related admissions are increasing .
a significant number of these admissions are attributable to a small number of complex patients with other comorbidities who do not engage well with mainstream services .
assertive outreach teams have been used in the field of psychiatry to engage patients who are poorly compliant .
this study examines whether an alcohol assertive outreach team ( aaot ) can engage with this group and reduce hospital admissions.designthe aaot is a multidisciplinary team with medical , psychiatric , substance misuse , psychology , nursing and social work specialists .
the team worked with patients with the highest number of alcohol - related admissions and case managed in a community setting for 6 months .
the admission and emergency department attendances of the cohort were compared for the 3-month period before and after the intervention .
christo inventory for substance misuse services ( ciss ) scores were determined pre and post the intervention period.results54 patients were case managed .
the total number of admissions in 3 months fell from 151 prior to the intervention period to 50 following the intervention .
emergency department attendances also fell from 360 in 3 months to 146 following the intervention period .
ciss scores fell from 11 preintervention to eight postintervention.conclusionsan aaot model appears to reduce hospital admissions and emergency department attendances in a complex group of patients that display high alcohol - related admissions .
|
Objective
Design
Results
Conclusions
Introduction
Methods
Results
Discussion
|
according to the world oral health report , dental caries remains a major public health problem in most countries , affecting 6090% of school going children and a vast majority of adults , and this may be due to the changing lifestyles , dietary habits , increased sugar consumption and inadequate exposure to fluorides .
the primary teeth are more susceptible to caries development than permanent teeth because of lower mineral and higher organic content of enamel . compared to adults ,
the demineralization potential at low oral ph is greater while the remineralization potential at normal ph is lower in children .
hence , the progression of caries will be faster , and reversal will be slower in children , as they depend upon the balance between demineralization and remineralization .
fluorides have the ability to remineralize early carious lesions , and can be used as active anticariogenic agents , which are available in the form of dentifrices , mouth rinses , varnishes , gels , and foams .
fluoride dentifrices are the most widely used products that deliver topical fluoride to the oral environment .
most fluoride dentifrices contain fluoride predominantly in the form of sodium fluoride ( naf ) or sodium monofluorophosphate ( mfp ) and other formulations are with amine fluoride or stannous fluoride .
the content of fluoride varies between 500 and 1500 ppm and are categorized into low - fluoride ( < 600 ppm f ) , standard ( 1000 ppm f ) or high - fluoride ( 1500 ppm f ) dentifrices .
the daily use of a fluoridated dentifrice will provide sufficient fluoride to maintain appropriate levels in saliva and plaque to actively influence remineralization , but the major drawback of fluoride dentifrices is the risk of dental fluorosis in children .
fluoride toothpastes contribute approximately 57% of the total daily amount of fluoride ingested by 4- to 7-year - old children , which occurs because of less control over swallowing especially in preschool children .
preventive measures to reduce the ingestion of fluorides from toothpastes are necessary , such as reducing the amount of toothpaste used , supervised brushing in preschool children and developing low - fluoride toothpastes .
the american academy of pediatric dentistry recommends low - fluoride dentifrices for children aged 26 years twice daily .
the amount to be used should be of a small pea or of smear size .
many studies were conducted to test the de / remineralizing efficacy of fluoridated and nonfluoridated dentifrices on the enamel of permanent teeth , with only a few studies conducted on the de / remineralization efficacy of low - fluoridated dentifrices on carious lesions in primary teeth . hence , there is an increasing need to know the effect of fluoride dentifrices on the carious lesions of the enamel in primary teeth .
therefore , the present study was undertaken to evaluate and compare the de / remineralization potential of different child formula dentifrices on artificial carious lesions in primary teeth using a 7 days ph - cycling model .
cheerio gel : a fluoride dentifrice manufactured by group pharmaceuticals limited , malur and marketed by dr .
reddy 's laboratories ltd , solan , himachal pradesh , india ( contents : 0.35% sodium mfp usp in a flavored gel base)colgate pokmon toothpaste : a fluoride dentifrice manufactured by colgate palmolive , usa ( contents : 0.11% naf , sorbitol , silica abrasive)children natural toothpaste : a nonfluoridated dentifrice manufactured by pigeon company , korea ( contents : calcium phosphate , glycerine , maltilol , carrageenan , flavor ) , which served as a negative control .
cheerio gel : a fluoride dentifrice manufactured by group pharmaceuticals limited , malur and marketed by dr .
reddy 's laboratories ltd , solan , himachal pradesh , india ( contents : 0.35% sodium mfp usp in a flavored gel base ) colgate pokmon toothpaste : a fluoride dentifrice manufactured by colgate palmolive , usa ( contents : 0.11% naf , sorbitol , silica abrasive ) children natural toothpaste : a nonfluoridated dentifrice manufactured by pigeon company , korea ( contents : calcium phosphate , glycerine , maltilol , carrageenan , flavor ) , which served as a negative control .
it contained 2.2 mm cacl2 , 2.2 mm kh2po4 , and 0.05 m acetic acid .
the remineralizing solution contained 1.5 mm cacl2 , 0.9 mm nah2 po4 , 0.15 m kcl and ph of 7 .
the dentifrice supernatants were prepared by thoroughly mixing a 3:1 ratio ( by weight ) of deionized water and dentifrice , which was then centrifuged at 4000 rpm for 20 min .
twenty - one sound primary teeth indicated for extraction due to preshedding mobility were collected , and soft tissue debris was cleaned and then stored in 0.2% thymol solution .
the teeth were inspected for cracks , hypoplasia and white spot lesions and then coated with an acid resistant nail varnish , leaving a narrow window , approximately 1 mm wide on the sound , intact surface of the buccal or lingual enamel .
then , they were immersed in demineralizing solution for 96 h to produce artificial carious lesions of 150200 m deep .
the teeth were embedded in self - cure acrylic resin blocks . a hard tissue microtome ( leica 1600 saw microtome , germany )
was used to section the teeth longitudinally through the lesions to produce enamel specimens of approximately 100150 m thick .
the damaged specimens were discarded , and the rest of the specimens were randomly assigned for each of the three groups ( groups a , b and c ) . polarizing light microscopy was utilized to record the depth of the lesions .
the sections were painted under a stereomicroscope with acid resistant nail varnish leaving the lesion surface exposed for exposure to experimental solutions .
the specimens were suspended with dental floss in a beaker containing deionized water and sealed with paraffin wax to achieve 100% humidity until usage .
all of the specimens in a particular group were placed in the ph - cycling system on an orbital shaker ( kemi company , kadavil electromechanical industries , ernakulam , kerala ) for a period of 7 days .
each cycle involved 3 h of demineralization twice daily , with 2 h of remineralization between periods of demineralization .
dentifrice supernatant was treated for 60 s before the first demineralization and both before and after the second demineralization .
the demineralizing , remineralizing solutions and dentifrice supernatants were freshly prepared for each cycle and stored in separate containers designed for each group throughout the experimental period . before a topical treatment with supernatant solutions , the teeth were removed from the de-/remineralizing solutions and thoroughly washed with deionized water . the de-/remineralizing solutions and supernatant solutions
were changed daily to prevent depletion or saturation of the solutions and accumulation of enamel dissolution products .
the sections were then studied under polarized light microscopy to evaluate the lesion depth before and after 7 days . for clear demarcation between sound and carious enamel ,
the specimens were imbibed in water and then recorded using polarizing light microscope both before and after ph - cycling , to evaluate qualitatively the lesion depth in each enamel section [ figures 13 ] .
the depths of the lesions were measured with a computerized calculation method using a software program ( progres , germany ) .
polarized light micrographs of enamel lesions before ( a ) and after ( b ) ph cycling for group a polarized light micrographs of enamel lesions before ( a ) and after ( b ) ph cycling for group b polarized light micrographs of enamel lesions before ( a ) and after ( b ) ph cycling for group c
cheerio gel : a fluoride dentifrice manufactured by group pharmaceuticals limited , malur and marketed by dr .
reddy 's laboratories ltd , solan , himachal pradesh , india ( contents : 0.35% sodium mfp usp in a flavored gel base)colgate pokmon toothpaste : a fluoride dentifrice manufactured by colgate palmolive , usa ( contents : 0.11% naf , sorbitol , silica abrasive)children natural toothpaste : a nonfluoridated dentifrice manufactured by pigeon company , korea ( contents : calcium phosphate , glycerine , maltilol , carrageenan , flavor ) , which served as a negative control .
cheerio gel : a fluoride dentifrice manufactured by group pharmaceuticals limited , malur and marketed by dr .
reddy 's laboratories ltd , solan , himachal pradesh , india ( contents : 0.35% sodium mfp usp in a flavored gel base ) colgate pokmon toothpaste : a fluoride dentifrice manufactured by colgate palmolive , usa ( contents : 0.11% naf , sorbitol , silica abrasive ) children natural toothpaste : a nonfluoridated dentifrice manufactured by pigeon company , korea ( contents : calcium phosphate , glycerine , maltilol , carrageenan , flavor ) , which served as a negative control .
it contained 2.2 mm cacl2 , 2.2 mm kh2po4 , and 0.05 m acetic acid .
the remineralizing solution contained 1.5 mm cacl2 , 0.9 mm nah2 po4 , 0.15 m kcl and ph of 7 .
the dentifrice supernatants were prepared by thoroughly mixing a 3:1 ratio ( by weight ) of deionized water and dentifrice , which was then centrifuged at 4000 rpm for 20 min .
twenty - one sound primary teeth indicated for extraction due to preshedding mobility were collected , and soft tissue debris was cleaned and then stored in 0.2% thymol solution .
the teeth were inspected for cracks , hypoplasia and white spot lesions and then coated with an acid resistant nail varnish , leaving a narrow window , approximately 1 mm wide on the sound , intact surface of the buccal or lingual enamel .
then , they were immersed in demineralizing solution for 96 h to produce artificial carious lesions of 150200 m deep .
the teeth were embedded in self - cure acrylic resin blocks . a hard tissue microtome ( leica 1600 saw microtome , germany )
was used to section the teeth longitudinally through the lesions to produce enamel specimens of approximately 100150 m thick .
the damaged specimens were discarded , and the rest of the specimens were randomly assigned for each of the three groups ( groups a , b and c ) . polarizing light microscopy was utilized to record the depth of the lesions .
the sections were painted under a stereomicroscope with acid resistant nail varnish leaving the lesion surface exposed for exposure to experimental solutions .
the specimens were suspended with dental floss in a beaker containing deionized water and sealed with paraffin wax to achieve 100% humidity until usage .
all of the specimens in a particular group were placed in the ph - cycling system on an orbital shaker ( kemi company , kadavil electromechanical industries , ernakulam , kerala ) for a period of 7 days .
each cycle involved 3 h of demineralization twice daily , with 2 h of remineralization between periods of demineralization .
dentifrice supernatant was treated for 60 s before the first demineralization and both before and after the second demineralization .
the demineralizing , remineralizing solutions and dentifrice supernatants were freshly prepared for each cycle and stored in separate containers designed for each group throughout the experimental period . before a topical treatment with supernatant solutions , the teeth were removed from the de-/remineralizing solutions and thoroughly washed with deionized water . the de-/remineralizing solutions and supernatant solutions
were changed daily to prevent depletion or saturation of the solutions and accumulation of enamel dissolution products .
the sections were then studied under polarized light microscopy to evaluate the lesion depth before and after 7 days .
for clear demarcation between sound and carious enamel , the specimens were imbibed in water and then recorded using polarizing light microscope both before and after ph - cycling , to evaluate qualitatively the lesion depth in each enamel section [ figures 13 ] .
the depths of the lesions were measured with a computerized calculation method using a software program ( progres , germany ) .
polarized light micrographs of enamel lesions before ( a ) and after ( b ) ph cycling for group a polarized light micrographs of enamel lesions before ( a ) and after ( b ) ph cycling for group b polarized light micrographs of enamel lesions before ( a ) and after ( b ) ph cycling for group c
for clear demarcation between sound and carious enamel , the specimens were imbibed in water and then recorded using polarizing light microscope both before and after ph - cycling , to evaluate qualitatively the lesion depth in each enamel section [ figures 13 ] .
the depths of the lesions were measured with a computerized calculation method using a software program ( progres , germany ) .
polarized light micrographs of enamel lesions before ( a ) and after ( b ) ph cycling for group a polarized light micrographs of enamel lesions before ( a ) and after ( b ) ph cycling for group b polarized light micrographs of enamel lesions before ( a ) and after ( b ) ph cycling for group c
seven sections of group a were treated with cheeriogel * and the mean score , and standard deviation ( sd ) was 180 26 before ph - cycling and 175 22 after ph - cycling ( p = 0.01 ) .
similarly in seven sections of group b treated with colgate pokeman * , the mean score and sd was 191 2l before ph - cycling and 173 16 after ph - cycling ( p = 0.03 ) . in seven sections of group c treated with children 's natural , the mean score and sd was 183 30 before ph - cycling and 201 18 after ph - cycling ( p = 0.04 ) [ table 1 ] .
in all the groups , there exists a statistical significance ( p < 0.05 ) .
comparison of the depth of the lesion before and after ph cycling in group a ( cheerio gel ) , group b ( colgate paokeman ) and group c ( children 's natural ) inter group comparison revealed that , the mean and sd in groups a and b after ph cycling had decreased than the mean and sd before ph cycling . only mean and sd for group c had increased after ph cycling .
the means and sd 's of the pretreatment lesion depths between groups a , b and c were not significantly different from each other ( p = 0.745 ) as shown in table 2 .
this shows that , even though , the specimens were sectioned from different primary teeth , the variations among the teeth did not show a major effect on the progress of demineralization .
comparison of the depth of the lesion among the pretreatment groups a , b and c using anova on comparison , the posttreatment lesion depths of groups a , b and c showed mean and sd as 175 22 , 173 16 and 201 18 respectively , which was found to be statistically significant ( p = 0.012 ) as shown in table 3 .
comparison of the depth of the lesion among the posttreatment groups a , b and c using anova on overall comparison , the lesion depths in groups a and b decreased by 3% and 10% respectively , while group c demonstrated an increase in lesion depth by 9% [ table 1 ] .
comparisons using anova and paired t - test showed that groups a and b were significantly different from group c , but there was no statistical significance between groups a and b. on using duncan 's multiple range test , it was observed that group a differ from group c ( p < 0.05 ) but not with group b ( p > 0.05 ) , whereas group b differs significantly from group c ( p < 0.05 ) .
the caries process is a continuum resulting from an imbalance between many cycles of demineralization and remineralization rather than a unidirectional demineralization process .
fluoride has been shown to have a greater inhibitory effect on caries progression than on caries initiation .
the levels needed to significantly reduce caries or , at a mechanistic level to shift the balance from caries initiation and progression to caries reversal are apparently in the sub ppm range .
hence , the present study was undertaken to evaluate and compare the remineralization effects of different child formula dentifrices on artificial carious lesions in primary teeth using a 7 days ph - cycling model .
artificial early caries - like lesions of the enamel showed all the principal histological features of natural caries and had been successfully used to study the remineralization of enamel in vitro .
these artificial lesions of the enamel were more homogenously reproducible than natural lesions and thus provide a reliable experimental model , hence ; carious lesions were artificially produced in the present study . extracted or naturally exfoliated primary teeth ( molars , canines , and incisors )
though there are variations in the morphology of individual teeth , it was hypothesized that these variations among the teeth do not play a significant role in caries formation , and in the present study too , the depths in the pretreatment test groups were not statistically different .
this implies that , even though , the specimens were sectioned from different teeth , the variations among the teeth did not show a major effect on the progress of demineralization .
single - section model , as used in this study had the advantage that a single section was fully evaluated prior to the experimental period and then again after the exposure period .
the de / remineralizing solutions and supernatant solutions were changed daily to prevent depletion or saturation of the solutions and accumulation of enamel dissolution products .
the concept of in vitro ph cycling was first proposed by ten cate and duijsters in 1982 , in experiments where they exposed artificial carious lesions in enamel to a combination of remineralizing and demineralizing solutions .
two types of ph - cycling models are used , the 7-day ph - cycling and the 10-day ph - cycling .
a 10-day ph - cycling model can be used on the enamel of permanent teeth whereas a 7-day ph - cycling or 10-day cycling with added 0.25 ppm fluoride can be used for primary teeth . in the present study ,
ph - cycling was done for 7 days without the addition of fluoride , because the addition of fluoride could have interfered with the hypothesis being tested .
naf and sodium mfp contain fluoride in chemically distinct forms , and they will differ in their mode of action with respect to caries reduction .
the reason for greater retention of oral fluoride from naf than from mfp could be due to : ( 1 ) fluoride ions diffuse faster from naf than mfp , by a factor of 1000 in dental enamel ; ( 2 ) there is no mfp analogue of calcium fluoride , which is important in oral fluoride retention ; ( 3 ) fluoride ions from mfp bind to a lesser extent to tooth mineral and plaque bacteria than naf .
an extensive series of in vitro and clinical trials have tested the anticaries efficacy of dentifrices containing naf or mfp .
many in vitro studies suggest that dentifrices containing naf perform better than dentifrices containing mfp whereas a study concluded no statistically significant difference between the two .
the efficacy of fluoride toothpastes in clinical trials is potentially influenced by several factors , namely : fluoride concentration , frequency of use , amount used and rinsing behavior whereas in the in vitro studies , the sole factor that plays key role is the fluoride concentration used .
this might be the reason for less effectiveness of mfp in the in vitro studies than in vivo studies .
this again might be due to the absence of a key mechanistic step in the in vitro studies in determining the clinical efficiency of mfp , namely the hydrolysis of mfp to fluoride ions .
these are in accordance with the present study where , both the mfp and naf dentifrices showed decrease in the lesion depths and the difference between the two was not statistically significant .
but the naf had showed a greater decrease in lesion depth confirming its superior anticaries efficacy over mfp .
based on the results obtained from the present study , we could conclude that the child formula dentifrices containing naf and sodium monofluorophosphate have the ability to remineralize the initial carious lesions in the primary teeth as both reduced the depth of the artificial carious lesions .
but , it is also important to emphasize other preventive methods in the prevention and/or reversal of caries as the child formula dentifrices could not completely remineralize the carious lesions .
|
aim : to evaluate the remineralizing potential of child formula dentifrices on primary teeth using an in vitro 7 days ph cycling model.materials and methods : twenty - one primary teeth were placed in demineralizing solution for 96 h to produce artificial carious lesions ; then cut longitudinally into 100150 m thick sections and randomly assigned to three groups .
sections in group a were treated with dentifrice containing 458 ppm monofluorophosphate ( mfp ) and sections in group b with 500 ppm sodium fluoride ( naf ) .
group c sections were treated with a nonfluoridated dentifrice.results:group a ( 458 ppm mfp ) and group b ( 500 ppm naf ) showed significant decrease in lesion depth , whereas group c ( non f ) showed a significant increase in depth ( p 0.05 , paired t - test).conclusion : though dentifrices containing 458 ppm mfp and 500 ppm naf demonstrated remineralization of carious lesions , it was not complete . therefore , it is also important to emphasize on other preventive methods in the prevention and/or reversal of carious lesions .
|
Introduction
Materials and Methods
Dentifrices used
De/and remineralizing solutions
Dentifrice supernatants
Lesion formation
pH-cycling model
Evaluation techniques
Polarizing light microscopy measurements
Results
Discussion
Conclusion
|
bacterial infections
as well as the emergence and spread of antibiotic
resistance in human pathogens are serious public health problems in
hospital and community settings across the globe .
new strategies to prevent and treat bacterial infections
are needed , including methods to overcome antibacterial resistance
that results from the outer membrane permeability barrier in gram - negative
organisms and targeting approaches that afford species- or pathogen - specific
therapeutics .
almost
all
bacterial species have a metabolic iron requirement and therefore
employ various strategies to acquire this metal ion when colonizing .
siderophores are high - affinity fe(iii ) chelators that are
produced by bacteria under conditions of iron limitation , such as
those encountered in the vertebrate host , to scavenge this metal ion
from the environment .
siderophore producers also
express dedicated ferric siderophore import machinery and employ
various mechanisms to release siderophore - bound iron following
cellular uptake ( e.g. , reductive and/or hydrolytic release mediated
by reductases and/or esterases , respectively ) .
numerous studies support the importance of siderophore - based
iron acquisition during bacterial infections .
thus , the potential of using siderophores , or targeting siderophore biosynthetic and transport machineries , in therapeutic development continues to attract significant interest .
of particular relevance to the advances described herein are prior
investigations pertaining to the development of siderophore
this strategy has received particular attention for the delivery
of antibiotics into gram - negative bacteria because these organisms
are inherently less sensitive to many antibiotics used in the clinic
as a result of the outer membrane permeability barrier .
both native siderophores and synthetic siderophore mimics
have been evaluated as platforms for therapeutic development . in the clinic , the native siderophore desferrioxamine
b is used for iron - chelation therapy in patients with iron overload .
several antibiotic small molecules found in nature called sideromycins
provide inspiration for synthetic siderophore
the sideromycins are secondary
metabolites comprised of a siderophore moiety and a toxic cargo ;
the siderophore portion targets sideromycins to bacterial strains
expressing the appropriate siderophore receptor .
antibiotic conjugate produced by a clinical
isolate of klebsiella pneumoniae , is an 84-residue
antibacterial peptide with a glucosylated enterobactin ( ent , figure 1 ) derivative attached to its c - terminus that exhibits
enhanced antibacterial activity against strains expressing the enterobactin
receptor fepa . from the standpoints of
antibacterial activity and therapeutic potential , studies of synthetic
siderophore
antibiotic conjugates have provided the
community with mixed results , causing some skepticism about the potential
of siderophore - based approaches despite the successful utilization
of such molecules by nature .
structures of enterobactin ( 1 ,
ent ) and a generalized
enterobactin cargo conjugate .
many of the failures encountered with early and recent studies
of siderophore - based antibiotic delivery may be attributed ,
at least in part , to ( i ) use of non - native siderophores with
relatively low fe(iii ) affinities and/or compromised receptor recognition ; ( ii ) modification of antibiotics such that the
antibacterial activity is attenuated or lost completely ; ( iii ) bridging the siderophores and antibiotics with problematic
linkers , including linkers designed for drug release that are either
too stable or too labile , the latter of which promotes premature release ; and ( iv ) antibiotic resistance .
nevertheless ,
the lessons of many unsuccessful studies highlight the complexity
of siderophore - based therapeutic development and provide a foundation
for inventing improved next - generation approaches .
many of the issues
described above may be overcome by careful molecular design and biological
evaluation .
in particular , the selection of appropriate native siderophore
platforms and modification of these platforms in ways that do not
compromise iron binding or receptor recognition , installation of an
antibacterial cargo in such a manner that antibacterial activity is
retained , and the development and application of assays that afford
insight into the fate of siderophore antibiotic conjugates
are critical to the overall success of this approach .
along such lines ,
a recent and insightful study by pfizer addressed complications associated
with using relatively low - molecular - weight siderophore mimics in vivo .
their results indicate
that competition between the siderophore - conjugated monobactam
mb-1 and native siderophores resulted in poor in vivo efficacy against pseudomonas aeruginosa and provide
support for designing and evaluating siderophore
one recent
and successful example based on a native siderophore platform
is a mycobactin
artemisinin conjugate that exhibits enhanced
antibacterial activity against mycobacterium tuberculosis compared to unmodified artemisinin .
enterobactin ( ent , figure 1 ) is a triscatecholate
siderophore biosynthesized by enteric bacteria and used for
iron acquisition in the vertebrate host . motivated by the importance of ent in the host / microbe interaction
as well as the decades of investigations pertaining to its ( bio)synthesis ,
coordination chemistry , and biology , in prior work we reported a synthetic
route to monofunctionalized ent platforms .
moreover , we established that the native ent platform , when monofunctionalized
at the c5 position of one catecholate ring ( figure 1 ) , affords delivery of nontoxic small - molecule cargo across
the outer membrane of gram - negative organisms that express ent uptake
machinery ( e.g. , fepabcdg of escherichia coli ) .
as described herein , this proof - of - concept study
motivated us to demonstrate that ent effectively delivers antibacterial
cargo to organisms that utilize ent for iron acquisition , thereby
providing antibiotic targeting to specific sub - populations and a means
to address antibiotic resistance that results from the gram - negative
outer membrane permeability barrier . in this work
, we present
the syntheses and characterization of
siderophore antibiotic conjugates based on the native
ent platform that harbor the clinically relevant -lactam antibiotics
ampicillin ( amp ) and amoxicillin ( amx ) .
these antibiotics block cell
wall biosynthesis by inhibiting transpeptidases , also named
penicillin binding proteins ( pbps ) , located in the periplasm of e. coli .
we report that the ent--lactam conjugates
exhibit significantly enhanced antibacterial activity ( up to 1000-fold )
against pathogenic e. coli and provide more rapid
cell - killing than the parent -lactams as a result of ent - mediated
delivery to the periplasm . moreover ,
in proof - of - concept studies for
species - specific killing , these conjugates selectively kill e. coli in the presence of staphylococcus aureus , a gram - positive organism that is more susceptible to the parent
-lactams .
these studies support the notion that native siderophore
platforms provide an effective means to target molecular cargo to
siderophore - utilizing organisms and to hijack siderophore
uptake machinery to deliver cargos , including antibiotics , across
the outer membrane permeability barrier of gram - negative microbes .
dimethylformamide ( dmf ) and dichloromethane
( ch2cl2 ) were obtained from a vac solvent purification
system ( vacuum atmospheres ) .
anhydrous dimethyl sulfoxide ( dmso ) was
purchased from sigma - aldrich and used as received .
l - ent 1 , the d - enantiomer of benzyl - protected
ent - co2h 2 , and the benzyl - protected ent - peg3-n33 were synthesized according to
previously reported procedures .
11-azido-3,6,9-trioxaundecan-1-amine
was purchased from fluka . all other chemicals and solvents were purchased
from sigma - aldrich or alfa aesar in the highest available purity and
used as received .
benzyl - protected ent - azide 3 ( 80 mg , 55 mol ) and pentamethylbenzene ( pmb , 147 mg ,
990 mol ) were dissolved in 5 ml of anhydrous ch2cl2 to give a light yellow solution .
this solution was
cooled to 78 c in an acetone / dry ice bath under n2 , and bcl3 ( 660 l of 1 m solution in ch2cl2 , 660 mol ) was added slowly along the
flask wall .
after the solution was stirred for 1.5 h , dipea ( 300 l ,
1.73 mmol ) was added to the flask , followed by meoh ( 2 ml ) to quench
the reaction . the reaction was then warmed to room temperature , and
the solvents were removed under reduced pressure . the resulting white
solid was dissolved in 5:3 meoh/1,4-dioxane and purified by preparative
hplc ( 33% b for 5 min and 3360% b over 20 min , 10 ml / min ) .
the product eluted at 17 min and was lyophilized to yield compound 4 as white solid ( 13.9 mg , 28% ) .
h nmr ( dmso - d6 , 500 mhz ) : 3.353.57 ( 16h ,
m ) , 4.384.41 ( 3h , m ) , 4.634.69 ( 3h , m ) , 4.894.96
( 3h , m ) , 6.74 ( 2h , dd , j = 7.5 , 8.0 hz ) , 6.97 ( 2h ,
d , j = 7.5 hz ) , 7.35 ( 2h , d , j =
8.0 hz ) , 7.46 ( 1h , s ) , 7.94 ( 1h , s ) , 8.338.35 ( 1h , m ) , 9.12
( 2h , d , j = 6.0 hz ) , 9.29 ( 1h , d , j = 6.0 hz ) , 9.44 ( 2h , bs ) , 9.76 ( 1h , bs ) , 11.6 ( 2h , bs ) , 11.9 ( 1h ,
bs ) .
c nmr ( cdcl3 , 125 mhz ) : 50.1 ,
51.5 , 63.6 , 69.1 , 69.4 , 69.8 , 69.8 , 69.9 , 69.9 , 115.3 , 115.4 , 115.4 ,
117.7 , 118.5 , 118.7 , 119.4 , 125.2 , 145.9 , 146.3 , 148.7 , 148.7 , 150.8 ,
166.0 , 168.4 , 169.1 , 169.6 , 169.7 .
ir ( kbr disk , cm ) : 3389 , 2954 , 2928 , 2868 , 2111 , 1754 , 1645 , 1589 , 1535 , 1460 , 1384 ,
1329 , 1266 , 1176 , 1132 , 1074 , 992 , 846 .
11-azido-3,6,9-trioxaundecan-1-amine
( 36 l , 181 mol ) and d - bn6ent - cooh
( 2 , 177 mg , 142 mol ) were dissolved in 5 ml of
dry ch2cl2 .
pyaop ( 147 mg , 283 mol ) and
dipea ( 98.5 l , 568 mol ) were added to give a light yellow
solution .
the reaction was stirred for 4 h at room temperature and
concentrated , and the crude product was purified by preparative tlc
( 50% etoac / ch2cl2 ) to afford 5 as
white foam ( 159 mg , 77% ) .
h nmr ( dmso - d6 , 500 mhz ) : 3.33
( 2h , j = 5.2 hz ) , 3.623.69 ( 14h , m ) , 4.024.06
( 3h , m ) , 4.154.18 ( 3h , m ) , 4.914.94 ( 3h , m ) , 5.045.21
( 12h , m ) , 6.96 ( 1h , s ) , 7.117.45 ( 36h , m ) , 7.657.67
( 2h , m ) , 7.857.85 ( 1h ,
m ) , 7.977.97 ( 1h , m ) , 8.508.54
( 3h , m ) .
c nmr ( cdcl3 , 125 mhz ) : 25.6 ,
29.5 , 38.8 , 40.0 , 45.3 , 51.3 , 51.4 , 63.9 , 64.1 , 69.8 , 39.8 , 70.0 ,
70.3 , 70.4 , 70.4 , 71.2 , 71.2 , 76.3 , 76.3 , 116.8 , 117.5 , 120.4 , 123.0 ,
124.3 , 125.4 , 126.1 , 126.2 , 127.6 , 127.6 , 127.9 , 128.2 , 128.3 , 128.4 ,
128.4 , 128.5 , 128.5 , 128.6 , 128.6 , 128.8 , 128.8 , 128.9 , 129.0 , 130.1 ,
135.4 , 135.7 , 135.9 , 136.0 , 136.1 , 146.8 , 146.9 , 149.1 , 151.6 , 151.8 ,
164.3 , 164.9 , 164.9 , 165.8 , 168.9 , 169.0 , 169.1 , 176.2 . ir ( kbr disk ,
cm ) : 3357 , 3062 , 3032 , 2958 , 2923 , 2859 , 2104 ,
1751 , 1551 , 1576 , 1515 , 1455 , 1375 , 1345 , 1299 , 1264 , 1204 , 1126 ,
1082 , 1040 , 1018 , 957 , 915 , 854 , 811 . hrms ( esi ) :
compound 6 was synthesized
from 5 ( 153 mg , 105 mol ) following the same procedure
as for compound 4 .
the crude reaction was purified by
preparative hplc ( 33% b for 5 min and 3360% b over 20 min ,
10 ml / min ) .
the product eluted at 17.0 min and was lyophilized to
yield compound 6 as white solid ( 31 mg , 33% ) .
h nmr ( cdcl3 , 500 mhz ) : 3.343.56 ( 16h ,
m ) , 4.394.41 ( 3h , m ) , 4.614.66 ( 3h , m ) , 4.884.94
( 3h , m ) , 6.74 ( 2h , dd , j = 7.8 , 7.8 hz ) , 6.96 ( 2h ,
d , j = 7.8 hz ) , 7.33 ( 2h , d , j =
7.8 hz ) , 7.44 ( 1h , s ) , 7.91 ( 1h , s ) , 8.318.33 ( 1h , m ) , 9.129.13
( 2h , m ) , 9.279.28 ( 1h , m ) , 9.50 ( 2h , bs ) , 9.84 ( 1h , bs ) , 11.6
( 2h , bs ) , 11.9 ( 1h , bs ) .
c nmr ( cdcl3 , 125
mhz ) : 50.1 , 51.5 , 63.6 , 69.1 , 69.4 , 69.8 , 69.8 , 69.9 , 69.9 ,
115.3 , 115.4 , 115.4 , 117.7 , 118.5 , 118.7 , 119.4 , 125.2 , 145.9 , 146.3 ,
148.7 , 148.7 , 150.8 , 166.0 , 168.4 , 169.1 , 169.6 , 169.7 .
ir ( kbr disk ,
cm ) : 3390 , 2958 , 2925 , 2863 , 2110 , 1754 , 1645 ,
1589 , 1535 , 1460 , 1384 , 1342 , 1262 , 1176 , 1117 , 1074 , 841 , 800 .
hrms
( esi ) : [ m+na]m / z calcd
936.2506 , found 936.2512 .
5-hexynoic acid ( 113 l , 1.00
mmol ) and thionyl chloride ( 1.00 ml , 13.8 mmol ) were combined and
refluxed for 1 h. the reaction was cooled to room temperature and
concentrated under reduced pressure , and the resulting crude acyl
chloride was dissolved in acetone ( 0.5 ml ) and carried on to the next
step without purification .
ampicillin sodium salt ( 186 mg , 0.500 mmol )
was dissolved in a solution of nahco3 ( 210 mg , 2.5 mmol )
in 4:1 water / acetone ( 2.5 ml ) and cooled on ice , to which the acyl
chloride was added slowly with stirring .
the reaction was subsequently
warmed to room temperature and stirred for 1 h. water ( 3 ml ) was added
to the reaction , and the aqueous phase was washed with etoac ( 2
10 ml ) , acidified to ph 2 by addition of hcl , and extracted with etoac
( 20 ml ) .
the resulting organic phase was washed with cold water ( 2
5 ml ) , dried over na2so4 , and concentrated
under reduced pressure . the crude reaction was triturated with hexanes ,
which afforded a yellow solid ( 180 mg , 77% ) .
h nmr ( dmso - d6 ,
500 mhz ) : 1.41 ( 3h , s ) , 1.55 ( 3h , s ) , 1.641.69 ( 2h ,
m ) , 2.132.16 ( 2h , m ) , 2.292.32 ( 2h , m ) , 2.772.78
( 1h , m ) , 4.20 ( 1h , s ) , 5.39 ( 1h , d , j = 4.0 hz ) ,
5.52 ( 1h , dd , j = 4.0 , 8.0 hz ) , 5.70 ( 1h , d , j = 8.0 hz ) , 7.257.43 ( 5h , m ) , 8.57 ( 1h , d , j = 8.0 hz ) , 9.11 ( 1h , d , j = 8.0 hz ) .
c nmr ( dmso - d6 , 125 mhz ) :
17.4 , 24.4 , 26.6 , 30.4 , 33.8 , 55.5 , 58.1 , 63.7 , 67.3 , 70.3 , 71.5 ,
84.2 , 127.2 , 127.6 , 128.2 , 138.2 , 169.0 , 170.2 , 171.5 , 173.5 .
ir ( kbr
disk , cm ) : 3297 , 3058 , 3023 , 2970 , 2937 , 2863 ,
2626 , 2526 , 2120 , 1780 , 1688 , 1518,1455 , 1437 , 1390 , 1373 , 1324 , 1295 ,
1208 , 1139 , 1027 , 1001 , 843 .
compound 8 was synthesized
as described for compound 7 except that amoxicillin ( 760
mg , 2.1 mmol ) was used instead of ampicillin sodium salt .
compound 8 was obtained as light yellow solid ( 533 mg , 56% ) after trituration
and employed without further purification .
h nmr ( dmso - d6 , 500 mhz ) :
1.42 ( 3h , s ) , 1.56 ( 3h , s ) , 1.631.68 ( 2h , m ) , 2.122.15
( 2h , m ) , 2.252.29 ( 2h , m ) , 2.762.78 ( 1h , m ) , 4.19
( 1h , s ) , 5.39 ( 1h , d , j = 4.0 hz ) , 5.515.54
( 2h , m ) , 6.69 ( 2h , d , j = 8.5 hz ) , 7.19 ( 2h , d , j = 8.5 hz ) , 8.41 ( 1h , d , j = 8.0 hz ) ,
8.93 ( 1h , d , j = 8.0 hz ) , 9.40 ( 1h , bs ) .
c nmr ( dmso - d6 , 125 mhz ) : 17.6 ,
24.5 , 26.8 , 30.4 , 34.0 , 55.4 , 58.2 , 63.9 , 71.6 , 71.7 , 84.4 , 115.1 ,
128.4 , 128.7 , 128.8 , 157.0 , 169.2 , 170.9 , 171.7 , 173.8 . ir ( kbr disk ,
cm ) : 3356 , 3294 , 3045 , 2970 , 2928 , 1770 , 1738 ,
1650 , 1615 , 1515 , 1457 , 1373 , 1208 , 1009 , 945 , 839 , 815 . hrms ( esi ) :
[ m+na]m / z calcd 460.1537 ,
found 460.1534 .
compound 7 ( 60 mg , 0.13
mmol ) was dissolved in 1:1 h2o / mecn ( 5 ml ) , and tfa was
added to a final concentration of 1% .
the solution was incubated at
37 c for 24 h and purified by preparative hplc ( 2050%
b over 25 min , 10 ml / min ) , which afforded a white powder ( 12 mg , 25% ) .
the white powder is a diastereomeric mixture of products , and no further
separation was performed .
h nmr ( dmso - d6 , 500 mhz ) : ( mixture of two diastereomers ) 1.221.23
( 3h , pair of s ) , 1.541.58 ( 3h , pair of s ) , 1.621.68
( 2h , m ) , 2.122.14 ( 2h , m ) , 2.282.30 ( 2h , m ) , 2.77
( 1h , s ) , 3.183.24 ( 0.5h , m ) , 3.303.36 ( 0.5h , m ) , 3.443.49
( 0.5h , m ) , 3.553.60 ( 0.5h , m ) , 3.92 ( 0.5h , s ) , 4.01 ( 0.5h ,
s ) , 4.67 ( 0.5h , dd , j = 6.7 , 6.5 hz ) , 4.78 ( 0.5h ,
dd , j = 5.2 , 5.2 hz ) , 5.455.48 ( 1h , m ) , 7.277.40
( 5h , m ) , 8.508.61 ( 2h , m ) .
c nmr ( dmso - d6 , 125 mhz ) : ( mixture of two diastereomers )
18.1 , 25.0 , 27.6 , 28.1 , 28.4 , 29.5 , 34.4 , 42.2 , 56.8 , 56.8 , 72.1 ,
72.2 , 84.8 , 127.9 , 128.2 , 128.9 , 139.2 , 139.4 , 171.2 , 171.4 , 172.0 .
ir ( kbr disk , cm ) : 3297 , 3071 , 3041 , 2967 , 2938 ,
2535 , 2124 , 1734 , 1653 , 1527 , 1456 , 1427 , 1375 , 1299 , 1199 , 1137 ,
1070 , 1027 , 836 . hrms ( esi ) :
compound 10 was synthesized
as described for compound 9 except that compound 8 was used instead of 7 ( 60 mg , 0.13 mmol ) .
the
product was purified by preparative hplc ( 2050% b over 25
min , 10 ml / min ) , and obtained as white powder ( 14.5 mg , 24% ) .
h nmr ( dmso - d6 , 500 mhz ) :
( mixture of two diastereomers ) 1.26 ( 3h , s ) , 1.551.59 ( 3h ,
pair of s ) , 1.611.67 ( 2h , m ) , 2.122.14 ( 2h , m ) , 2.242.27
( 2h , m ) , 2.77 ( 1h , s ) , 3.203.26 ( 0.5h , m ) , 3.343.39
( 0.5h , m ) , 3.423.47 ( 0.5h , m ) , 3.563.60 ( 0.5h , m ) ,
4.03 ( 0.5h , s ) , 4.12 ( 0.5h , s ) , 4.68 ( 0.5h , dd , j = 6.5 , 6.5 hz ) , 4.79 ( 0.5h , dd , j = 5.5 , 5.5 hz ) ,
5.285.31 ( 1h , m ) , 6.69 ( 2h , d , j = 8.5 hz ) ,
7.17 ( 2h , d , j = 8.5 hz ) , 8.368.50 ( 3h , m ) .
c nmr ( dmso - d6 , 125 mhz ) :
( mixture of two diastereomers ) 17.5 , 24.3 , 26.9 , 27.5 , 27.7 , 28.8 ,
33.8 , 41.4 , 55.7 , 55.8 , 71.4 , 71.6 , 84.2 , 114.8 , 115.0 , 128.5 , 128.7 ,
128.8 , 156.9 , 158.3 , 158.6 , 171.2 , 171.3 .
ir ( kbr disk , cm ) : 3301 , 3071 , 3028 , 2973 , 2928 , 2548 , 2111 , 1737 , 1662 , 1606 , 1593 ,
1515 , 1435 , 1377 , 1197 , 1139 , 837 . hrms ( esi ) :
ampicillin - alkyne 7 ( 120 l
of an 80 mm solution in dmso , 9.6 mol ) and
ent - peg3-n34 ( 250 l of
a 13 mm solution in 1,4-dioxane , 3.3 mol ) were combined , and
400 l of dmso was added .
cuso4 ( 100 l of
a 90 mm solution in water , 9.0 mol ) and tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine ( tbta , 200 l of
a 50 mm solution in dmso , 10 mol ) were combined , and 100 l
of dmso was added to give a blue - green solution , to which naasc ( 400
l of a 90 mm solution in water , 36.0 mol ) was added .
this solution became light yellow and was immediately added to the
alkyne / azide solution .
the reaction was shaken on a benchtop rotator
for 2 h at room temperature , diluted by 3- to 4-fold with 1:1 mecn / water ,
centrifuged ( 13,000 rpm 10 min , 4 c ) , and purified by
semi - preparative hplc ( 20% b for 5 min and 20%50% b over 11
min , 4 ml / min ; 0.005% tfa was used in the solvent system to prevent
decomposition of the -lactam ) .
the hplc fractions containing 11 were collected manually and flash frozen in liquid n2 immediately after collection to prevent -lactam decomposition .
h nmr ( dmso - d6 , 500 mhz ) : 1.40
( 3h , s ) , 1.54 ( 3h , s ) , 1.781.81 ( 2h , m ) , 2.27 ( 2h , t , j = 6.8 hz ) , 2.58 ( 2h , t , j = 6.5 hz ) ,
3.48 ( 12h , m ) , 3.76 ( 2h , s ) , 4.19 ( 1h , s ) , 4.384.44 ( 5h , m ) ,
4.644.66 ( 3h , m ) , 4.914.92 ( 3h , m ) , 5.39 ( 1h , d , j = 3.5 hz ) , 5.515.52 ( 1h , m ) , 5.72 ( 1h , d , j = 7.5 hz ) , 6.74 ( 2h , dd , j = 7.8 , 7.8
hz ) , 6.96 ( 2h , d , j = 7.5 hz ) , 7.267.35 ( 5h ,
m ) , 7.427.45 ( 3h , m ) , 7.81 ( 1h , s ) , 7.92 ( 1h , s ) , 8.33 ( 1h ,
s ) , 8.55 ( 1h , d , j = 7.5 hz ) , 9.12 ( 3h , d , j = 7.0 hz ) , 9.29 ( 1h , d , j = 6.5 hz ) ,
9.42 ( 2h , bs ) , 9.74 ( 1h , s ) , 11.6 ( 2h , s ) , 11.9 ( 1h , bs ) , 13.35 ( 1h ,
bs )
. hrms ( esi ) : [ m+na]m / z calcd 1379.4021 , found 1379.4046 .
compound 12 was
synthesized as described for 11 except that compound 8 was used instead of compound 7 .
h nmr ( dmso - d6 , 500 mhz ) : 1.40 ( 3h , s ) , 1.54 ( 3h ,
s ) , 1.781.80 ( 2h , m ) , 2.23 ( 2h , t , j = 6.5
hz ) , 2.57 ( 2h , t , j = 6.5 hz ) , 3.47 ( 12h , m ) , 3.76
( 2h , bs ) , 4.18 ( 1h , s ) , 4.394.43 ( 5h , m ) , 4.634.65
( 3h , m ) , 4.90 ( 3h , bs ) , 5.38 ( 1h , s ) , 5.525.56 ( 2h , m ) , 6.68
( 2h , d , j = 8.5 hz ) , 6.73 ( 2h , dd , j = 7.8 , 7.8 hz ) , 6.96 ( 2h , d , j = 7.5 ) , 7.19 ( 2h ,
d , j = 8.5 hz ) , 7.33 ( 2h , d , j =
7.5 hz ) , 7.44 ( 1h , s ) , 7.80 ( 1h , s ) , 7.92 ( 1h , s ) , 8.338.39
( 2h , m ) , 8.94 ( 1h , d , j = 8.0 hz ) , 9.119.12
( 2h , m ) , 9.29 ( 1h , bs ) , 9.389.43 ( 3h , m ) , 9.75 ( 1h , s ) , 11.6
( 2h , bs ) , 11.9 ( 1h , bs ) .
compound 13 was
synthesized as described for 11 except
that compound 6 was used instead of compound 4 .
hrms ( esi ) :
[ m+na]m / z calcd 1379.4021 ,
found 1379.4022 .
compound 14 was synthesized as described for 12 except
that compound 6 was used instead of compound 4 .
hrms ( esi ) :
[ m+na]m / z calcd 1395.3970 ,
found 1395.3995 .
compound 15 was synthesized as described for 11 except that compound 9 was used instead of compound 7 .
hrms ( esi ) : [ m+h]m / z calcd 1331.4409 , found
1331.4389 .
compound 16 was synthesized as described for 12 except that compound 10 was used instead of compound 8 .
hrms ( esi ) : [ m+na]m / z calcd 1369.4177 , found
1369.4191 .
general microbiology materials and methods , including
details of
ent - amp / amx stock solution preparation and storage , are provided as supporting information .
overnight cultures of
the bacterial strains ( table s1 ) were prepared
in 15-ml polypropylene tubes by inoculating 5 ml of lb media with
the appropriate freezer stock .
the overnight cultures were incubated
at 37 c for 1618 h in a tabletop incubator shaker set
at 150 rpm and housing a beaker of water .
each overnight culture was
diluted 1:100 into 5 ml of fresh lb media containing 200 m
2,2-dipyridyl ( dp ) and incubated at 37 c with shaking
at 150 rpm until od600 reached 0.6 .
g / l ) with or without 200 m
dp to achieve an od600 value of 0.001 .
a 90-l aliquot
of the diluted culture was combined with a 10-l aliquot of
a 10 solution of the antibiotic or ent - antibiotic conjugate
in a 96-well plate , and the covered plate was wrapped in parafilm
and incubated at 30 c with shaking at 150 rpm for 19 h in a
tabletop incubator housing a beaker of water .
bacterial growth was
determined by measuring od600 ( end point analysis ) using
a biotek synergy ht plate reader .
each well condition was prepared
in duplicate and at least three independent replicates using two different
synthetic batches of each conjugate were conducted on different days .
the resulting mean od600 values are reported , and the error
bars are the standard error of the mean ( sem ) obtained from the independent
replicates .
these assays were performed with e. coli atcc
35218 and k. pneumoniae atcc 13883 following the
general procedure except that sulbactam ( sb ) or potassium clavulanate
( pc ) were mixed with ampicillin or amoxicillin and the ent - amp or
ent - amx conjugates , respectively .
the molar ratios of the inhibitor/-lactam
mixtures were sulbactam / amp or ent - amp , 1.5:1 , and potassium clavulanate / amx
or ent - amx , 0.9:1 .
these assays were
performed with e. coli k-12 and
cft073 following the general procedure except that varying concentrations
( 1100 m ) of synthetic l - ent were mixed with
ent - amp / amx .
this assay is based on a published protocol and was conducted with e. coli cft073 .
each overnight culture was serially diluted into
m9 minimal medium to provide 1010 cfu / ml .
lipocalin 2 was diluted in pbs to a concentration of 10 m upon
arrival , aliquoted , and stored at 20 c until use .
a
90-l aliquot of the diluted culture was added to each well
of a 96-well plate that contained varying concentrations of lipocalin
2 , ent - amp , and ent , and the final volume was adjusted to 100 l
with sterile pbs .
the 96-well plate was incubated at 37 c for
24 h in a tabletop incubator set at 150 rpm , and bacterial growth
was determined by measuring od600 using a plate reader .
each well condition was repeated at least three times independently
on different days and with different batches of lipocalin 2 .
the resulting
mean od600 is reported , and the error bars are the sem .
a 5-ml overnight culture of e. coli k-12 or cft073 was grown in lb ( vide supra ) and diluted 1:100 into 5 ml of fresh lb media containing 200 m
dp , and this culture was incubated at 37 c with shaking at 150
rpm in a tabletop incubator housing a beaker of water until od600 reached 0.3 .
the culture was centrifuged ( 3000
rpm 10 min , rt ) , and the resulting pellet was washed twice
by resuspension in 50% mhb and centrifugation ( 3000 rpm 10
min , rt ) .
the resulting pellet was resuspended in 50% mhb with or
without dp , and the od600 was adjusted to 0.3 .
a 90-l
aliquot of the resulting culture was mixed with a 10-l aliquot
of a 10 solution of amp / amx or the ent - amp / amx in a 96-well
plate , which was covered , wrapped in parafilm , and incubated at 37
c with shaking at 150 rpm .
the od600 values were
recorded at t = 0 , 1 , 2 , and 3 h by using a plate
reader . in a parallel experiment ,
a 10-l aliquot of the culture
was taken at t = 0 , 1 , 2 , and 3 h , serially diluted
by using sterile phosphate - buffered saline ( pbs ) , and plated on lb - agar
plates for colony counting ( cfu / ml ) .
the resulting
mean od600 or cfu / ml is reported , and the error bars are
the sem .
a 5 ml overnight culture of each
bacterial strain was grown in lb , diluted 1:100 into 5 ml of fresh
lb media containing 200 m dp , and incubated at 37 c with
shaking at 150 rpm in a tabletop incubator housing a beaker of water
until od600 reached 0.6 .
each mid - log - phase culture was
diluted to 10 cfu / ml in 50% mhb with or without 200 m
dp . for experiments requiring a mixture of two species ,
a 1:1 mixture
was prepared ( 10 cfu / ml for each strain ) in 50% mhb with
or without 200 m dp from the mid - log - phase cultures . to confirm
cfu / ml of each culture ,
the single- and double - species cultures were
serially diluted by using sterile pbs , and aliquots were plated on
a chrom - uti plate ( starter - culture plate ) .
for each
cell - killing experiment , a 90-l aliquot of each culture was
combined with a 10-l aliquot of a 10 m solution of the
antibiotic or ent - antibiotic conjugate in a 96-well plate , which was
covered , wrapped in parafilm , and incubated at 30 c with shaking
at 150 rpm for 19 h. bacterial growth was assayed both by measuring
od600 using the plate reader and by plating on chrom - uti
plates after serial dilution ( assay plate ) .
each well
condition was repeated at least three times independently on different
days .
the resulting mean od600 is reported , and the error
bars are the sem .
we aimed to harness our enterobactin - mediated
cargo delivery strategy to enable the transport of toxic cargo across
the outer membrane of e. coli . to address this goal ,
we linked the -lactam antibiotics ampicillin ( amp ) and amoxicillin
( amx ) to a monofunctionalized ent scaffold where ent is derivatized
at the c5 position of one catechol ring via a flexible and stable
peg3 linker .
we selected amp and amx as antibacterial cargo
for several reasons : these molecules are commercially available and
amenable to synthetic modification , retain antibacterial activity
when appropriately modified , possess periplasmic targets in gram - negative
bacteria and must cross the outer membrane to be active against these
species , and have relatively low molecular weights .
we selected low - molecular - weight
antibiotics because our prior studies of ent - mediated cargo transport
indicated that the ent transport machinery of e. coli k-12 imports ent - cargo conjugates harboring relatively small cargos
( e.g. , cyclohexane , naphthalene , phenylmethylbenzene )
to the cytosol readily , whereas large cargos ( e.g. , vancomycin ) are
not transported to the cytosol .
moreover ,
in prior studies , various -lactams including amp and amx have
been linked to simple catechols and more complex catechol - containing siderophores
or mimics thereof , which provides the
opportunity to compare the outcomes obtained for different siderophore - inspired
design strategies . in scheme 1
, we present the syntheses of
ent - amp 11 and ent - amx 12 , which feature
installation of alkyne - modified -lactam warheads onto ent - azide 4 via copper - catalyzed azide - alkyne cycloaddition ( hereafter
click reaction ) in the final step .
the catechol moieties of benzyl - protected
ent - azide 3 were deprotected by using bcl3 at 78 c to achieve ent - azide 4 as a white
powder in 28% yield following purification by reverse - phase preparative
hplc . catalytic hydrogenation using hydrogen gas and a pd / c catalyst
is typically employed to deprotect ent catechols ; however , we observed that amp / amx decompose under these conditions
and poison the pd / c catalyst .
moreover , deprotection of the enterobactin
catechols prior to installing the -lactams requires preservation
of the azide moiety , and we therefore employed bcl3 for
this reaction .
initial attempts at assembling ent - amp / amx using standard
conditions for the copper - catalyzed click reactions with ent - azide 4 and the alkyne - modified -lactams 7/8 , prepared by thionyl chloride coupling of 5-hexynoic acid
to the amino group of amp / amx , failed because of copper - mediated -lactam
decomposition .
this problem was overcome
by including the metal - ion chelator tris[(1-benzyl-1h-1,2,3-triazol-4-yl)methyl]amine ( tbta ) in the click reactions , and ent - amp and ent - amx were obtained as
white powders in high purity and yields of 66% and 76% , respectively ,
following semi - preparative hplc purification .
it was necessary to
perform hplc purification with eluents containing only 0.005% tfa
to prevent decomposition of the acid - sensitive -lactam moieties .
this synthetic route was likewise employed to prepare the d - enantiomers of ent - amp / amx 13 and 14 ( scheme 1 ) .
deprotected ent - azide 4 enables alkyne - functionalized
molecules to be covalently linked to ent via a click reaction , including
molecules that are incompatible with reaction conditions required
to deprotect the ent catechols .
moreover , the deprotected ent - azide
may be employed to append ent to surfaces , other materials , or biomolecules
harboring alkyne groups .
indeed , very
few examples employing the copper(i)-catalyzed click reaction with
fused -lactams are reported in the literature , and it is likely that this paucity stems from the fact that -lactams
are incompatible with standard conditions for copper - catalyzed azide - alkyne
cycloaddition .
the conditions defined
in this work employing tbta allow for copper - catalyzed triazole formation
and preserve the -lactam warhead . in the absence of fe(iii ) ,
ent - amp / amx exhibit an absorption band centered at 316 nm resulting
from catecholate absorption and the solutions are colorless ( meoh ,
rt ) .
addition of 1.0 equiv of fe(iii ) to methanolic solutions of ent - amp
and ent - amx causes the solution to immediately change from colorless
to purple - red , and a broad absorption feature in the 400700
nm range appears ( figure s1 ) , confirming
that both ent - amp / amx readily chelate fe(iii ) . to ascertain whether ent - amp / amx provide antibacterial activity
against e. coli , including pathogenic strains , we preformed antimicrobial activity assays using
six strains ( table s1 , figures 2 , s2s7 ) .
e. coli atcc 25922 is a laboratory susceptibility test strain
originally obtained as a clinical isolate .
coli uti89 and
cft073 are both pathogens of the human
urinary tract ( upec).e .
coli atcc 35401 ( serotype o78:h11 ) is an enterotoxigenic ( etec ) strain
that was isolated from human feces .
e. coli atcc
43895 ( serotype o157:h7 ) is an enterohemorrhagic ( ehec ) strain that
was isolated from raw hamburger meat implicated in a hemorrhagic colitis
outbreak .
both etec and ehec strains produce virulence factors and
toxins and cause diarrhea in humans .
all e. coli strains biosynthesize ent for iron acquisition and
express the ent receptor fepa .
some e. coli strains
employed in this work also have the capacity to produce and utilize
salmochelins , c - glucoyslated ent derivatives .
these molecules are produced by salmonella spp .
and pathogenic e. coli strains for iron acquisition .
the iroa gene cluster ( irobcden )
encodes proteins required for the biosynthesis and transport of salmochelins ,
and iron is the outer membrane receptor for salmochelins encoded by
the iroa cluster .
studies with salmonella indicate that iron has the ability to transport
ent as well as its glucosylated forms . of the strains considered in this work , e. coli cft073 and uti89 harbor the iroa gene cluster .
e. coli h9049 does not produce salmochelins , and a blast search using available e.
coli genomes reveals that the e. coli 43985
genome does not contain the iroa cluster .
the genome
for e. coli atcc 25922 is unpublished ; however , this
strain is reported to be sensitive to lipocalin-2 ( vide infra ) , which suggests that its genome does
not encode the iroa cluster .
lastly , it should be noted that e. coli cft073 is
celebrated for having redundant iron import machineries , and this
strain also harbors the iha gene , which encodes the
outer membrane ent receptor iha that is distinct from fepa .
antibacterial activity of ent - amp / amx against various e.
coli strains that include human pathogens .
all assays
were performed in 50% mhb medium supplemented with 200 m dp
to provide iron - limiting conditions ( mean sem , n 3 ) .
the data for assays performed in the absence of dp
are presented in figures s2s7 .
we performed antibacterial activity
assays using a 10-fold dilution
series to compare the abilities of ent - amp / amx and unmodified amp / amx
to kill e. coli ( figures 2 , s2s7 ) .
these assays were conducted
in 50% mhb and in the absence or presence of 200 m dp .
the
latter growth conditions provide iron limitation and result in expression
of the ent uptake machinery fepabcdg .
amp / amx exhibit minimum inhibitory
concentration ( mic ) values of 10 m against these e.
coli strains regardless of the presence of dp in the growth
medium ( figures 2 , s2s7 ) .
all six e. coli strains are more susceptible
to ent - amp / amx than amp / amx under conditions of iron limitation ( figure 2 ) .
based on the 10-fold dilution series , ent - amp / amx
are 100-fold more potent against e. coli 25922 , uti89 ,
and h9049 under iron - limiting conditions .
although the mic values
for 35401 and 43895 are only ca . 10-fold higher than for amp / amx in
this assay , a significant reduction in growth is observed at 100 nm
ent - amp / amx , whereas this concentration of amp / amx affords no growth
inhibition .
the enhanced sensitivity of e. coli cft073
to ent - amp / amx is remarkable .
this strain exhibits the greatest sensitivity
to ent - amp / amx , providing a 1000-fold decreased mic value ( 10 nm ) ,
and growth inhibition in the presence of 1 nm of the conjugate .
moreover , in the absence of dp , e. coli cft073 exhibits
the greatest susceptibility to ent - amp / amx ( figures
s2s7 ) .
a noteworthy characteristic of cft073 is its
multiple mechanisms for iron acquisition , and we hypothesize that
the presence of multiple receptors that recognize and transport ent
( fepa , iron , iha ) contributes to this enhanced sensitivity .
cft073
and uti89 both express iron , which may indicate that iha is responsible
for the enhanced susceptibility of cft073 ; however , we can not rule
out the possibility that the conserved receptors function differently
or exhibit different expression levels depending on the strain . moreover
,
other unappreciated mechanisms may contribute to the potent bactericidal
action exhibited by ent - amp / amx against cft073 and other strains . to gain further insight into the mechanism
of ent - amp / amx antibacterial action
, we performed a series of experiments
with the standard laboratory strain e. coli k-12 .
we employed the same 10-fold dilution series to compare the activities
of ent - amp / amx and amp / amx against k-12 . in the absence of dp ,
ent - amp / amx
and amp / amx exhibit comparable mic values , with complete killing observed
at 10 m . at lower concentrations , ent - amp / amx exhibit
slightly greater antibacterial activity than unmodified amp / amx ( figure 3a ) .
this phenomenon is most evident at a conjugate / drug
concentration of 1 m , where ent - amp / amx inhibit e.
coli k-12 growth to varying degrees and amp / amx do not affect
bacterial growth . under conditions of iron limitation ,
a 100-fold
reduction in mic value ( 10 m to 100 nm ) for ent - amp / amx is
observed , and ca .
50% growth inhibition occurs at 10 nm of each conjugate
( figure 3b ) .
these trends are comparable to
those observed for e. coli atcc 25922 , h9049 , and
uti89 ( figures 2 , s2 , s3 ,
and s5 ) .
when the antibacterial activity assay was performed
using a 1:1 ratio of native l - ent and amp / amx , no reduction
of amp / amx mic values was observed ( figure 3c ) , which suggests the conjugation between ent and the -lactams
is required for the enhanced bactericidal action
. moreover , treatment
of e. coli k-12 with the iron - bound forms of ent - amp / amx ,
obtained by pre - incubating each conjugate with 1 equiv of ferric chloride ,
afforded the same mic values as observed for apo ent - amp / amx .
these
results indicate that the enhanced antibacterial activity does not
result from iron chelation in the growth media ( figure 3d ) . in total
, the data obtained for e. coli k-12 as well as the six other e. coli strains demonstrate
that the antibacterial activity of ent - amp / amx against e.
coli k-12 is enhanced under conditions of iron limitation
and support a model of ent - mediated delivery of antibacterial cargo
to the e. coli periplasm . antibacterial activity
of ent - amp / amx against wild - type and mutant e. coli k-12 .
( a , b ) growth inhibition of e. coli k-12 by
amp / amx and ent - amp / amx in the absence ( a ) and presence
( b ) of dp .
( c ) growth inhibition of e. coli k-12
treated with a 1:1 molar ratio of ent / amp and ent / amx .
( d ) growth
inhibition of e. coli k-12 treated with ferric ent - amp / amx .
( e g ) growth inhibition of fepa- ( e ) , fepc- ( f ) , and fes- ( g ) by amp / amx and
ent - amp / amx .
( h , i ) growth of e. coli k-12 in the
presence of 1 m ent - amp / amx ( conjugate ) and
mixtures of ent - amp / amx ( 1 m ) and 1 , 5 , or 20 equiv of exogenous
ent in the absence ( h ) and presence ( i ) of dp . the * * indicates od600 < 0.01 .
all assays were performed in 50% mhb medium
with or without 200 m dp ( see panels ) ( mean sem ,
the data for additional assays performed
in the absence of dp are presented in figure s8 . to evaluate transport of ent - amp / amx
into e. coli , we investigated the antimicrobial activity
of ent - amp / amx and amp / amx against three single - gene knockout e. coli k-12 strains obtained from the keio collection , fepa- , fepc- , and fes- ( figures 3e
we selected these mutants to ascertain
how components of the enterobactin transport and processing machinery
contribute to ent - amp / amx antibacterial activity .
e. colifepa- lacks the outer membrane ent receptor fepa
that allows periplasmic delivery , fepc- lacks the
atpase component of the inner membrane ent permease that transports
ent into the cytosol , and e. colifes- lacks the cytoplasmic esterase fes responsible for hydrolysis of
the ent macrolactone for iron release . on the basis of our studies
with wild - type k-12 and other e. coli strains , we
hypothesized that the activity of ent - amp / amx would be attenuated
for the fepa- mutant .
moreover , we questioned whether
loss of fepc or fes would modulate the antimicrobial activity .
overnight cultures of fepa- and fepc- reached od600 values ( 0.15 ) similar to that observed
for wild - type k-12 when grown in 50% mhb supplemented with 200 m
dp .
in contrast , the fes- strain exhibited a severe
growth defect under these conditions ( od600 0.04 ) .
treatment of fepa- with ent - amp / amx afforded the
same mic values as for amp / amx ( figure 3e )
and hence a 100-fold reduction in activity as compared to wild - type
k-12 .
iron deprivation is deleterious to e. coli ,
and we contend that the growth inhibition observed at 10 m
ent - amp / amx results from iron starvation rather than an antibacterial
activity of the amp / amx cargo .
analysis of 50% mhb by inductively
coupled plasma optical emission spectroscopy ( icp - oes ) revealed a
total iron concentration of ca .
4 m ( table
s2 ) , which can be compared to 10 m of a high - affinity
extracellular iron chelator .
indeed , we previously observed similar
growth inhibition of fepa- with 10 m of an
ent - vancomycin conjugate ( extracellular iron chelation ) and also 10
m d - ent ( iron chelator that can not be used for iron
acquisition ) under these growth conditions .
these data confirm that fepa is essential for the potent antibacterial
activity of ent - amp / amx against e. coli k-12 .
in contrast to fepa- , the growth of fepc- and fes- was completely inhibited with 100 nm ent - amp / amx
under iron - limiting conditions ( figure 3f , g ) ,
comparable to what was observed for the wild - type strain . the targets
of -lactam antibiotics are penicillin binding proteins ( pbps ) ,
which are located in the periplasm of gram - negative bacteria .
after
crossing the outer membrane through fepa , ent - amp / amx enter the periplasm
where covalent capture by the pbps presumably occurs .
thus , it is
reasonable that the downstream ent transport and processing steps
involving fepcdg and fes do not affect the antimicrobial activity
of the conjugates if they are trapped in the periplasm as a result
of pbp binding .
although it is possible that the ent uptake machinery
( e.g. , periplasmic binding protein fepb ) competes with the pbps for
ent - amp / amx , no improved antibacterial activities were observed for
the fepc- and fes- mutants compared
to wild - type k-12 .
this observation indicates that ent - amp / amx bind
to pbps and are trapped in the periplasm .
it should be noted that
the fepb- mutant , which lacks the periplasmic binding
protein , was also considered in this work ; however , this strain exhibited
a severe growth defect and afforded inconsistent results . to probe
interaction between fepa and ent - amp / amx , we performed
growth inhibition assays employing mixtures of ent - amp / amx and varying
concentrations of unmodified ent ( figure 3h
when these assays were performed under conditions of iron limitation ,
the presence of exogenous ent attenuated the antibacterial activity
of ent - amp / amx .
a 1:1 molar ratio of ent - amx / ent afforded an od600 value comparable to that of the untreated control , whereas
higher equivalents of ent were required to block the antibacterial
action of ent - amp .
the origins of this difference are unclear and
may indicate that the hydroxyl group of amx has a negative effect
on the transport efficiency of the conjugate . in total , these ent
addition assays suggest that competition for ent - amp / amx and ent occurs
at the receptor(s ) and that the conjugates are delivered into the
bacteria via the same uptake machinery as ent .
l - serine is a biosynthetic
building block for ent , and a role
for chiral recognition in ent transport has been probed in prior studies . in one series of investigations ,
e. coli fepa was
found to bind ferric l - ent and ferric d - ent with
similar affinities ( kd = 21 and 17 nm ,
respectively ; ascertained by measuring the binding of fe - loaded siderophores to e. coli bn1071 cells ) .
a lack of transport of ferric d - ent
into e. coli bn1071 was also reported in this work .
a later study probed ent uptake in bacillus subtilis , and transport of both l- and d - ent analogues
was observed to occur with similar efficiency .
2457 t to hydrolyze l - ent and d - ent was
evaluated , and fes did not accept d - ent as a substrate . as
a result of the transport studies in b. subtilis and
enzymatic activity assays with s. flexneri fes , a
model in which both ent enantiomers are transported and chiral
taken together , these studies suggest that the ability
to transport d - ent may vary between species and even between
strains of a given species , and more studies are required to address
such possibilities .
based on the observation that e. coli fepa binds d - ent and that b. subtilis transports d - ent analogues , we synthesized the d - enantiomers of
the ent--lactam conjugates ( 13 and 14 , scheme 1 ) and evaluated the antibacterial
activity of these conjugates against e. coli k-12 ,
25922 , cft073 , 35401 , and 43895 ( figures s9s13 ) .
d - ent - amp / amx exhibited reduced antibacterial activity
relative to ent - amp / amx for k-12 , 25922 , cft073 , and 43895 . under
conditions of iron limitation ,
complete growth inhibition was observed
with 1 m d - ent - amp / amx for e. coli k-12 , 25922 , and 43895 , compared to 100 nm for ent - amp / amx .
likewise ,
a 10-fold reduction in antibacterial activity was observed for e. coli cft073 , where 100 nm d - ent was required
to inhibit growth completely .
in contrast , a negligible difference
in antibacterial activity of the l- and d - isomers
was observed for e. coli 35401 .
regardless of enantiomer ,
all four ent - amp / amx conjugates provide enhanced antibacterial activity
against these e. coli strains relative to amp / amx .
nevertheless , these data suggest that d - ent - amp / amx are less
readily transported into various e. coli strains
than the l - isomers .
although this modification provides no
appreciable benefit for this -lactam delivery system with periplasmic
targets , it is possible that ent - antibiotic conjugates based on d - ent may be desirable for delivering cargos to the cytosol ,
precluding concomitant delivery of nutrient fe(iii ) . with support for ent - mediated delivery of ent - amp /
amx
to the e. coli periplasm , we sought to confirm the
essentiality of the -lactam warheads in antibacterial action .
we therefore designed and prepared hydrolyzed ent - amp / amx analogues 15 and 16 ( scheme 2 ) where
the -lactam structure is destroyed .
hydrolysis of the amp / amx - alkynes 7 and 8 was achieved in the presence of 1% tfa
with heating at 37 c , and the decomposition products 9 and 10 were obtained as diastereomeric mixtures ( scheme 2 ) .
the formation of these species followed the reported
degradation pathways for ampicillin , where hydrolysis and subsequent
decarboxylation occur .
the diastereomeric
mixtures were employed to prepare the hydrolyzed conjugates ent - hydro - amp 15 and ent - hydro - amx 16 via a copper - catalyzed
click reaction .
( a , b ) antibacterial activity assays against e. coli k-12 ( a ) and cft073 ( b ) using ent - amp / amx and ent - hydro - amp / amx .
( c ) antibacterial activity assays against e. coli atcc 35218 , which expresses a class a serine -lactamase ,
using ent - amp / amx in the absence and presence of the -lactamase
inhibitors potassium clavulanate ( pc ) and sulbactam ( sb ) .
all assays
were performed in 50% mhb supplemented with 200 m dp ( mean
sem , n 3 ) .
we employed ent - hydro - amp / amx in antibacterial activity assays
against e. coli k-12 and observed negligible growth
inhibition ( figure 4a ) . when e. coli cft073 was treated with ent - hydro - amp / amx in the presence of dp
this result indicates that ent - hydro - amp / amx
are transported into the cytoplasm of e. coli cft073 ,
where nutrient iron is released .
we also performed a series
of antibacterial activity assays with e. coli atcc
35218 , a strain that expresses a class a serine
-lactamase .
similar to unmodified amp / amx , ent - amp / amx were
inactive against e. coli atcc 35218 ( mic > 10
m )
in the absence and presence of dp ( figures 4c and s14 ) .
slight growth inhibition was
observed at 10 m under conditions of iron limitation , which
may be attributed to iron chelation .
the addition of -lactamase
inhibitors restored the activities of ent - amp / amx and amp / amx , and
the conjugates exhibited greater antibacterial activity than the parent
antibiotics ( figures 4c and s14 ) . in total , the assays with ent - hydro - amp / amx and strains
expressing -lactamase demonstrate that an intact -lactam
is required for the antibacterial activity of ent - amp / amx .
moreover ,
these studies indicate that the -lactams retain their original
function and inhibit pbps when conjugated to ent .
the remarkable sensitivity of e. coli cft073 to
ent - amp / amx ( figure 2c ) motivated us to investigate
the relative cell - killing kinetics
of ent - amp / amx and amp / amx to determine whether these conjugates kill e. coli cft073 more rapidly than the unmodified drugs . for
comparison between e. coli strains ,
ent - amp / amx
provide more rapid cell death than unmodified amp / amx ( figure 5 ) , and this behavior is most apparent for e. coli cft073 , where the od600 value was almost
reduced to the baseline value after 1 h incubation with 5 m
ent - amp / amx , corresponding to a 2-fold log reduction in cfu / ml . in
contrast , the change in od600 and cfu / ml for e.
coli cft073 treated with 50 m unmodified amp / amx is
negligible over this time period .
the time - kill kinetics for e. coli k-12 , conducted with 50 m of both unmodified
and modified -lactams , indicate a slight increase in kill kinetics
for ent - amp / amx relative to amp / amx , and that the kinetics of cell - killing
are slower for k-12 than cft073 ( figure 5 ) .
these results support a model whereby ent modification facilitates
uptake of amp / amx relative to the unmodified drugs .
this effect is
more dramatic for e. coli cft073 than k-12 , which
is in accordance with the enhanced antibacterial activity observed
for cft073 relative to the other e. coli strains
considered in this work .
time - kill kinetic assays for treatment of e. coli k-12 ( top panel ) and cft073 ( bottom panel ) with
amp / amx and ent - amp / amx .
e. coli k-12 ( 10 cfu / ml ) was treated
with 50 m of amp / amx or 50 m ent - amp / amx .
e.
coli cft073 ( 10 cfu / ml ) was treated with
50 m of amp / amx or 5 m ent - amp / amx .
the assays were
conducted in 50% mhb medium containing 200 m dp at 37 c
( mean sem , n = 3 ) . to determine whether ent - amp / amx exhibit broad - spectrum or species - selective
activity , we performed antibacterial activity assays with two additional
gram - negative and two gram - positive species in both the absence and
presence of dp .
these species include klebsiella pneumoniae atcc 13883 , pseudomonas aeruginosa pao1 , s. aureus atcc 25923 , and bacillus cereus atcc 14579 .
k. pneumoniae is a gram - negative species
that biosynthesizes and utilizes ent for iron acquisition .
p. aeruginosa is a gram - negative bacterium that captures
ent as a xenosiderophore and expresses two ent receptors
pfea and pira.s .
aureus and b. cereus are both gram - positive bacterial species , and the ability to utilize
ferric ent as an iron source is reported for both species .
in contrast to gram - negative bacteria , where the pbps are located
in the periplasm , the targets of -lactam antibiotics are in
the extracellular peptidoglycan of gram - positive organisms .
k. pneumoniae atcc 13883 has a chromosomally encoded
class a -lactamase ( shv-1 ) and lacks sensitivity to amp / amx .
we observed no effect of 100 m amp / amx on k. pneumoniae growth under our assay conditions ( figure s15 ) .
only 50% growth inhibition was observed when k.
pneumoniae was treated with high concentrations ( 10 m )
of ent - amp / amx in the absence of dp , and the ent - amp conjugate provided
the greatest activity under conditions of iron limitation with 90%
growth inhibition at 10 m .
when -lactamase inhibitors
were included in the assays , k. pneumoniae exhibited
greater sensitivity to amp / amx ( mic = 100 m ) and ent - amp / amx
( mic = 10 m ) ; however , we observed some growth inhibitory activity
of the -lactamase inhibitor sulbactam alone under these assay
conditions ( 100 m sb , figure s15 ) .
thus , the possibility of a synergistic effect from the inhibitors
and conjugates can not be ruled out completely .
the lack of activity
of ent - amp / amx against k. pneumoniae atcc 13883 is
reminiscent of results obtained during investigations of amp / amx - functionalized
tripodal triscatecholate ligands .
these
compounds were inactive against k. pneumoniae , and
the behavior was attributed to either an inability of the k. pneumoniae iron transport machinery to import the conjugates
or the development of resistance over the course of the assay .
an
alternative explanation is that -lactamase expression by k. pneumoniae resulted in inactivation of the -lactams .
the p. aeruginosa pao1 strain employed in this
work exhibited little sensitivity to both amp / amx and the conjugates
under the antibacterial assay conditions ( figure
s16 ) .
amp / amx exhibited no activity up to 100 m , whereas
ent - amp / amx provided growth inhibition at 10 m in both the
absence and presence of dp . whether these results indicate that ent - amp / amx
will be ineffective against multiple p. aeruginosa strains is unclear .
p. aeruginosa strains exhibit
different phenotypes , and highly variable and strain - dependent mic
values have been reported for triscatecholate-lactam
conjugates against p. aeruginosa .
we previously reported that p. aeruginosa pao1 imports ent - cargo conjugates , and
we speculate that the lack of activity observed for this strain stems
from its inherent insensitivity to amp / amx .
b. cereus atcc 14579 was also insensitive to amp / amx ,
which only afforded growth inhibition at 100 m .
some growth
inhibition was observed for b. cereus treated with
10 m ent - amp / amx , which may result from iron sequestration
( figure s17 ) .
s. aureus atcc 25923 is susceptible to amp / amx , with complete growth inhibition
observed at 1 m . in this case , a 10-fold reduction in antibacterial
activity was observed for ent - amp / amx relative to unmodified amp / amx
( figure 6 ) . although the origins of this attenuation
are unclear , we speculate that ent - amp / amx may have trouble penetrating
the thick peptidoglycan of s. aureus .
an alternative
possibility is that recognition of ent - amp / amx by the s. aureus ent receptor diverts the -lactams from the pbps . in
total , the results from these assays indicate that ent - amp / amx
exhibit antibacterial activity enhancements that are species - selective ,
providing increased potency against e. coli strains
and not for the other strains evaluated in this work . we therefore
reasoned that ent - amp / amx , at low concentrations , should selectively
kill e. coli in the presence of other less sensitive
species .
we treated co - cultures of e. coli cft073
and s. aureus with ent - amp / amx or amp / amx and analyzed
the species composition following a 19-h incubation using harty - uti
plates .
these agar plates are employed in medical microbiology laboratories
for the diagnosis of urinary tract infections and provide species
identification by the colony color . when grown on harty - uti plates ,
s. aureus are off - white and e. coli are
purple - pink . in figure 6 , we present representative
images of the colonies that resulted from treating co - cultures of e. coli and s. aureus with amp / amx or ent - amp / amx .
in the absence of antibiotic ,
the cultures provide a mixture of off - white
and purple - pink colonies , indicating that both e. coli and s. aureus grow when cultured together .
when
the co - cultures are treated with 1 m amp / amx , only purple - pink
colonies are present , which reveals that only e. coli survives .
in contrast , treatment of the co - cultures with 1 m
ent - amp / amx results in only off - white colonies from s. aureus .
these comparisons demonstrate that ent - amp / amx selectively kill e. coli in the presence of s. aureus and
that the siderophore modification reverses the inherent species
selectivity of the parent antibiotics .
achieving such species - selective
and single - pathogen antibiotic targeting is an important goal and
unmet need for pharmaceutical development that will allow for treating
disease with minimal perturbation to the commensal microbiota .
ent - amp / amx
selectively kill e. coli cft073 in
the presence of s. aureus atcc 25923 .
( a , b ) antimicrobial
activity assays against s. aureus atcc 25923 in the
absence ( a ) and presence ( b ) of 200 m dp .
( c , d ) bacterial growth
monitored by od600 for cultures of e. coli only , s. aureus only , and 1:1 e. coli / s .
aureus mixtures treated with amp / amx or ent - amp / amx
in the absence ( c ) and presence ( d ) of 200 m dp .
( e ) representative photographs of colonies
from mixed cultures of e. coli cft073 and s. aureus atcc 29523 treated with ent - amp / amx ( 1 m )
or amp / amx ( 1 m ) in the presence of 200 m dp .
all assays
were conducted in 50% mhb medium ( t = 19 h , 30 c )
( mean sem , n 3 for a d ) .
we evaluated the cytotoxicity of ent - amp
against the human t84 colon
epithelial cell line .
cell survival was evaluated by mtt assay after
a 24 h treatment with apo or iron - bound ent - amp , amp , or ent .
the
iron - bound forms were assayed to determine whether iron chelation
in the growth medium is a factor .
no cytoxicity was observed for amp
or ent - amp , whereas apo ent itself decreased the survival of t84 cells
by approximately 30% at the highest concentration evaluated . when
pre - loaded with fe(iii ) ,
percent cell survival quantified by mtt assay after
a 24 h treatment with apo or iron - bound ent , ent - amp , and the parent
antibiotic amp in the absence and presence of 1 equiv of fe(iii ) ( mean
sem , n = 3 ) .
lipocalin-2
( lcn2 , also known as siderocalin or ngal ) is a 22-kda protein produced
and released by neutrophils and epithelial cells .
it has a hydrophobic
binding pocket and coordinates ferric ent with sub - nanomolar affinity . by sequestering ferric ent
, this host - defense protein contributes
to the metal - withholding response and prevents bacterial acquisition
of this essential nutrient . to determine whether lcn2 also binds ent - amp / amx
and thereby blocks antibacterial activity ,
we performed antibacterial
activity assays with e. coli cft073 in m9 minimal
medium supplemented with lcn2 or bovine serum albumin ( bsa ) . under
these conditions , up to 1 m of lcn2 had no effect on the growth
of e. coli cft073 . addition of 1 m lcn2 to
the medium rescued the growth of e. coli cft073 treated
with 100 nm of ent - amp ( figure 8) whereas addition
of 1 m bsa had no effect on ent - amp cell killing .
these results
suggest that lcn2 binds ent - amp and blocks its recognition and uptake .
to ascertain whether lcn2 binds ent - amp in the presence of exogenous
ent , we performed a series of experiments where e. coli cft073 were treated with fixed concentrations of ent - amp ( 100 nm )
and lcn2 ( 1 m ) and the concentration of ent was varied ( 0 ,
0.5 , and 1 m ) .
moreover , when
ent - amp was combined with a 1:1 molar ratio of ent and lcn2 at 10-fold
excess over the conjugate , no e. coli growth was
observed , which suggests that lcn2 preferentially binds ent .
prior
work demonstrated that lcn2 can not bind glucosylated ent , which was
attributed to a steric clash between the glucose moieties and the
ent binding site of the protein , and
decreased hydrophobicity of the siderophore may also be a factor .
thus , our data suggest that the nature of linker attachment at c5
and the peg3 moiety of ent - amp / amx do no abrogate lcn2
binding as effectively as the glucose moieties exhibited by the salmochelins
. antibacterial
activity of ent - amp against e. coli cft073 in the
presence of lcn2 or bsa .
( a ) e. coli cft073 treated
with 100 nm ent - amp and varying concentrations of
lcn2 or bsa control .
( b ) e. coli cft073 treated with
ent - amp , varying concentration of ent , and varying concentrations
of lcn2 or bsa control .
the assays were performed in m9 minimal medium
( 24 h , 37 c ) ( mean sem , n 3 ) .
ent - amp / amx are two siderophore-lactam
conjugates
based on the native enterobactin scaffold .
these molecules hijack
siderophore - based iron uptake pathways and provide potent antibacterial
activity against various e. coli strains , including
human pathogens .
our investigations of ent - amp / amx establish the following :
( i ) ent - amp / amx provide up to 1000-fold enhanced antibacterial activity
against e. coli strains ; ( ii ) ent - amp / amx are transported
into e. coli by fepa and potentially other catecholate
siderophore receptors ( iron , iha ) employed by pathogenic ctf073
and uti98 ; ( iii ) ent - amp / amx are captured by pbps in the periplasm ,
which results in pbp inhibition and cell death ; ( iv ) selective killing
of e. coli in the presence of less susceptible organisms
such as s. aureus is achieved because of the enterobactin
scaffold ; ( v ) ent - amp / amx exhibit negligible cytotoxicity to human
t84 intestinal epithelial cells ; and ( vi ) although lcn2 has the ability
to bind ent - amp / amx , this siderophore - scavenging protein prefers
to capture native ent . in total
, these studies demonstrate that modification
of antibiotic cargo with the native enterobactin platform provides
many desirable features for antibiotic delivery and efficacy .
the
large molecular weight of the conjugates resulting from the native
ent scaffold ( as opposed to a smaller mimic ) enhances rather than
diminishes uptake for gram - negative e. coli .
moreover ,
we observed no evidence for the development of resistance to ent - amp / amx
over the course of the antibacterial activity assays performed during
these investigations .
our studies confirm that the enhanced
antibacterial activity observed
for ent - amp / amx requires both enterobactin recognition by outer membrane
receptors and an intact -lactam moiety .
the results are reminiscent of the dramatic
antibacterial activity enhancements observed for albomycin , a secondary
metabolite produced by actinomyces subtropicus .
albomycin
is comprised of the siderophore ferrichrome and a trna synthetase
inhibitor , and it exhibits antimicrobial activities that are 30,000-fold
greater than those of the unmodified trna synthetase inhibitor against e. coli and s. aureus . nonetheless , the ent - amp / amx cell - killing mechanism may
be more complex than only more efficient -lactam delivery across
the gram - negative outer membrane .
binding of ent - amp / amx to the pbps
presumably results in accumulation of ferric enterobactin in the e. coli periplasm for some period of time , which may have
deleterious consequences .
a recent study of an e. colitolc- mutant revealed that enterobactin accumulation
in the periplasm affords growth defects and abnormal cellular morphologies .
a fascinating observation that stems
from our current work is the
variable susceptibilities and responses of different e. coli strains to ent - amp / amx , which contrast the effects of unmodified
amp / amx .
such differences are manifest in the mic values to some degree
and time - kill kinetics ; however , the results presented in figure 2 indicate that mic values alone do not provide a
full description of how ent - amp / amx susceptibility differs between e. coli strains .
these results suggest underlying complexity
in microbial physiology related to iron - uptake pathways that can not
be fully explained by the presence or absence of a gene for a particular
receptor ( i.e. , fepa , iron ) .
the heightened sensitivity of uropathogenic e. coli cft073 is particularly noteworthy , and it will be
interesting to decipher the physiological origins of this effect as
well as the differential behavior of various e. coli pathogens toward ent - antibiotic conjugates . there is a clear
and unmet need for new antibacterial agents to
treat bacterial infections in humans , including antibiotics that target
specific bacterial sub - populations . preventing undesirable consequences of antibiotic treatment on the
commensal microbiota , which contributes to human health in beneficial
ways , is a challenge that needs to be addressed .
such targeted therapeutics will be valuable not only for treating
bacterial infections when the causative agent is known ( e.g. , urinary
tract infection and e. coli , cystic fibrosis lung
infection and p. aeruginosa ) but also for other pathologies
that involve microbial dysbiosis , such as irritable bowel disease .
our studies of ent - amp / amx provide one step
toward addressing species - specific antibiotic targeting as well as
overcoming gram - negative outer membrane permeability . from the standpoint
of the host environment , commensal e. coli employ
ent for acquiring iron in the host , and thus further elaboration of
this strategy to specifically target pathogenicity and evade host
responses ( e.g. , lcn2 ) is desirable .
|
the design , synthesis , and characterization
of enterobactin
antibiotic
conjugates , hereafter ent - amp / amx , where the -lactam antibiotics
ampicillin ( amp ) and amoxicillin ( amx ) are linked to a monofunctionalized
enterobactin scaffold via a stable poly(ethylene glycol ) linker are
reported . under conditions of iron limitation ,
these siderophore - modified
antibiotics provide enhanced antibacterial activity against escherichia coli strains , including uropathogenic e. coli cft073 and uti89 , enterohemorrhagic e. coli o157:h7 , and enterotoxigenic e. coli o78:h11 , compared to the parent -lactams .
studies with e. coli k-12 derivatives defective in ferric enterobactin
transport reveal that the enhanced antibacterial activity observed
for this strain requires the outer membrane ferric enterobactin transporter
fepa . a remarkable 1000-fold decrease in minimum inhibitory concentration
( mic ) value is observed for uropathogenic e. coli cft073 relative to amp / amx , and time - kill kinetic studies demonstrate
that ent - amp / amx kill this strain more rapidly at 10-fold lower concentrations
than the parent antibiotics .
moreover , ent - amp and ent - amx selectively
kill e. coli cft073 co - cultured with other bacterial
species such as staphylococcus aureus , and ent - amp
exhibits low cytotoxicity against human t84 intestinal cells in both
the apo and iron - bound forms .
these studies demonstrate that the native
enterobactin platform provides a means to effectively deliver antibacterial
cargo across the outer membrane permeability barrier of gram - negative
pathogens utilizing enterobactin for iron acquisition .
|
Introduction
Experimental Section
Results and Discussion
Summary and Perspectives
|
retinal changes associated with the development of age - related macular degeneration ( amd ) have become even more important than once described .
even prompt therapy of the wet form of amd with antivascular , endothelial , growth factor drugs has been shown to ameliorate vision loss .
if diagnosis is established earlier during the dry form of amd , there is a better chance that visual acuity can be maintained .
rheohemapheresis ( formally known as rheopheresis ) has been investigated over the past decade as a possible method of positively affecting amd in its dry - form stage .
rheohemapheresis is a method of double plasma filtration performed in order to eliminate high - molecular - weight substances especially proteins such as fibrinogen , 2-macroglobulin , immunoglobulin m ( igm ) , thrombomodulin , and low - density ( ldl ) cholesterol [ 36 ] .
this method leads to the improvement of rheological parameters ( reduction of plasma and whole blood viscosity ) , as well as the improvement of erythrocyte aggregation and their flexibility [ 6 , 7 ] .
it can also lead to a significant improvement of blood flow in the choroid , which is reduced in patients with amd .
when treating our patients suffering from the dry form of amd accompanied by reticular drusen or even drusenoid detachment of the retinal pigment epithelium , we noticed positive morphological changes of the retina after rheohemapheresis treatment , including the preservation of the photoreceptor inner / outer segment ( is / os ) junction , as well as stabilisation or even improvement of retinal function : best - corrected visual acuity ( bcva ) and electroretinography results . a description of the above changes and their correlation is the subject of this work .
at our department , we have seven years of experience in treating amd using rheohemapheresis .
so far , 61 patients with the dry form of amd , soft drusen , confluent soft drusen , and drusenoid pigment epithelium detachment ( dped ) have been treated .
we perform long - term monitoring of morphological and functional changes in the retina [ 12 , 13 ] .
the interrelationship of morphological changes in the photoreceptor inner and outer segment ( is / os ) junction retinal layer as well as changes in visual acuity and retinal function ( electroretinography ) was evaluated in the group of 24 patients ( 40 eyes ) who were long - term followed ( for 2.5 years or longer ) .
all patients had the dry form of amd with the presence of drusenoid pigment epithelium detachment .
twelve of these patients ( 22 eyes ) with an average age of 64.3 years ( range : 6483 years ) were treated with rhf .
twelve controls ( 18 eyes ) with an average age of 65.6 years ( range : 6483 years ) were randomized .
ophthalmologic inclusion criteria were high - risk , preangiogenic form of amd with soft drusen , reticular drusen , confluent soft drusen , and dped in accordance with the eureye study and ability to complete the series of 8 rheohemapheresis procedures within 10 weeks .
exclusion criteria were any retinal or choroidal disorders other than amd , optic nerve disorders , glaucoma , conditions limiting the examination of the fundus , and acute bleeding in the studied eye .
general exclusion criteria for rheohemapheresis treatment were the usual exclusion criteria of extracorporeal circulation or therapeutic hemapheresis and the absence of peripheral veins suitable for establishing an extracorporeal circuit .
rheohemapheresis was performed in the treatment group , as described in detail in our previous work [ 3 , 13 ] .
after plasma separation ( blood - cell separator , cobe spectra or optia , terumo , lakewood , co , usa ) , the separated plasma was pumped through the rheofilter ( evaflux , kawasumi , tokyo , japan ) to remove high - molecular - weight factors . using this filter , we repeatedly ( 8 times over the period of 10 weeks ) removed a precisely defined spectrum of high - molecular - weight substances , such as fibrinogen , ldl cholesterol , and 2-macroglobulin , from the blood of the patient , thus reducing blood viscosity in an attempt to improve the perfusion of the retina and the choroid .
fundus photography and fluorescein angiography were performed using a digital fundus camera ( zeiss ff 450 , jena , germany ) .
the dped area was measured in mm by fundus photography using visupac software ( zeiss meditec ag , jena , germany ) , which is acceptable for measuring the area of retinal affections .
spectral domain- ( sd- ) oct ( cirrus hd - oct , zeiss meditec , jena , germany ) with an axial resolution of 6 m was used to evaluate central retinal thickness .
thickness in the central point of the 1 mm fixation zone was evaluated using 6 radial scans .
sd - oct enabled us to distinguish between dped and the vascular type of retinal pigment epithelium detachment . a detailed image of the photoreceptor is / os junction was obtained using a 5-line raster scan , which precisely shows the shape , coherence , and defects of the photoreceptor is / os junction as well as the detachment of this layer from the retinal pigment epithelium ( rpe )
multifocal erg ( mferg ; reti - port plus mferg system , roland consult gmbh , brandenburg , germany ) was performed according to the standards of the international society for clinical electroretinography and vision ( iscev ) [ 16 , 17 ] .
mferg traces were recorded from the central 60 of the retina using a resolution of 61-scaled hexagons .
we evaluated the amplitudes of the positive peak component from the first - order kernel analysis of the central element , representing the foveal response ( 01.8 ) of the four rings centered on the fovea : 1st ring ( 1.87.0 ) , 2nd ring ( 513 ) , 3rd ring ( 1122 ) , and 4th ring ( 1730 ) .
all examinations were performed at baseline and at 2.5-year follow - up . for statistical analysis
, we used nonparametric tests ( the kruskal - wallis test , mann - whitney u test , and chi - square approximation ) .
the study protocol was approved by the institutional ethics committee and the reported investigations were in accordance with the principles of the current version of the helsinki declaration .
median best - corrected visual acuity ( bcva ) before rheohemapheresis in the treatment group was 74.0 letters ( 56.2 to 81.3 letters ; 95% ci ) and increased to 79.0 letters ( 57.3 to 83.4 letters ; 95% ci ) , ( p = 0.187 ) after 2.5 years . in the control group ,
mean bcva decreased from 74.0 letters ( 25.2 to 82.6 letters ; 95% ci ) at baseline to 72.5 letters ( 23.4 to 83.1 letters ) after 2.5 years ( p = 0.041 ) .
while the difference in bcva between the treatment groups was insignificant at baseline ( p = 0.457 ) , bcva was significantly higher in the treatment group after 2.5 years ( p = 0.021 ) . at baseline , mean dped was 3.68 4.45 mm in treated patients and 4.12 6.64 mm in controls , reaching the central fovea in all cases .
after 2.5 years , the mean dped area decreased significantly to 0.71 1.27 mm in the rheohemapheresis group ( p < 0.001 ) , whereas it increased significantly to 9.19 9.51 mm in the controls ( p < 0.01 ) .
the differences in size of the dped area were only insignificant at baseline ( p = 0.605 ) and significant after 2.5 years ( p < 0.001 ) .
reduction of the size of the dped area after rheohemapheresis was found in 19/22 rheohemapheresis - treated eyes ( 86.4% ) , as opposed to only 3/18 eyes ( 16.7% ) in the control group .
enlargement of the dped area occurred as part of the natural progress of amd over the same period of 2.5 years in 15/18 ( 83.3% ) eyes in the control group , compared to only 3/22 eyes ( 13.6% ) of treated patients .
the study and control groups were comparable.in the group of treated patients , the photoreceptor is / os junction was attached to the dped in 6/22 ( 27.3% ) eyes .
( dped was accompanied by a detachment of the photoreceptor is / os junction in the remaining 16/22 eyes , i.e. , 72.7% , without defect in 15/22 eyes and with defect in 1 eye).in the control group , the photoreceptor is / os junction was attached to the dped in 6/18 ( 33.3% ) ( dped was accompanied by a detachment of the photoreceptor is / os junction in the remaining 12/18 eyes ( 66.6% ) , without defect in 4 eyes and with defect in 8 eyes ) . in the group of treated patients , the photoreceptor is / os junction was attached to the dped in 6/22 ( 27.3% ) eyes .
( dped was accompanied by a detachment of the photoreceptor is / os junction in the remaining 16/22 eyes , i.e. , 72.7% , without defect in 15/22 eyes and with defect in 1 eye ) . in the control group ,
the photoreceptor is / os junction was attached to the dped in 6/18 ( 33.3% ) ( dped was accompanied by a detachment of the photoreceptor is / os junction in the remaining 12/18 eyes ( 66.6% ) , without defect in 4 eyes and with defect in 8 eyes ) .
( 1 ) the group of patients . in the group of treated patients , where the photoreceptor is / os junction was intact in 6/22 ( 27.3% ) eyes , it remained without defect even after 2.5 years .
however , in 7 cases of the attached is / os junction , there was a certain residual defect .
in one additional eye with the is / os junction at baseline , the defect of this layer significantly improved after attachment .
overall , is / os junction defects were demonstrated in 8/22 eyes ( 31.8% ) .
defects reached the central foveola in 4 eyes ( 8.8% ) , thus negatively affecting photoreceptor function and vision .
integrity of the is / os junction layer was preserved in only 3/18 eyes ( 16.7% ) after 2.5 years and is / os junction defects were diagnosed in 15/18 eyes ( 83.3% ) , 12 of which reached the foveolar region and adversely affected vision .
it is important to note that none of the treated patients progressed into the wet form during the 2.5-year follow - up . in the control group ,
6 eyes with detachment of the is / os junction at baseline developed choroidal neovascularization ( cnv ) ( confirmed by fluorescein angiography ) .
the amplitudes of foveal responses and responses in the most peripheral areas of the retina , as shown on the multifocal erg , changed only slightly during the entire follow - up period in both groups of patients . in the parafoveal and paramacular regions of eccentricity between 1.8 and 13
the differences were statistically nonsignificant between groups of patients in both periods of examination , except for the activity in the region of eccentricity between 1.8 and 7 , which was significantly higher in treated patients ( p = 0.04 ) at 2.5-year follow - up .
the implicit times of the majority of responses increased after 2.5 years in all patients . at baseline , they were significantly longer in the controls ( p values ranging from < 0.05 to < 0.01 ) , with the exception of the foveal response . at 2.5 years , the foveal response was significantly longer in the control group ( p = 0.035 ) .
in general , retinal activity remained stable or even improved in treated patients with early decrease or complete disappearance of dped and detachment of the photoreceptor is / os junction , along with preservation of its integrity or development of only small defects in the parafoveal region ( figures 2(a ) , 2(b ) , 2(c ) , 3(b ) , and 3(c ) ) .
in contrast , in patients with long - lasting or persistent dped with detachment of the is / os junction and development of its defects or even development of cnv , retinal activity was even reduced .
the photoreceptor is / os junction layer is currently receiving attention from many researchers [ 10 , 1822 ] . according to baba et al .
, the state of this thin hyper - reflective layer , which lies above the rpe layer , directly correlates with bcva after successful macular hole repair .
bcva deterioration has proved to be an early indicator of transformation to the wet form of amd .
we have verified the direct correlation of the state of the is / os photoreceptor junction and bcva on patients with stargardt disease .
disruption of the is / os junction is associated with poor vision in uveitic macular edema and retinitis pigmentosa [ 22 , 23 ] . as a result of our findings
, we can add the is / os junction as another indicator of the risk of transformation to the wet form of amd .
the detachment of the photoreceptor is / os junction ( figure 1 ) is usually located at the top of the area affected by drusenoid retinal pigment epithelium detachment ( dped ) .
sikorski et al . found that rupture of is / os photoreceptor junction detachment is directly associated with the emergence of submacular neovascularization .
schuman et al . reported thinning of the is / os photoreceptor layer and the progress of its changes over drusen and dped . in the present study
, we found reattachment of the photoreceptor is / os junction layer in the study group .
after the 2.5-year follow - up , 8 eyes were reattached without is / os junction defect after rhf ; the is / os junction defect was partially reattached in another 8 of the treated eyes . however , in only 4 of these eyes ( 18.1 % of the entire sample ) , the defect reached the central fovea . the fact that , after rheohemapheresis , patients only rarely developed the photoreceptor is / os junction defect affecting the central foveola and that mean visual acuity
actually slightly improved in the majority of these patients is considered an obvious interdependence . in control patients , we did not observe full restoration ( reattachment ) of a photoreceptor is / os junction layer when detached at the baseline .
drusenoid retinal pigment epithelium detachment usually progresses ( which was observed in 7 eyes ) ; otherwise , rupture occurs caused by submacular neovascularization development , as has also been observed by other authors [ 24 , 25 ] .
the photoreceptor is / os junction layer rupture is usually apparent in patients with neovascularization .
the remaining fluid as well as the fluid under the detached drusenoid retinal pigment epithelium then spreads into the inner retinal layers and can cause macular edema . in the control group , we observed 6 cases ( 33.3% ) of submacular choroidal neovascularization .
the following is one possible mechanism of its development : the existing dped was later accompanied by a detached layer adjoined to it from above ( i.e. , by detachment of the photoreceptor is / os junction ) .
dped and is / os layer detachment gradually grew in size and eventually ruptured due to the development of submacular choroidal neovascularization .
as evidence of progression to the wet form of amd , fluid inside and underneath the inner retinal layer appeared on sd - oct .
these findings are supported by the findings of occult submacular choroidal neovascularization using fluorescein angiography .
patients with preservation of photoreceptor is / os junction integrity or development of only small , paracentral defects by early decrease or complete disappearance of the dped area also exhibited slightly increased retinal activity , which led to significantly higher parafoveal activity in treated patients using mferg ( see section 3.4 ) .
we found a significantly higher amplitude of parafoveal responses in eccentricity between 1.8 and 7 in treated patients 2.5 years after initiation of the treatment ( p = 0.04 ) .
in contrast , in patients with development of is / os junction defects or even progression of the disease to its wet form , central retinal activity and visual acuity were even reduced .
development of photoreceptor is / os junction defects in the control group probably contributed to the slight decrease of electrical retinal activity in the parafoveal and paramacular regions of eccentricity between 1.8 and 13 using mferg .
this may be due to the long - term accumulation of fluid between the rpe and is / os junctions , which causes stretching of photoreceptor outer segments , thus leading to their malfunction even without the development of associated rpe atrophy .
the question of treating the dry form of amd by rheohemapheresis has not yet been fully answered .
case series , two controlled trials and five completed randomized controlled trials , have reported the efficacy of rheohemapheresis in treating dry amd .
these studies ( inclusive of our two [ 3 , 26 ] ) have shown an improvement in the number of lines that can be read on etdrs charts , improvement in the pepper visual skills for reading tests , a decrease in viscosity parameters , shortening of arteriovenous passage times , and improvement in electroretinograms .
the studies have shown improvements shortly after completion of treatment , lasting up to four years [ 6 , 18 ] .
considerable confusion has been aroused by the preliminary results from the mira 1 study an extensive , sham - controlled , randomized , multicenter trial conducted in the usa .
its encouraging first results were later challenged and a clear positive effect was not proved [ 27 , 28 ] .
however , analysis revealed that 37% of treated patients and 29% of control patients were protocol violators who did not fulfill the trial 's inclusion criteria of amd leading to bias in the study 's final outcome . excluding those subjects who had vision loss due to other causes
, this trial demonstrated significant improvement with treatment but the trial was underpowered by us fda licensure .
two hundred and seventy - nine patients with dry amd were treated and compared to 55 untreated controls . in the treated group , visual acuity gain greater than or equal to one etdrs line
vision loss greater than or equal to one etdrs line was seen in 17% of the treated patients versus 40% of controls .
the asfa ( american society for apheresis ) issues directives ( guidelines ) for individual procedures in various diseases ; according to the latest criteria from 2013 , rheohemapheresis is newly classified as a category ib treatment ( first line therapy ) .
the second limitation of this study is obviously the small number of patients , despite the relatively long follow - up .
the clinical impression of the importance of the is / os layer was established at the beginning of our research 7 years ago , but only patients with follow - up times longer than 2.5 years , that is , in the case of 40 eyes ( 22 eyes in the study group and 18 control eyes ) , were randomized for assessment of morphological and functional changes to the is / os layer .
our results will need to be confirmed on a larger number of patients in the future .
with the use of rheohemapheresis , preservation of photoreceptor is / os junction layer coherence in the fovea of patients with high - risk dry amd can be achieved , even when drusenoid retinal pigment epithelium detachment is already present .
after rheohemapheresis , there was a significant morphological improvement of the damaged is / os layer , as evidenced by reduction of the scope of the defect or reattachment of the detached is / os layer either with or even without the defect of this layer . after 2.5 years of follow - up , better visual acuity , reduced dped size , and improvement of some functional parameters ( electroretinography findings ) were observed in the study group .
|
aim . to evaluate the long - term effect of rheohemapheresis ( rhf ) treatment of age - related macular degeneration ( amd ) on photoreceptor is / os junction status
. methods . in our study
, we followed 24 patients with dry amd and drusenoid retinal pigment epithelium detachment ( dped ) for a period of more than 2.5 years .
twelve patients ( 22 eyes ) were treated by rhf and 12 controls ( 18 eyes ) were randomized .
the treated group underwent 8 rhf standardized procedures .
we evaluated best - corrected visual acuity , is / os junction status ( sd oct ) , and macular function ( multifocal electroretinography ) at baseline and at 2.5-year follow - up . results .
rhf caused a decrease of whole - blood viscosity / plasma viscosity at about 15/12% .
bcva of treated patients increased insignificantly ( p = 0.187 ) from median 74.0 letters ( 56.2 to 81.3 letters ) to median 79.0 letters ( 57.3 to 83.4 letters ) , but it decreased significantly from 74.0 letters ( 25.2 to 82.6 letters ) to 72.5 letters ( 23.4 to 83.1 letters ) in the control group ( p = 0.041 ) .
the mferg responses in the region of eccentricity between 1.8 and 7 were significantly higher in treated patients ( p = 0.04 ) .
conclusions .
rhf contributed to sparing of photoreceptor is / os junction integrity in the fovea , which is assumed to be a predictive factor for preservation of visual acuity .
|
1. Introduction
2. Methods and Subjects of the Study
3. Results
4. Discussion
5. Conclusion
|
congenital myasthenic syndromes ( cmss ) are a group of rare genetic disorders affecting neuromuscular junction transmission .
the subtype of cms depends on whether the defect is presynaptic , synaptic , or postsynaptic . in the past 33 years , at least 23 genes encoding proteins of the neuromuscular junction have been identified containing causative mutations .
cmss are clinically heterogeneous and characterized by fatigable weakness of skeletal muscle that occurs between infancy and adulthood .
ptosis and extraocular muscle , facial , bulbar , and generalized weakness are the common presentations .
subtypes exist with onset later in childhood that exhibit morbid muscle fatigability with difficulty in running or climbing stairs .
clinical manifestation , severity , and course of cms vary , even between patients from the same family .
although myasthenic symptoms may be mild , respiratory insufficiency may occur in patients with cms if respiratory muscles are severely affected .
it has been reported that 75% of cms cases are postsynaptic subtype , and a genetic deficiency of acetylcholine receptor ( achr ) tends to be the most frequent etiological basis .
cmss that show decreased synaptic response to acetylcholine are referred to as fast channel ; conversely , those with an increased acetylcholine response are referred to as slow channel . in patients with slow - channel cms ( sccms ) , electrophysiological examination has revealed that prolonged opening activity of the achr channel causes depolarization block , which contributes to muscle weakness and fatigability .
progressive spinal deformities or scoliosis result from paraspinal muscle weakness and are usually found in adult sccms patients .
patients with cms have been reported worldwide , including in east asia , but there have been only a few published cases of cms in china .
herein , we identified three patients with sccms from a chinese family who presented with muscle weakness during early or late childhood . in these patients
, we found autosomal dominant inheritance of a heterozygous mutation in acetylcholine receptor epsilon - subunit ( chrne ) gene .
the proband was referred to the department of neurology , the first affiliated hospital of chongqing medical university in march 2014 .
detailed medical history was obtained from each family member , and the affected family members underwent laboratory tests and physical and electrophysiological examination , including repetitive nerve stimulation ( rns ) and nerve conduction velocity studies . for low - frequency stimuli , decrements were compared between 1 and the 4 stimuli . for high - frequency stimuli ,
laboratory tests on the proband consisted of assaying for blood biochemicals , endocrine hormones , antibodies against nuclear antigens , and antibodies against achr .
patients included in this study provided written informed consent , and the study was approved by medical ethics committee of the first affiliated hospital of chongqing medical university .
genomic dna was extracted from peripheral leukocytes of fresh blood samples from the proband ( iii-3 ) , elder sister of proband ( iii-2 ) , and mother of proband ( ii-2 ) , using standard methods of proteinase k digestion and phenol - chloroform extraction .
we used next - generation gene sequencing to screen the proband dna for genes associated with neuromuscular diseases , including genes that have been reported to encode factors involved in myasthenic syndromes .
the identity of the mutated chrne gene detected by this screen was verified by sanger sequencing .
all exons of the chrne gene were amplified by polymerase chain reaction ( pcr ) using the geneamp pcr system 9700 thermal cycler ( perkin elmer , shelton , ct , usa ) .
the promoter region and the entire coding sequence of the chrne gene ( genbank accession number af105999/gi4580858 ) were determined as described .
each 25 l pcr reaction contained 50 ng genomic dna , 10 pmol of each forward and reverse primers , 5 mmol dntps , and 2.5 u of taq polymerase in taq reaction buffer ( takara biotechnology , dalian , china ) .
thermal cycling consisted of a denaturation step at 94c for 5 min , and then 35 cycles at 94c for 3040 s , 5763c for 30 s , and 72c for 30 s , followed by a final elongation step at 72c for 10 min .
the pcr products were purified by 1.5% agarose gel electrophoresis and directly sequenced with an abi prism 3730xl dna analyzer ( applied biosystems , foster city , ca , usa ) .
the data were analyzed with chromas 2.22 chromatogram file editor software ( technelysium pty ltd .
the base mutations in chrne gene were described and numbered according to criteria provided by the ensembl genome browser ( http://www.ensembl.org ) .
the proband was referred to the department of neurology , the first affiliated hospital of chongqing medical university in march 2014 .
detailed medical history was obtained from each family member , and the affected family members underwent laboratory tests and physical and electrophysiological examination , including repetitive nerve stimulation ( rns ) and nerve conduction velocity studies . for low - frequency stimuli , decrements were compared between 1 and the 4 stimuli . for high - frequency stimuli ,
laboratory tests on the proband consisted of assaying for blood biochemicals , endocrine hormones , antibodies against nuclear antigens , and antibodies against achr .
patients included in this study provided written informed consent , and the study was approved by medical ethics committee of the first affiliated hospital of chongqing medical university .
genomic dna was extracted from peripheral leukocytes of fresh blood samples from the proband ( iii-3 ) , elder sister of proband ( iii-2 ) , and mother of proband ( ii-2 ) , using standard methods of proteinase k digestion and phenol - chloroform extraction .
we used next - generation gene sequencing to screen the proband dna for genes associated with neuromuscular diseases , including genes that have been reported to encode factors involved in myasthenic syndromes .
the identity of the mutated chrne gene detected by this screen was verified by sanger sequencing .
all exons of the chrne gene were amplified by polymerase chain reaction ( pcr ) using the geneamp pcr system 9700 thermal cycler ( perkin elmer , shelton , ct , usa ) .
the promoter region and the entire coding sequence of the chrne gene ( genbank accession number af105999/gi4580858 ) were determined as described .
each 25 l pcr reaction contained 50 ng genomic dna , 10 pmol of each forward and reverse primers , 5 mmol dntps , and 2.5 u of taq polymerase in taq reaction buffer ( takara biotechnology , dalian , china ) .
thermal cycling consisted of a denaturation step at 94c for 5 min , and then 35 cycles at 94c for 3040 s , 5763c for 30 s , and 72c for 30 s , followed by a final elongation step at 72c for 10 min .
the pcr products were purified by 1.5% agarose gel electrophoresis and directly sequenced with an abi prism 3730xl dna analyzer ( applied biosystems , foster city , ca , usa ) .
the data were analyzed with chromas 2.22 chromatogram file editor software ( technelysium pty ltd .
the base mutations in chrne gene were described and numbered according to criteria provided by the ensembl genome browser ( http://www.ensembl.org ) .
the proband ( iii-3 ) , 21-year - old man , had generalized muscle weakness at 4 years of age .
his ability to run and jump was lower compared with his same - age peers . he had experienced fatigue while walking since 4 years old .
symptoms of weakness fluctuated in severity , and exacerbated transiently by physical activity , but slightly relieved during rest breaks .
the symptoms progressed gradually , and the upper limbs became affected at the age of 9 .
symptoms exacerbated several times each year , with each episode lasting a few days to a month , especially during winter .
the symptoms gradually progressed in the last 12 months and he experienced difficulty in climbing stairs ; however , he did not experience diplopia , dysphagia , respiratory insufficiency , or muscle twitching .
bilateral facial weakness was observed , and the bulbar muscles were slightly involved , manifesting as nasal speech ; however , swallowing difficulties were not present , and chewing problems were not marked .
examination showed mild weakness of the neck flexor muscles , bilateral muscles of the tibialis anterior and gastrocnemius , and finger extensor muscles .
the mother of the proband ( ii-2 ) , a 47-year - old woman , had difficulties in grasping and finger extension at 10 years of age . at age 12 , she presented marked muscular weakness and fatigability , especially in the lower limbs .
interestingly , symptoms stopped progressing and she began to recover gradually , though not to normal levels , but she was now able to complete daily housework and perform mildly taxing farm work .
she had bilaterally limited eye movements without ptosis , symmetric bilateral facial weakness , and mild weakness of neck flexor and bilateral muscles of the limbs ( grade 4/5 on the medical research council scale for the proximal muscles and 4/5 for the distal muscles of the limbs ) .
the affected elder sister ( iii-2 ) was 24 years old and had symptoms similar to the proband .
she began to have difficulty in walking and extending fingers at 11 years of age .
facial weakness , muscle wasting in bilateral forearm and interosseous muscles , and bilateral ophthalmoplegia without ptosis were also found .
muscle strength on the medical research council scale was 3/5 for the neck flexor muscles and 4/5 for the proximal muscles .
she was neither able to extend her fingers nor able to walk using either the toe or heel .
the daughter of the elder sister ( iv-1 ) , a 4-year - old girl , however , had no symptoms or complaints .
the laboratory tests for proband showed that the levels of creatine kinase and thyroid and sex hormones were normal , and antibodies to achr were not present .
a neostigmine test showed no recovery for muscle weakness . with the proband 's consent , we prescribed fluoxetine at a dosage of 20 mg / d as treatment .
after no improvement was observed in his condition for 2 weeks , we increased the dosage to 40 mg daily ; 10 days later , fatigue and weakness were mildly improved .
when the elder sister underwent the same fluoxetine treatment , symptoms also were relieved ; however , after 6 months of therapy , neurological examination showed no changes for either patient .
motor nerve conduction studies revealed a repetitive - compound muscle action potential ( r - cmap ) after a single stimulation in the median , ulnar , and peroneal nerves in affected patients from the pedigree [ figure 2a2c ] , which even included the asymptomatic 4-year - old girl although she did not complain of weakness or fatigue [ figure 2d ] .
however , compared with the recordings from the proband [ iii-3 ; figure 2a ] and his elder sister [ iii-2 ; figure 2b ] , the r - cmap of the peroneal nerve of their mother [ ii-2 ; figure 2c ] was more noticeably affected .
after fluoxetine therapy , r - cmap was still present in the proband ( iii-3 ) and his elder sister ( iii-2 ) .
( a ) iii-3 : two peaks , the second peak of the second wave overlaps the first peak of the first wave .
rns induced cmap decrements in several nerves of the affected patients in response to low- or high - frequency rns , as summarized in table 1 and shown in figure 3 .
for the proband 's mother ( ii-2 ) , no decrements were detected in response to low - frequency rns .
decrements of the proband ( iii-3 ) were more obvious than those of the other family members , despite similar degrees of illness .
decrements in response to high - frequency rns were more significant than those to low - frequency rns for all affected members except the 4-year - old girl ( iv-1 ) , who did not receive high - frequency rns to avoid inflicting unnecessary pain .
for the proband ( iii-3 ) , fluoxetine treatment partially restored the decrease in cmap for the facial and peroneal nerves in response to low - frequency rns [ figure 3a and 3e ] , peroneal nerve ] .
however , significant improvements in the decrements were not observed for any of the tested nerves for the elder sister ( iii-2 ) , especially in response to low - frequency rns [ figure 3b and 3f , ulnar nerve ] . for the 4-year - old girl ( iv-1 ; daughter of iii-2 ) , although she did not have symptoms at the time , we measured a decrease in cmap for the ulnar nerve [ figure 3d , ulnar nerve ] . in the elder sister ( iii-2 ) ,
a significant decrease in the median nerve response at higher frequency rns was not found [ figure 3 g ] ; however , a significant decrease in the ulnar nerve response at higher frequency rns was observed even after fluoxetine treatment [ figure 3h ] .
repetitive nerve stimulation tests in the affected patients of the pedigree ( % amplitude ) na : not available because ii-2 did not receive fluoxetine treatment ; nd : decrement was not detected ;
proband ( iii-3 ; e ) and his elder sister ( iii-2 ; f ) monitored for the effects of fluoxetine treatment at low frequency .
the proband 's mother ( ii-2 ; g ) and elder sister ( iii-2 ; h ) tested at higher 30-hz stimulation .
next - generation sequencing revealed a mutation in the chrne gene of the proband ( iii-3 ) .
proband 's dna was amplified and sequenced . a heterozygous missense mutation c.865c > t in exon 8 was found [ figure 4 ] , resulting in a substitution from leucine to phenylalanine at position 289 ( l289f ) .
the mother ( ii-2 ) and elder sister ( iii-2 ) [ figure 4 ] carried the same heterozygous mutation .
dna of the 4-year - old girl ( iv-1 ) was unavailable because her mother refused it .
the mutation c.865c > t appears to have been initially reported as c.805c > t , leading to the changed l269f ( i.e. , a different residue number ) .
this variant is located in chromosome 17 ( focus : 4804140 g > a ) . the ensembl transcript i d is enst00000293780 , leading to a cdna.865c > t change . according to the latest mrna sequence of genbank ( mrna sequence nm_000080 ) ,
these two residue positions are in fact identical , and the current accepted numbering is l289 ; accordingly , the mutation site has been updated as c.865c > t . in the human gene mutation database ( hgmd ) , this mutation c.865c > t has been reported as a known disease mutation ( hgmd i d cm960300 ) .
the heterozygous missense mutation ( arrows ) , c.865c > t , was identified in the proband ( iii-3 ) , his elder sister ( iii-2 ) , and his mother ( ii-2 ) .
the potential functional impacts of mutations within chrne gene were predicted using polymorphism phenotyping 2 ( polyphen-2 ) software ( http://geneics.bwh.harvard.edu/pph2/ ) , sorting intolerant from tolerant ( sift ) software ( http://sift.jcvi.org/ ) and mutationtaster ( http://www.mutationtaster.org/ ) .
the p. leu289phe substitution was predicted by the polyphen-2 software to be probably damaging and disruptive of the function of chrne .
mutationtaster and sift predicted that the p. leu289phe mutation is functionally disease causing and damaging , respectively .
with mutationtaster software , this mutation is predicted to result in splice site changes and that protein features might be affected .
it has been reported that the mutation can lead to an unusually high rate of spontaneous achr channel openings and a 9-fold increase in affinity for acetylcholine .
this mutation has been found to be absent in the 100 healthy controls ( 50% male ) .
the proband ( iii-3 ) , 21-year - old man , had generalized muscle weakness at 4 years of age .
his ability to run and jump was lower compared with his same - age peers . he had experienced fatigue while walking since 4 years old .
symptoms of weakness fluctuated in severity , and exacerbated transiently by physical activity , but slightly relieved during rest breaks .
the symptoms progressed gradually , and the upper limbs became affected at the age of 9 .
symptoms exacerbated several times each year , with each episode lasting a few days to a month , especially during winter .
the symptoms gradually progressed in the last 12 months and he experienced difficulty in climbing stairs ; however , he did not experience diplopia , dysphagia , respiratory insufficiency , or muscle twitching .
bilateral facial weakness was observed , and the bulbar muscles were slightly involved , manifesting as nasal speech ; however , swallowing difficulties were not present , and chewing problems were not marked .
examination showed mild weakness of the neck flexor muscles , bilateral muscles of the tibialis anterior and gastrocnemius , and finger extensor muscles .
the mother of the proband ( ii-2 ) , a 47-year - old woman , had difficulties in grasping and finger extension at 10 years of age . at age 12 , she presented marked muscular weakness and fatigability , especially in the lower limbs .
interestingly , symptoms stopped progressing and she began to recover gradually , though not to normal levels , but she was now able to complete daily housework and perform mildly taxing farm work .
she had bilaterally limited eye movements without ptosis , symmetric bilateral facial weakness , and mild weakness of neck flexor and bilateral muscles of the limbs ( grade 4/5 on the medical research council scale for the proximal muscles and 4/5 for the distal muscles of the limbs ) .
the affected elder sister ( iii-2 ) was 24 years old and had symptoms similar to the proband .
she began to have difficulty in walking and extending fingers at 11 years of age .
facial weakness , muscle wasting in bilateral forearm and interosseous muscles , and bilateral ophthalmoplegia without ptosis were also found .
muscle strength on the medical research council scale was 3/5 for the neck flexor muscles and 4/5 for the proximal muscles .
she was neither able to extend her fingers nor able to walk using either the toe or heel .
the daughter of the elder sister ( iv-1 ) , a 4-year - old girl , however , had no symptoms or complaints .
the laboratory tests for proband showed that the levels of creatine kinase and thyroid and sex hormones were normal , and antibodies to achr were not present .
a neostigmine test showed no recovery for muscle weakness . with the proband 's consent , we prescribed fluoxetine at a dosage of 20 mg / d as treatment .
after no improvement was observed in his condition for 2 weeks , we increased the dosage to 40 mg daily ; 10 days later , fatigue and weakness were mildly improved .
when the elder sister underwent the same fluoxetine treatment , symptoms also were relieved ; however , after 6 months of therapy , neurological examination showed no changes for either patient .
motor nerve conduction studies revealed a repetitive - compound muscle action potential ( r - cmap ) after a single stimulation in the median , ulnar , and peroneal nerves in affected patients from the pedigree [ figure 2a2c ] , which even included the asymptomatic 4-year - old girl although she did not complain of weakness or fatigue [ figure 2d ] .
however , compared with the recordings from the proband [ iii-3 ; figure 2a ] and his elder sister [ iii-2 ; figure 2b ] , the r - cmap of the peroneal nerve of their mother [ ii-2 ; figure 2c ] was more noticeably affected .
after fluoxetine therapy , r - cmap was still present in the proband ( iii-3 ) and his elder sister ( iii-2 ) .
( a ) iii-3 : two peaks , the second peak of the second wave overlaps the first peak of the first wave .
rns induced cmap decrements in several nerves of the affected patients in response to low- or high - frequency rns , as summarized in table 1 and shown in figure 3 .
for the proband 's mother ( ii-2 ) , no decrements were detected in response to low - frequency rns .
decrements of the proband ( iii-3 ) were more obvious than those of the other family members , despite similar degrees of illness .
decrements in response to high - frequency rns were more significant than those to low - frequency rns for all affected members except the 4-year - old girl ( iv-1 ) , who did not receive high - frequency rns to avoid inflicting unnecessary pain .
for the proband ( iii-3 ) , fluoxetine treatment partially restored the decrease in cmap for the facial and peroneal nerves in response to low - frequency rns [ figure 3a and 3e ] , peroneal nerve ] .
however , significant improvements in the decrements were not observed for any of the tested nerves for the elder sister ( iii-2 ) , especially in response to low - frequency rns [ figure 3b and 3f , ulnar nerve ] .
for the 4-year - old girl ( iv-1 ; daughter of iii-2 ) , although she did not have symptoms at the time , we measured a decrease in cmap for the ulnar nerve [ figure 3d , ulnar nerve ] . in the elder sister ( iii-2 ) ,
a significant decrease in the median nerve response at higher frequency rns was not found [ figure 3 g ] ; however , a significant decrease in the ulnar nerve response at higher frequency rns was observed even after fluoxetine treatment [ figure 3h ] .
repetitive nerve stimulation tests in the affected patients of the pedigree ( % amplitude ) na : not available because ii-2 did not receive fluoxetine treatment ; nd : decrement was not detected ; : signal not detected .
proband ( iii-3 ; e ) and his elder sister ( iii-2 ; f ) monitored for the effects of fluoxetine treatment at low frequency .
the proband 's mother ( ii-2 ; g ) and elder sister ( iii-2 ; h ) tested at higher 30-hz stimulation .
next - generation sequencing revealed a mutation in the chrne gene of the proband ( iii-3 ) .
a heterozygous missense mutation c.865c > t in exon 8 was found [ figure 4 ] , resulting in a substitution from leucine to phenylalanine at position 289 ( l289f ) .
the mother ( ii-2 ) and elder sister ( iii-2 ) [ figure 4 ] carried the same heterozygous mutation .
dna of the 4-year - old girl ( iv-1 ) was unavailable because her mother refused it .
the mutation c.865c > t appears to have been initially reported as c.805c > t , leading to the changed l269f ( i.e. , a different residue number ) .
this variant is located in chromosome 17 ( focus : 4804140 g > a ) .
the ensembl transcript i d is enst00000293780 , leading to a cdna.865c > t change . according to the latest mrna sequence of genbank ( mrna sequence nm_000080 ) , these two residue positions are in fact identical , and the current accepted numbering is l289 ; accordingly , the mutation site has been updated as c.865c > t . in the human gene mutation database ( hgmd ) , this mutation c.865c > t has been reported as a known disease mutation ( hgmd i d cm960300 ) .
the heterozygous missense mutation ( arrows ) , c.865c > t , was identified in the proband ( iii-3 ) , his elder sister ( iii-2 ) , and his mother ( ii-2 ) .
the potential functional impacts of mutations within chrne gene were predicted using polymorphism phenotyping 2 ( polyphen-2 ) software ( http://geneics.bwh.harvard.edu/pph2/ ) , sorting intolerant from tolerant ( sift ) software ( http://sift.jcvi.org/ ) and mutationtaster ( http://www.mutationtaster.org/ ) .
the p. leu289phe substitution was predicted by the polyphen-2 software to be probably damaging and disruptive of the function of chrne .
mutationtaster and sift predicted that the p. leu289phe mutation is functionally disease causing and damaging , respectively .
with mutationtaster software , this mutation is predicted to result in splice site changes and that protein features might be affected .
it has been reported that the mutation can lead to an unusually high rate of spontaneous achr channel openings and a 9-fold increase in affinity for acetylcholine .
this mutation has been found to be absent in the 100 healthy controls ( 50% male ) .
sccms is a progressive disorder and may present with ophthalmoplegia , ptosis , facial paralysis , weakness , fatigue of trunk and limb muscles , and spinal deformity such as scoliosis .
a pathological gain of function of achr located in the postsynaptic membrane has been observed in sccms , which was caused by mutations in the achr alpha - subunit ( chrna ) gene and chrne gene .
however , mutations of the chrne gene can also lead to the fast - channel variation of csm .
the clinical phenotype can range from mild to severe and vary among sccms patients with different mutations and even among patients with the same mutation .
the mutation c.865c > t / l289f of chrne gene identified in our study was first identified in 1995 .
the mutation identified in 1995 came from a family that was initially described in 1982 .
at least seven patients with this mutation in chrne gene have been reported ; however , no patient with this mutation has been found in asia until now . compared with other patients
reported to have the same mutation , certain characteristics were different in our chinese family .
for example , all members in the family experienced childhood onset and none presented ptosis . respiratory failure did not occur , hence ventilator - assisted breathing was not used . however , severe symptoms have been reported in other patients carrying this identical mutation , including onset in infancy and severe effects on respiratory muscles that caused respiratory failure requiring mechanical ventilation in two cases .
interestingly , the proband in our family experienced episodes of weakness and deterioration in winter for unknown reasons , the influence of climate on the disease has not been reported previously .
we also discovered that , in the mother of the proband , the weakness improved gradually with an increase in age , which suggested that even without medications , this disease might gradually improve with age during adulthood .
the role of the mutation c.865c > t in the pathogenesis of cms has been investigated in several studies .
this mutation was definitively demonstrated to be a causative factor in the development of sccms and was localized to chrne and more precisely to the pore , within the m2 domain , which forms a part of the achr channel .
mutations in this m2 domain have more severe phenotypic consequences than those in the extracellular domain .
indeed , patch clamp studies revealed an unusually high rate of spontaneous achr channel openings and a 9-fold increase in affinity for acetylcholine resulting from this mutation , leading to pathological gain of function .
in addition , ultrastructural studies showed that endplate myopathy occurs in the postsynaptic muscle fiber .
after fluoxetine treatment , our patients reported mild improvement of muscular weakness although neurological signs did not change .
electrophysiological studies showed that r - cmap remained after fluoxetine treatment in both the proband ( iii-3 ) and his elder sister ( iii-2 ) , which were consistent with a previous study .
varying degrees of decrements in response to rns were found in the affected family members in our study , especially in response to high - frequency stimulation , which indicated that high - frequency rns is a more sensitive indicator than low - frequency rns for this disease .
in contrast , decrements were not observed in the mother ( ii-2 ) in response to low - frequency rns .
heterogeneities in the family 's electrophysiological data were also found , but we could not fully address the issue , at least not yet , as to whether the magnitude of the cmap decrease observed in the rns test positively correlated with the severity of illness .
the 4-year - old girl ( iv-1 ) did not have any symptoms at present ; however , decrements were observed when she received low - frequency rns , especially with 3 hz , which indicated that she would likely suffer from this disorder in the future .
furthermore , decrements in response to low - frequency rns of the ulnar nerve of this girl ( iv-1 ) were more pronounced than those in her mother ( iii-2 ) .
in addition , although the decrease in rns was greater in the proband ( iii-3 ) than the elder sister ( iii-2 ) , the severity of the clinical symptoms was nearly same . therefore , we hypothesized that the extents of decrements in response to rns did not necessarily correlate with the severity of illness .
after fluoxetine treatment , decrements in response to rns were observed to improve in a subset of the nerves of one patient but not in the others receiving the same treatment , although they both reported clinical relief of symptoms .
electrophysiological heterogeneities in cms due to chrne gene mutations have been reported . a patient with c.855c >
t mutation in chrne gene showed a mild but significant decrement in all the muscles except for the tibialis anterior .
the greatest decrement was observed in the anconeus muscle ( 15% in amplitude ) . a mild decrement ( 10% )
was found in a patient due to the mutation ( epsilon1369delg ) of chrne gene , but another patient with the same mutation had no decrement .
patients due to duplication mutations 123_127dupctcac in exon 2 of the chrne gene showed decrements .
for sccms patients whose illness can be attributed to the same mutation as in this study , a decremented electromyographic response was observed on 2 to 3 hz of stimulation ; however , r - cmap was not found .
rns test revealed 30%70% decrements in different muscles of a spanish boy with the same mutation l269f
cms may be misdiagnosed as myasthenia gravis or congenital muscular dystrophy or myopathy , such as ulrich congenital muscular dystrophy , which could lead to incorrect or delayed treatment .
for example , pyridostigmine treatment for sccms patients can induce endplate myopathy , and it is thus contraindicated .
the presence of r - cmap indicates a diagnosis of either sccms or acetylcholine esterase deficiency , which is caused by mutations in the acetylcholinesterase - associated collagen ( colq ) gene ; hence , the identification of gene mutations underlying sccms will be necessary for further differential diagnosis .
|
background : congenital myasthenic syndromes are a group of rare disorders that are clinically and genetically heterogeneous and caused by mutations in the genes encoding proteins of the neuromuscular junction . here
, we described a chinese family that presented with phenotypes of classic slow - channel congenital myasthenic syndrome ( sccms).methods : clinical characteristics and electrophysiological features of three patients from a chinese family were examined , and next - generation sequencing followed by direct sequencing was carried out.results:the patients revealed variability in clinical and electrophysiological features .
however , weakness , scoliosis , and repetitive - compound muscle action potential were found in all affected members in the family .
a heterozygous c > t missense mutation at nucleotide 865 in acetylcholine receptor epsilon - subunit ( chrne ) gene that causes a leucine - to - phenylalanine substitution at position 289 ( l289f ) was found.conclusions:we reported a sccms family of chinese origin . in the family , classical clinical phenotype with phenotypic variability among different members
was found .
genetic testing could help diagnose this rare disease .
|
I
M
Patients
Mutational analysis
R
Clinical findings
Electrophysiological examination
Genetic studies
D
Financial support and sponsorship
Conflicts of interest
|
we identified isolates for sequencing from 29 invasive gas cases diagnosed in patients in a northern arizona hospital during january
july 2015 and randomly selected an additional 99 gas isolates from a repository of > 2,000 arizona gas isolates collected during 20022006 ( no isolates from patients in arizona were available for 20072014 ) .
four additional isolates from central arizona identified in 2015 were included in the analysis ( technical appendix table ) .
all isolates were grown on 5% sheep blood tryptic soy agar plates ( hardy diagnostics , santa maria , ca ) , and incubated at 37c with 5% co2 .
dna was extracted by using a dneasy blood and tissue kit ( qiagen , valencia , ca , usa ) following manufacturer s protocol .
genomic dna libraries were prepared by using the nextera xt library prep kit ( illumina , san diego , ca ) and sequenced with paired - end reads ( 250 bp ) on an illumina miseq instrument , as previously described ( 9 ) . the finished genome of the emm59 canadian clone mgas15252 ( genbank accession no .
cp003116 ) and high - quality publicly available sequence - read data from 44 us isolates , from ncbi short read archive ( bioproject # prjna194066 ) , were included in the subsequent phylogenetic analyses .
the final core genome ( all nucleotide loci found in all genomes ) for single - nucleotide polymorphism ( snp ) detection was 1,636,024 bp ( 98.6% of reference ) .
we used nasp snp analysis pipeline ( http://tgennorth.github.io/nasp/ ) for whole - genome snp typing as previously described ( 10 ) .
we used mega version 5.2.2 software ( 11 ) to generate maximum parsimony phylogenetic trees .
regions of high snp density were identified as possible regions of recombination and were further analyzed for impact on the consistency index .
gas emm subtypes were assigned by using blast ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) , querying the study genome assemblies against the centers for disease control and prevention s ( cdc ) emm type - specific sequence database ( http://www.cdc.gov/streplab/m-proteingene-typing.html ) .
we resolved dual emm - type hits using cdc s emm typing sanger sequencing primers ( http://www.cdc.gov/streplab/protocol-emm-type.html ) as a blast query and noting hit locations .
we identified 18 of the 29 contemporary northern arizona isolates as subtype emm59 ; the remaining isolates were composed of 6 additional emm types : emm1 ( n = 2 ) , emm5 ( n = 2 ) , emm58 ( n = 1 ) , emm81 ( n = 2 ) , emm83 ( n = 1 ) , emm89 ( n = 2 ) , and emm94 ( n = 1 ) .
the 99 historical and 4 contemporary background arizona isolates included 25 distinct emm types ( technical appendix table ) .
no emm59 isolates were identified in this background set , and none had been previously reported in arizona .
an emm59-only phylogenetic analysis demonstrated the apparent presence of multiple lineages of emm59 in the 2015 arizona isolates ( figure 1 ) .
a distinct clone consisting of 14 of the 18 emm59 isolates were separated from each other by only 04 snps , genomically supporting the presence of an ongoing outbreak ; > 8 of these patients were epidemiologically linked to physical contact , cohabitation , or both with 1 other person ( data not shown ) .
the additional emm59 isolates make up additional lineages separated from one other by 828 snps .
a relatively large number of snps and indels were seen within an approximate 23-kilobase region ( figure 1 ) .
this region has been previously reported to contain mutational hotspots associated with virulence ( 12,13 ) . considering the presumptive positive selective force on this region , snps within the region
phylogenetic single - nucleotide polymorphism ( snp ) tree of emm59 isolates from a northern arizona hospital displaying distribution of mutations in a 23 kb positively selected region during invasive group a streptococcus outbreak , southwestern united states .
maximum parsimony tree of all snp loci ( n = 58 ) in emm59 isolates ( n = 18 ) from arizona , 2 recent new mexico isolate genomes , and the canadian clone reference isolate mgas15252 .
branch lengths represent numbers of snps between isolates ; unit bar is in the figure .
numbered circles distinguish lineages of selected mutations in scpa , enn , sfbl , mga , sfbx , and sof genes in a 23-kb hotspot mutational region .
when compared with all other publicly available us emm59 isolate genomes , nearly all the genomes identified in the united states were closely related to each other and to the canadian clone mgas15252 ; individual isolate snp branch lengths ranged from 0 to 10 ( figure 2 ) .
the arizona outbreak isolates were separated from 2 new mexico isolates by 4 and 5 snps each ; these isolates fell within the overall arizona clade and were subsequently included in the arizona - only phylogenetic analysis ( figure 1 ) .
conversley , the isolate from patient m appears more distant from the larger arizona population .
the arizona clades , with the exception of that of the isolate from patient m , all appear to arise from the large minnesota polytomy .
the previously estimated 1.32.1 snps / year mutation rates for gas ( 14,15 ) further support the arizona outbreak as being caused by a single clone , likely originating from new mexico and being spread over 612 months .
phylogenetic single - nucleotide polymorphism ( snp ) tree of emm59 isolates from arizona during invasive group a streptococcus outbreak in the southwestern united states , previously analyzed us emm59 isolates , and the canadian clone .
maximum parsimony tree of all 177 snp loci ( 44 parsimony informative snps ) in emm59 isolates from arizona ( n = 18 ) , minnesota ( n = 29 ) , oregon ( n = 8) , new mexico ( n = 3 ) , colorado ( n = 2 ) , and california ( n = 1 ) and the canadian clone reference isolate mgas15252 . tree has regions of recombination removed and is rooted with minnesota isolate srr11574570 .
the emm59 subtype of gas , the etiologic agent of a substantial nationwide outbreak of invasive gas in canada during 20062009 ( 4 ) , is now present in arizona , causing at least 1 outbreak of epidemiologically and genomically linked cases and several additional epidemiologically unrelated cases .
the lack of emm59 in background isolates in arizona from the previous decade , along with its low genetic diversity , suggests that emm59 emerged recently in arizona .
following the emm59 epidemic in canada , this subtype was subsequently seen in a few us states ; a retrospective analyses of the centers for disease control and prevention active bacterial core surveillance ( abcs ) system ( http://www.cdc.gov/abcs/reports-findings/survreports.pdf ) identified 40 us emm59 isolates during 20002009 ( 6 ) and an additional 67 isolates during 20102012 ( 7 ) . of note , only 5 ( of the 40 emm59 isolates from 20002009 ( 2 from minnesota , 2 from california , and 1 from oregon ) appeared to be closely related to the canadian clone ( defined by the authors as being separated by < 16 snps ) ( 6 ) ; in contrast , all of the strains from the 20102012 survey appeared to be more closely related to the canadian clone .
the more recent abcs analysis identified an increasing number of southwestern isolates , including 4 from colorado and 6 from new mexico ( 7 ) , although no outbreaks were specifically described in these states ( arizona is not included in the abcs system ) .
( 7 ) , in an analysis of 60 mn emm59 isolates from case - patients with identified race , determined that 25 ( 42% ) were from native americans ; of 5 isolates from new mexico in that study , 3 were from native americans .
given the apparent distal nature of the arizona / new mexico isolates to the minnesota population in our study , it is reasonable to propose an unidentified epidemiologic relationship between these case populations .
however , caution must be used in drawing conclusions regarding the relationships of isolates from disparate geographic regions because only limited comparable sequence data from previous emm59 studies in the united states ( 7 ) were publicly available to compare to the arizona isolates .
epidemiologic investigations , along with healthcare provider and patient education activities , are ongoing in arizona to further determine the extent of the current outbreak and the associated risk factors and to help mitigate effects and limit or prevent further spread to at - risk populations .
|
the hyper - virulent emm59 genotype of invasive group a streptococcus was identified in northern arizona in 2015 .
eighteen isolates belonging to a genomic cluster grouped most closely with recently identified isolates in new mexico .
the continued transmission of emm59 in the southwestern united states poses a public health concern .
|
The Study
Conclusions
None
|
juxtafacet cysts ( jfcs ) are uncommon intraspinal , extradural lesions that arise from the facet joints .
jfcs can be further distinguished as either synovial cysts , if histology confirms the presence of a synovial lining membrane , or alternatively as a ganglion cyst , if no such membrane is present .
however , few differences exist between the two in terms of diagnosis and management.1
2
3
4
5
6
7 their pathogenesis remains unclear , with most authors citing excessive joint mobility leading to synovial fluid herniation through joint capsule defects.2
4
7
calcification in the cyst walls is a common finding on imaging for jfcs , appearing as peripheral areas of high attenuation on computed tomography ( ct ) and peripheral hypointensity on t1- and t2-weighted magnetic resonance imaging ( mri).8
9
10 but although calcification of the cyst walls are commonly reported findings,9
10
11 complete calcification of the cyst is exceedingly rare.11
12
13 we present here an unusual case of a jfc presenting as a giant , multilobular , completely calcified mass with significant extraforaminal extension .
the patient was a 57-year - old woman who presented with a 2-year history of gradually worsening lower back and left leg pain .
she also reported numbness in her left heel and foot and subjective weakness in the left foot .
her past medical history was significant for hypothyroidism , hypertension , mild levoscoliosis , rheumatoid arthritis , and scleroderma managed on weekly methotrexate injection .
her past surgical history was negative for any previous lumbar surgeries , but the patient had received multiple cervical surgeries resulting in c3c7 fusion . on neurologic exam ,
the patient was found to have decreased strength in plantarflexion and dorsiflexion of the left lower extremity but was otherwise nonfocal .
the patient 's imaging was most notable for a large , multilobular mass , measuring 54 31 30 mm , arising from the left l5s1 facet joint that had caused destruction of the joint itself and that showed severe encroachment of the adjoining foramen , with coexisting grade i anterolisthesis of l5 on s1 .
the lesion was hyperdense on plain radiograph ( fig . 1 ) and on ct imaging ( fig
. 2a , 2b , and 2c ) and appeared hypointense on t1- and t2-weighted mri sequences ( fig .
preoperative plain film of the lumbar spine showing a large , completely calcified lesion extending out from the l5s1 intervertebral space .
( a ) axial computed tomography ( ct ) image taken preoperatively , showing the origin of the lesion from the left l5s1 facet joint .
( b ) a preoperative midsagittal ct image demonstrating grade 1 anterolisthesis of l5 on s1 . ( c ) a preoperative sagittal ct image demonstrating foraminal stenosis at l5s1 caused by the lesion .
( a ) t1-weighted magnetic resonance image ( mri ) of the same lesion , which appears uniformly hypointense . ( b )
the patient underwent an l5 laminectomy for excision of the mass along with an l5 to s1 fixation and fusion .
intraoperatively , the lesion was noted to consist of a pastelike material that was compressing the thecal sac and that extended ventrally out of the foramen .
histologic analysis identified the lesion as a mineralized simple pseudocyst , containing mineralized proteinaceous material that did not refract plane polarized light after ethanol fixation ( fig . 4a and 4b ) .
( a ) hematoxylin and eosin stained sections of the resection reveal dense membranous fibrous connective tissue separating spaces associated with dense calcifications as well as smaller loculated spaces filled with calcium ( 20 magnification ) .
( b ) higher magnification ( 40 ) reveals the wall of the spaces to be associated with granular calcifications and no obvious cellular lining .
the patient experienced no intraoperative or postoperative complications , and her postoperative hospital course was unremarkable . by postoperative day 2 , the patient had recovered full strength in her extremities but still had some residual numbness , and she was discharged to home in stable condition on postoperative day 3 . at 2-year follow - up ,
the patient continued to be free of her lower extremity pain and weakness and had not experienced any subsequent postsurgical complications ( fig . 5a and 5b ) .
( a ) postoperative plain film ( anterior - posterior ) showing removal of the mass and placement of fixation hardware .
mineralized , extradural lesions of the lumbar spine arising from the facet joint are uncommon causes of spinal pathology .
the differential diagnosis consists of several uncommon disorders , including synovial osteochondromatosis,14 tumoral calcinosis,15 tumoral calcium pyrophosphate dehydrate crystal deposition disease ( pseudogout),16 and lumbar presentation of ossification of the ligamentum flavum.17 the case we present here represents another potential consideration in that differential diagnosis .
completely calcified jfcs are rare , with only a handful of instances existing in the literature .
almefty et al presented four patients with multiple - level , bilateral , consistently calcified thoracic spinal cysts.12 these lesions were atypical due to their number , location , and mineralization .
kasliwal and deutsch presented on the case of a unilateral lumbar jfc at the l4l5 level.13 similar to our case , the patient presented with radicular symptoms in the setting of a lumbar lesion that appeared hypointense on t1- and t2-weighted mri .
it should be noted that calcification of jfcs can result from treatment as well , as mtellus et al noted a case of a cyst undergoing complete calcification after intracystic steroid injection therapy.11
the case presented here was remarkable not just for its mineralization but also for its size .
jfcs have typically been recorded to be between 10 and 20 mm in diameter at their widest dimension on imaging.10
18 the case we report here , however , measured 54 mm at its widest dimension , showing significant extension into nearby structures .
moreover , although jfcs frequently extend into the spinal canal and cause compression of the thecal sac,2 the majority of the lesion in our case existed outside the spinal canal .
this may explain how the cyst grew to be so large before the patient presented for evaluation .
it should also be noted that our patient 's medical history included rheumatoid arthritis and scleroderma , and systemic inflammatory diseases have been associated with jfcs in the past.1
19 moreover , inflammatory arthritides are known to contribute to the pathogenesis of synovial cysts outside of the spine.20 however , no studies to date have established systemic inflammation as a risk factor for jfc development , and the role of inflammatory joint disease in the pathogenesis jfcs remains unclear .
many methods of treating jfcs have been described , including simple laminotomy with cystectomy and the potential addition of a laminectomy , hemilaminectomy , mesial facetectomy , or foraminotomy.3
4 minimally invasive approaches with tubular retractors have also been described.19
21 in terms of treatment of this patient , it was presumed that the symptoms of back pain were secondary to facet dysfunction and that the left - sided plantar flexion weakness was due to foraminal stenosis . due to the size and mineralization of the lesion
, it was felt that a complete facetectomy was needed to adequately address the underlying pathology , necessitating a concurrent fusion .
our case highlights the significant variation that exists in the size , location , and content of jfcs .
although uncommon , jfcs should be considered in the differential diagnosis of lesions arising from the facet joint , even if they appear calcified and noncystic in nature .
|
study design case report .
objective to report the case of one patient who developed a giant , completely calcified , juxtafacet cyst .
methods a 57-year - old woman presented with a 2-year history of progressively worsening lower back pain , left leg pain , weakness , and paresthesias .
imaging showed a giant , completely calcified mass arising from the left l5s1 facet joint , with coexisting grade i l5 on s1 anterolisthesis .
the patient was treated with laminectomy , excision of the mass , and l5s1 fixation and fusion .
results the patient had an uncomplicated postoperative course and had complete resolution of her symptoms as of 1-year follow - up .
conclusions when presented with a solid - appearing , calcified mass arising from the facet joint , a completely calcified juxtafacet cyst should be considered as part of the differential diagnosis .
|
Introduction
Case Report
Discussion
|
the name of mucosa - associated lymphoid tissue ( malt ) lymphoma was first established in 1983 by isaacson and du . from the beginning , it was adopted well and is still used in an unchanged form .
marginal zone lymphoma of malt is , apart from diffuse large b - cell lymphoma , the most frequent type of lymphoma that occurs in the stomach .
what is important is that it can develop in almost every organ and tissue , for instance lungs , breast , thyroid gland , bladder , skin , or orbital adnexa .
it is an indolent type , but clinical outcomes and response to treatment vary among patients .
malt lymphoma arises from the extranodal sites reach in b - lymphocytes , which appears in response to chronic antigenic stimulation caused by infection ( helicobacter pylori ) or autoimmune process ( hashimoto disease ) .
this disorder is the best example of how infectious pathogens and genetic abnormalities lead to malignant transformation .
gastric malt lymphoma pathogenesis is a complex process including many gene alternations that result in cancer appearance .
better understanding of the background of the disease is crucial for discovering new prognostic factors , helpful in deciding when more aggressive treatment should be employed .
the incidence of malignant lymphomas is at the rate of 3%-4% of all malignancy worldwide and has been increasing during the last 50 years . lately
, some stabilization in the number of diagnosis was observed , but only in developed countries .
malignant lymphomas are observed to be more frequent in north america , australia , and europe than in asia and africa .
malt lymphomas determine almost 7% of all non - hodgkin 's lymphoma , and at least 40% is primarily located in stomach .
it is confirmed that gastric malt lymphoma occurs in younger patients than the rest of malignant lymphomas .
the malt lymphoma is mainly a disease of older adults , with a median age of 60 years .
, there is much higher proportions of malt lymphomas , which can be caused by more frequent prevalence of helicobacter pylori in this region of the world .
although 90% of population worldwide have confirmed bacteria colonization , only 2% will develop malignant lymphoma .
it was confirmed by weber et al . that almost 90% of patients with gastric malt lymphoma are infected with helicobacter pylori .
this curved bacillus , previously called campylobacter pyloridis , is a gram negative pathogen found in the stomach .
although over 80% of people are asymptomatic , chronic infection can lead to gastritis , gastric and duodenal ulcer , gastric adenocarcinoma , and malt lymphoma [ 5 , 6 ] .
nowadays , it is widely accepted that helicobacter pylori gastritis is crucial in an evolution of malt lymphoma localized in stomach .
it was confirmed by several studies that chronic gastric inflammation causes constant antigenic stimulation , which leads to clonal expansion of b - cell lymphocytes [ 7 , 8 ] . in the gastric mucosal cells ,
there are elevated levels of some cytokines , including proliferation - inducing ligand ( april ) , which belongs to the tumour necrosis factor ( tnf ) family .
april is produced by macrophages present in the gastric malt infiltrate , located close to the neoplastic cells .
april may also induce b - cells transformation and the progression to the diffuse large b - cell lymphoma ( figure 1 ) .
they come either from t cells or directly by the antigenic autostimulation of lymphoma cells .
gastric inflammation causes the appearance of a large number of macrophages , which , under a helicobacter pylori infection , release large amounts of april .
importantly , a number of april - producing macrophages significantly decrease in complete remission after eradication therapy .
other pathogens , are also suspected to play an important role in malt lymphoma pathogenesis .
there are bacteria such as campylobacter jejuni , borrelia burgdorferi , and chlamydia psittaci and viruses like hepatitis c virus ( hcv ) that are potentially responsible for oncogenesis .
these pathogens were found in histological material , but so far no strong evidences were established . patients with autoimmune disease have for sure higher risk of developing malt lymphoma .
autoreactive b cells infiltrate the healthy organs and create lymphoid infiltrate similar to normal malt tissue with huge amount of reactive clonal b lymphocytes .
this situation is observed in salivary gland in patients with diagnosis of sjgren syndrome and in the thyroid gland in hashimoto disease .
sjgren syndrome is associated with 44 times increased risk of lymphoma , whereas hashimoto 's thyroiditis causes 70 times increased risk of thyroid lymphoma .
some of them are proven to be strongly associated with the disease , but some are still not confirmed .
it is believed , that on the background of chronic inflammation not only reactive b - cells are stimulated but also activated neutrophils which can lead to production of oxygen species . as a result , this genotoxins provoke dna damages , which are responsible for mutations and transformations of genetic material .
it originates from a fusion of two proteins : apoptosis inhibitor 2 ( api2 ) and paracaspase malt lymphoma - translocation gene 1 ( malt1 ) .
what is more important is that while t(11;18)(q21;q21 ) is detected , no other chromosome abnormality can be found .
unfortunately , positive cases do not response to helicobacter pylori eradication , but , in contrast , they do not transform to more aggressive diffusive large b - cell lymphoma .
it is known that complete remission can be seen in at least 20% of patients with t(11;18)(q21;q21 ) .
the incidence of positivity for this translocation malt lymphoma is at approximately 20% in europe [ 16 , 17 ] but is not as common in the united states where only 5% are positive .
moreover , it is usually connected with an advanced stage of disease and poor outcomes .
bcl-10 gene is relocated from chromosome 1 to 14 , which in consequence triggers overexpression of bcl-10 protein also known as ciper , carmen , or me10 . in healthy organisms , higher expression is observed in lymph nodes , spleen , and testis .
so far , it is believed that bcl-10 protein expression is responsible for proliferative effects [ 19 , 20 ] .
the t(14;18)(q32;q21)(igh - bcl-2 ) is commonly present in follicular lymphoma , in about 20% of diffuse large b - cell lymphoma and sometimes in chronic lymphocytic leukemia .
although this aberration is extremely rare in other types of lymphomas , it can be found in some cases of gastric malt lymphoma .
bcl-2 is an antiapoptotic protein , which helps in survival and expansion of clonal b cells .
so far , the role of t(14;18 ) in gastric malt lymphoma is not fully understood .
overexpression of bcl-2 is found not only in translocation positive patients but also in the negative ones .
it is believed that similar to other types of lymphomas , t(14;18)(igh - bcl-2 ) must coexist with other genetic abnormalities in order to develop neoplasm .
t(3;14)(p14;q32)(igh - foxp1 ) is a newly described abnormality present in patients with malt lymphoma .
the first study showed that positivity for this translocation is approximately 10% of all malt lymphoma patients .
the most recent studies described the presence of t(3;14)(p14;q32 ) in diffuse large b - cell lymphoma , outside the lymph nodes especially [ 23 , 24 ] .
only one study , so far , confirmed the existence of this translocation in gastric malt lymphoma which involved bad clinical outcomes .
in pathogenesis of malt lymphoma , the above described translocation promotes oncogenesis by similar well - known mechanism .
the majority of them involve the same pathway , which leads to antigen receptor - mediated activation of nfb .
this is a crucial transcript factor which plays a key role in malt lymphogenesis [ 26 , 27 ] .
it regulates processes connected with b - cell development , growth , and survival by production of cytokines and growth factors , for example , tnf- family ( baff ) .
latest studies have shown that b - cell activation in malt lymphoma can be strictly connected with tnf family .
it can be also responsible for activation of cell apoptosis [ 28 , 29 ] .
it is observed that in patients with higher baff levels in serum , the prognosis and survival are much worse .
based on recent knowledge about genetic abnormalities in gastric malt lymphoma , there is a model of multistep pathogenesis . on
the background of chronic inflammation and antigenic stimulation occurs genetic instability . as a result , many possible translocation and unbalanced aberrations are observed .
gastric malt lymphoma can be long - time asymptomatic or associated with dyspepsia , abdominal pain , vomiting , diarrhea , obstruction , and nausea . sometimes bleeding from gastrointestinal tract or even perforation
may occur while extensive lesions are present . as a result , symptoms of anemia like paleness , weakness , or easy fatigue can be observed .
b symptoms ( weight loss , unexplained fever , and night sweats ) in gastric malt lymphoma are very rare , but the most common of the above is weight loss . a prompt diagnosis is crucial , but , unfortunately , it is usually made by incidence .
patients with early stage of disease have usually low tumor growth and minimal possibility to spread .
in contrast , patients with advanced stage of disease can undergo transformation to more aggressive lymphoma and may become resistant to treatment . not only symptoms but also endoscopic picture can be inconclusive . difficulties often arise to differentiate between chronic gastritis or ulcer from an early - stage lymphoma . in order to confirm the diagnosis ,
there always must be made pcr or fish analysis for t(11;18 ) , which is important to separate groups that will not respond to standard treatment .
characteristic for gastric malt lymphoma are lymphoepithelial lesions ( lel ) with the presence of mainly two types of cells : neoplastic centrocyte - like or small lymphoid .
there is no specific immunohistochemical profile typical for gastric malt lymphoma diagnosis . in 50% of patients , there is coexpression of cd43/bcl2 .
neoplastic cells are positive for cd-20 and negative for cd-10 , cd-23 , and cyclin d1 .
if it is negative in histochemistry , rapid urea breath test or fecal antigen test have to be made . another analysis
to prove absence of helicobacter pylori infection is serological test for caga antibodies and helicobacter pylori - igg antibodies .
sometimes , there is possibility to detect other helicobacter species , for example , heilmannii or felis .
before taking any decision on how aggressive the treatment should be , it is extremely important to perform a complete staging of the disease .
what is more important is that risk factors and individual parameters , which can affect later therapy , are crucial .
medical history must include information about the age , time of the first symptoms , the family history , and medical condition .
the most important factor that we rely on during choosing method of treatment is clinical stage of the patient . during physical examination ,
it is important to remember about waldeyer 's ring , which is mandatory in every gastric lymphoma patient . staging in gastric malt lymphoma is similar to that in other types of lymphomas . according to recent european society for medical oncology ( esmo ) recommendations , it should include morphology with basic biochemical studies .
if the blood cell count is lower , it can be caused by infiltration of bone marrow .
biochemical tests can detect liver or kidney problems , which can be important before the beginning of a chemotherapy .
lactate dehydrogenase ( ldh ) and 2-mikroglobulin are prognostic factors and will be abnormally high in patients with fast - growing tumor .
every newly diagnosed patient should be examined in case of certain viral infections that can affect treatment , such as hepatitis b and c or human immunodeficiency virus ( hiv ) . in every case ,
computed tomography ( ct ) scans of neck , chest , abdomen , and pelvis , which are crucial to evaluate enlarged lymph nodes , should be performed .
core needle biopsy of bone marrow is made to diagnose possible infiltration of neoplastic cells .
it was confirmed that 15% of gastric malt lymphoma patients have lymphoma cells in bone marrow .
positron emission tomography ( pet ) has still not confirmed clinical necessity , but it can be extremely helpful in controversial cases . moreover , during staging procedures of gastric malt lymphoma , gastroduodenal endoscopy must be made .
biopsies are taken from different sites of gastrointestinal tract ( e.g. , stomach , duodenum , and gastroesophageal junction ) and every location that looks suspicious .
most often , ann arbour staging is employed , which describe the extend of all types of non - hodgkin lymphoma in adults .
thus , staging of gastric lymphoma based upon the ann arbor system includes stage i e , which is disease limited to the stomach without nodal spread .
stage ii e1 is tumor in the stomach with spread to adjacent contiguous lymph nodes .
stage ii e2 is tumor in the stomach with spread to lymph nodes that are noncontiguous with the primary tumor .
moreover , if the spleen is affected , we add s. if the person has any of the b symptoms , we add letter b , and if is asymptomatic , we assign a ( table 1 ) .
prognostic factors in gastric malt lymphoma are similar to the value for non - hodgkin b - cells lymphoma .
factors that determine poor outcome are age , high level of ldh in serum , higher ecog performance status , stages iii and iv in ann - arbour scale , white blood count , and more than one extranodal site .
it was observed that patients with nodal invasion has difficulty with complete remission after eradication treatment .
for instance patients , with t(11;18)(q21;q21 ) especially , are resistant to the first line therapy , and remission rate was lower than that in patients of api2-malt1 negative ( 78% versus 22.2% ; p = 0.0001 ) .
only one study so far proved that the presence of t(3;14)(p14;q32 ) is connected with poor clinical outcomes of patients with gastric malt lymphoma .
while helicobacter pylori plays a main role in the pathogenesis of malt lymphoma , it is also crucial in approach to the treatment . according to current international guidelines , first line treatment for localized helicobacter
the treatment may be used with every highly effective antibiotics against helicobacter pylori , taking into consideration the locally expected antibiotic resistance . if there is no response to the therapy above , second line triple or quadruple therapy is used .
it was reported that after two lines of treatment , 99.8% of patients were cured from gastritis . in a large study of 1408 patients , remission after eradication treatment in early stage
unfortunately , in 5%10% of gastric malt lymphoma patients , we can not confirm helicobacter pylori infection .
moreover , more than 30% patients are resistant to first line treatment , and 30% of them have t(11;18)(q21;q21 ) .
treatment for this patients should be chosen individually depending on the clinical stage of disease . for those who have stable disease without any symptoms ,
radiotherapy , chemotherapy , and/or surgery can be considered after unsuccessful eradication treatment . further recommended
surgery is considered to be a standard therapy in therapy of patients with gastric malt lymphomas , but , recently , the value of this therapy has been not confirmed .
even if the lymphoma is localized at early stage , the gastrectomy should be rather extensive due to the nature of the disease .
moreover , it is a major surgery and can be associated with serious complications and worsen a quality of life .
german multicenter study group ( gmsg ) presented no difference between survival in patients treated with gastrectomy compared to eradication ( overall survival rate 82% to 84% ) .
what is more important is that there were observed 50% long - term complications were observed after surgery . in few studies
the use of a modest dose of involved fields was performed on resistant - to - eradication therapy patients with early - stage disease .
the dose was 2535 gy to the stomach and perigastric nodes for the period of 4 weeks [ 42 , 43 ] . compared to surgery , no serious long - term complications and toxicity were observed . only nausea and anorexia were present during the time of radiotherapy . for a long time
it was believed that gastric malt lymphoma is just a localized disease and that surgery and radiotherapy are the best treatment strategy .
now , when it is well known that it is disseminated disorder chemotherapy , it became more important .
still , there are no standard recommendations for relapse or progressive patients after therapy and for those with late stage of the disease from the beginning .
it was observed that chemotherapy alone is more effective than surgery apart from some cases with gastric obstruction .
complete remission ( cr ) after oral monochemotherapy with cyclophosphamide was 83% in a study by nakamura and coworkers .
unfortunately , patients with positive translocation t(11 ; 18 ) are resistant to second line therapy with oral monochemotherapy with alkylating agents .
nucleoside analogs are confirmed to be effective in treatment of different kinds of indolent lymphomas .
a polychemotherapy with fludarabine and mitoxantrone ( fm ) has a very good effect on patients with gastric malt lymphoma in both first and second line treatment .
the complete remission after 4 cycles achieved 84% of investigated and all of them reacted to the treatment .
after 2-cda , there were observed complications such as toxicities of 3 and 4 grade of who , mainly leukopenia , infections , and secondary neoplastic disease .
nowadays , immunotherapy became an extremely important part of treatment of non - hodgkin lymphomas .
it is a chimeric mouse / human monoclonal antibody specified to cd20 antigen expressed on the surface of b lymphocytes .
now it is widely used alone or in combination with chemotherapeutic drugs in many types of b - cell non - hodgkin lymphomas .
rituximab binds to cd20 antigen and activates the lysis of b cells by mediating cytotoxicity of complement dependent ( cdc ) and cell - mediated cytotoxicity antibody dependent ( adcc ) .
the role of this drug is still not clear in gastric malt lymphoma . in 2003 , there was a first - phase study by conconi et al .
the cr was observed in 29% and overall response rate ( orr ) was 64% .
the toxicity of this treatment was moderate or even mild , but the relapse rate was 36% .
an important fact is that patients with translocation t(11;18 ) are responsive to rituximab treatment [ 58 , 59 ] .
what is more important is that in a study by the international extranodal lymphoma study group ( ielsg ) , it was confirmed that chlorambucil in combination with rituximab was more effective than chlorambucil alone .
the conclusion is that rituximab may a benefit in individual patients , but for the majority it is not sufficient when used alone .
the efficacy of the combination of rituximab with chlorambucil was evaluated in a randomized study ( comparator was chlorambucil alone ) by the international extranodal lymphoma study group ( ielsg ) in gastric malt lymphomas that had failed antibiotics and in nongastric malt lymphomas .
the preliminary report showed that the 5-year event - free survival was significantly better for patients treated with chlorambucil plus rituximab .
there were also studies by raderer et al . with cycles generally used in more aggressive lymphomas .
twenty - six patients were administrated rituximab plus cyclophosphamide , doxorubicin or mitoxantrone , vincristine , and prednisone .
lately , bortezomib , the first therapeutic proteasome inhibitor , was examined by kiesewetter et al . in 2012 with cr in 33% and pr in 27.8% .
the results on phase ii studies with chemotherapy and immunotherapy are shown in table 2 .
outcomes in gastric malt lymphoma patients with progressive , disseminated disease are very comparable with outcomes in follicular lymphoma .
although gastric malt lymphoma has a very favorable outcome , it is still important to have a proper followup
. it is possible that the disease will return even after 5 years of complete remission .
the relapse can be due to reinfection of helicobcater pylori . in a study by zullo et al .
the followup is obligatory in patients with gastric malt lymphoma to identify early phase of the recurrence of the disease . to confirm a complete remission ,
although , there are no specified recommendations for a followup , the biopsy of gastric sites should be made every 6 months in first two years , and later once a year for the next five years .
systemic followup consist of blood tests and minimal adequate radiological and ultrasound and should be made at least once a year in the first 5 years .
the transformation in more aggressive lymphoma is low at the level of 0.05% , but there is a higher risk of occurrence of secondary neoplasm and gastric cancer .
these studies confirm that patients with gastric malt lymphoma need a long - term followup not only to detect early recurrence but also to find secondary disease .
recently , enormous progress has been made in better understanding of pathogenesis of gastric malt lymphoma .
it has a great influence on the development of new and more effective treatment strategy .
still , not enough clinical trials are performed due to rare expression and high effectiveness of first line treatment of gastric malt lymphoma .
what is more important is that early diagnosis of gastric malt lymphoma is extremely important . while the symptoms are unspecific or not , always during the endoscopic exam the complete histological biopsies must be taken to make diagnosis correctly . the less advanced the stage of the disease , the bigger the chances to achieve complete remission
|
nowadays , it is believed that the main role in the development of gastric mucosa - associated lymphoid tissue ( malt ) lymphoma plays helicobacter pylori infection .
this world - wide distributed bacteria is in charge of most cases of not only upper gastrointestinal tract disorders but also some of extragastric problems .
constant stimulation of the immune system causes a b - lymphocytes proliferation , which is considered to be responsible for the neoplastic transformation . on the other hand ,
there are 10%20% of patients who do not respond to helicobacter pylori eradication treatment .
this group has often a chromosome translocation , which suggests that there is another unknown , so far , pathogenetic mechanism of malt lymphoma .
majority of genetic abnormalities are connected with nuclear factor-b ( nf-b ) pathway , which activates the uncontrolled proliferation of neoplastic cells .
translocations already described in studies are t(11;18)(q21;q21 ) , which is the most common , t(14;18)(q32;q21 ) , t(14;18)(q32;q21 ) , and t(3;14)(p14.1;q32 ) .
this non - hodgkin 's lymphoma is an indolent type originated outside lymph nodes . in more than 50% of cases
, it occurs in the stomach .
occasionally , it can be found in salivary and thyroid gland , lung , breast , bladder , skin , or any other place in the human body . this paper is a review of the current knowledge on etiology , pathogenesis , treatment , and follow - up of gastric malt lymphoma .
|
1. Introduction
2. Epidemiology
3. Pathogenesis
4. Symptoms and Diagnosis
5. Staging and Risk Factors
6. Treatment
7. Followup
8. Conclusions
|
hardware removal is indicated for infection , nonunion , failure of fixation , pain , soft tissue irritation , and anticipated strenuous activity after fracture healing [ 14 ] . during removal cases
subsequent removal of broken hardware increases surgical time , and retained metalwork potentially complicates future surgeries ( figure 1 ) .
although there have been several articles that have discussed titanium implant failure , most have discussed this issue within the context of hardware failure during fracture healing , and not particularly during removal of hardware [ 69 ] . to our knowledge
, none have been specific to the elbow , which merits its own discussion due to its unique anatomy .
the distal humerus of the elbow is unique in that is has a high ratio of cortical to cancellous bone .
therefore , in this study we set out to investigate incidence of bone screw failure during hardware removal procedures and we were interested in comparing titanium and stainless steel bone screws because these are the most common types of metallic fracture implants in circulation .
in addition , we set out to determine whether the duration of implantation and the anatomic location of the bone screws about the elbow were associated with bone screw failure during removal procedures
. a better understanding of metallic hardware failure during removal procedures may help surgeons in the preoperative planning stages of these cases , in terms of surgical tool selection and staff availability .
after institutional review board ( irb ) approval , all cases performed by orthopaedic trauma or upper extremity surgeons between 1/1/2000 and 10/1/2009 at our level 1 trauma center were reviewed .
inclusion criteria were ( 1 ) deep implant removal cases , ( 2 ) hardware removed from the distal humerus or the proximal ulna , and ( 3 ) isolated elbow injuries .
the exclusion criteria were ( 1 ) cases that did not have relevant or inaccessible elbow x - rays , ( 2 ) single screw fragment extraction cases ( because in these cases the hardware had previously broke and was small in size , which we believe was not representative of the other screws being removed ) , ( 3 ) patients younger than 17 years , and ( 4 ) cases that were originally performed at an outside institution ( unavailable medical records ) . the factors considered were ( 1 ) whether or not the bone screws broke during removal and the type of implant metal used ( titanium alloy , ti6al4v or stainless steel ) , ( 2 ) the length of time between initial implantation and removal , where cases were divided into two groups based on a conservative estimate of the time period required for osseointegration of titanium implants [ 10 , 11 ] : one group was for cases where the duration of time between implantation and removal was less than 12 months and the second group was for cases where the duration between implantation and removal was 12 months or more ; and ( 3 ) anatomic location about the elbow ( distal humerus or proximal ulna ) .
the data was extracted from the medical record . due to the small sample size , fisher 's exact test was used to determine statistical differences between two sets of categorical data .
an independent t - test was used to compare the means of two independent groups .
differences that had less than 0.05 probability of occurring from chance were considered statistically significant .
we identified a total of 47 cases , of which 21 met the inclusion criteria .
we carried out an independent t - test to determine if there were any differences between the ages of patients that had broken screws and those that did not , and no statistical significance was found , p = 0.740 .
out of 21 cases , screws broke during removal in 5 cases ( 23.8% ) . in 16 out of 21 cases ,
the reasons for hardware removal were infection in 7/21 cases , symptomatic , prominent hardware in 7/21 cases , nonunion in 6/21 cases , and contracture in 1/21 cases .
14 involved titanium alloy and 7 involved stainless steel implants . within the titanium hardware group , in 10 cases removal was uneventful , and in 4 cases , fracture of at least one screw occurred . in comparison , out of the 7 stainless steel hardware removal cases , there was one case that resulted in one or more broken screws .
overall , compared to stainless steel , failure of titanium alloy screws during removal was not found to be statistically significant ( p = 0.61 ) . in order to determine whether there were any association between the duration of implantation and hardware failure during removal
, cases were divided into two groups : group ( 1 ) duration of hardware implantation was 12 months or less ( mean 7.7 , range two to 12 months ) , and group ( 2 ) : duration of implantation was more than 12 months ( mean 41.6 , range 16 to 74 months ) .
twelve cases had hardware removed within 12 months of implantation and nine cases had hardware removed after 12 months of initial implantation .
bone screws that were removed after 12 months of surgery were more likely to break during removal ( p = 0.046 ) .
when titanium screws were analyzed separately , those removed within 12 months of surgery were more likely to be removed intact as compared to those removed more than 12 months after implantation ( p = 0.003 ) . the small number of stainless steel cases ( seven ) did not warrant statistical calculations .
with respect to anatomic location , there were 12 distal humerus and 15 proximal ulna cases ( table 1 ) .
six cases involved the distal humerus only , nine cases involved the proximal ulna only , and six cases had simultaneous proximal ulna and distal humerus involvement . in one case where titanium screws broke and in one case where stainless steel screws broke , it was unclear where the location was and these cases were discarded from the analysis . in general , bone screw failure was equally likely to occur when removed from the distal humerus and the proximal ulna ( p = 0.28 ) .
hardware failure during removal cases is a commonly seen problem in orthopaedics ( figure 1 ) .
currently , there is no single hardware removal technique that is uniformly successful , and several different methods may be employed during the same case .
such techniques include the use of screw extractors , trephines , extraction bolts , pliers , and various other devices .
the purpose of this article was to determine the incidence of bone screw failure during hardware removal procedures , and we were interested in comparing titanium and stainless steel bone screws .
in addition , we set out to determine whether the duration of implantation and the anatomic location of the bone screws about the elbow had any association with bone screw failure during removal procedures .
we believe that prior knowledge of the type of metal implanted ( mainly titanium ) and the duration of implantation to be useful information that can help in the preoperative planning of hardware removal procedures .
firstly , this may allow surgeons to request hardware removal kits , thus saving precious operative time .
second , it is our experience that hardware removal procedures are often considered not technically demanding and are often delegated to less experienced surgical staff such as junior residents who may be more likely to break the hardware .
therefore , we believe that experienced staff surgeons should be available during procedures where titanium is being removed . having broken hardware in the elbow may complicate future surgeries in the same region of the limb . with regards to orthopaedic implants
, it is known that both titanium alloy and commercially pure titanium hardware are more predisposed to in situ fracture relative to stainless steel [ 69 ] . as compared to stainless steel ,
titanium alloy is lighter , has a lower modulus of elasticity , and has superior corrosion resistance and biocompatibility , but inferior ductility and notch sensitivity .
the literature search performed for this review did not reveal any previous studies that compare hardware removal from the elbow in vivo for titanium and stainless steel fracture implants .
in contrast to titanium implants remaining in situ for less than 12 months , we observed that the titanium implants remaining in situ for more than 12 months had a tendency to fail during extraction . in this series , it is likely that a combination of titanium alloy 's fatigue properties secondary to notch sensitivity and osseointegration were responsible for this observation .
the fatigue strength of titanium alloy is generally comparable to stainless steel 316l , but notch sensitivity in both commercially pure titanium and titanium alloy has been shown to significantly shorten the fatigue life of these implants in comparison to stainless steel [ 1214 ] .
osseointegration has been observed to occur within 310 months in titanium alloy [ 10 , 11 ] .
the degree of bone ingrowth and on - growth , however , continues to increase for years after initial implantation ( figure 2 ) .
although there have been studies showing evidence of stainless steel osseointegration , it is generally accepted that commercially pure titanium and titanium alloy are more biocompatible and more likely to osseointegrate than stainless steel . in our series , it is likely that as osseointegrataion became more complete , greater removal torques contributed to the failure of titanium alloy screws in this series . given these properties , we postulate that over longer periods and increased loading cycles , the development of micofractures and osseointegration contributed to screw breakage during implant removal .
secondly , the cases studied were not uniform ; there were a wide variety fractures and hardware systems involved .
in addition , due to the small number of cases it was necessary to include multiple surgeons .
in addition , not all x - rays were available for review ; therefore we were not able to account for the type of hardware , such as locking or non locking plate technology . in this study
, there appears to be a time - related association for bone screw failure during removal cases , and for titanium alloy in particular .
this is likely due to the increased bone ingrowth and the adverse effect of notch sensitivity on titanium alloy 's fatigue properties .
|
a retrospective review of 21 patients that underwent bone screw removal from the elbow was studied in relation to the type of metal , duration of implantation , and the location of the screws about the elbow .
screw failure during extraction was the dependent variable .
five of 21 patients experienced hardware failure during extraction .
fourteen patients had titanium alloy implants . in four cases ,
titanium screws broke during extraction .
compared to stainless steel , titanium screw failure during removal was not statistically significant ( p = 0.61 ) .
screw removal 12 months after surgery was more likely to result in broken , retained screws in general ( p = 0.046 ) and specifically for titanium alloy ( p = 0.003 ) .
bone screws removed from the distal humerus or proximal ulna had an equal chance of fracturing ( p = 0.28 ) .
there appears to be a time - related association of titanium alloy bone screw failure during hardware removal cases from the elbow .
this may be explained by titanium 's properties and osseointegration .
|
1. Introduction
2. Methods
3. Results
4. Discussion
|
gossypiboma ( retained surgical sponge ) describes a mass of cotton or sponge that is left behind in the body cavity during a surgical operation .
these foreign bodies can often mimic tumors or abscesses when detected clinically or with radiation .
in fact , surgical sponges without opaque markers are the leading cause for the difficulty of a correct diagnosis . here , we present a case in which a foreign body , gauze without radiopaque markers , was left behind during a procedure that involved the removal of an intrapelvic tumor . after a computed tomography ( ct )
scan had revealed information leading to an inaccurate diagnosis , an accurate diagnosis of gossypiboma was successfully made using endoscopic ultrasound - fine - needle aspiration ( eus - fna ) .
a 60-year - old female patient was referred with an unexpected detection of an intrapelvic tumor during a routine ct scan .
the patient 's medical history included a hysterectomy and a left side ovariectomy at the age of 27 .
the ct scan revealed a 45 mm tumor in the patient 's pelvis [ figure 1a and b ] , which was diagnosed as a gastrointestinal stromal tumor ( gist ) , a chronic expanding hematoma or leiomyoma . a forward - view eus ( tgf - uc260j , olympus optical co. , tokyo , japan ) identified a low echoic lesion when scanned from the sigmoid colon .
fna was performed with a 22-gauge needle ( ezshot2 , olympus optical co. , tokyo , japan ) by using the forward - view eus [ figure 2 ] .
examination of the fna specimen revealed a foreign body ( string of gauze ) displaying macrophage cells and necrotic tissue [ figure 3 ; hematoxylin and eosin , original magnification 40 ] . during the surgery ,
histological examination of the tissue adjacent to the foreign body revealed granuloma formations with fragmented silken threads [ figure 4 ; hematoxylin and eosin , original magnification , 100 and 200 ] .
a : sagittal section ( black arrow ) ; b : horizontal section ( black arrow ) .
endoscopic ultrasound ( eus)-fine - needle aspiration using forward - view eus a foreign body ( string of gauze ) obtained using the 22-gauge needle , with macrophage cells and necrotic tissue granuloma formations with fragmented silken threads
gossypiboma describes a mass of cotton or a sponge that is retained in the body after a surgery .
gossypiboma causes foreign body reactions of the surrounding tissue since they are inert and show no specific decomposition . in this case ,
radiopaque sponges were not used , and there were no adverse symptoms in the 30 years postsurgery .
the typical appearance of gossypiboma shows a spongiform pattern with gas bubbles . in typical cases ,
however , it can be difficult to diagnose the disorder with ct scans in cases without gas bubbles . in the case described in this paper ,
radiologists using findings from a ct scan , initially and incorrectly diagnosed the lesion as gist with fibrosis .
however , the disorder was correctly diagnosed by a team of gastroenterologists using eus - fna .
the eus - fna procedure revealed a segment of string in a fixed foreign body in the pelvis .
the application of eus using curved linear array ( cla ) endoscopes in the lower gi tract has been limited to the rectum and distal sigmoid colon because of the oblique - viewing optics .
there were case reports using oblique - viewing cla echoendoscopes to evaluate lesions proximal to the sigmoid colon , but advancement of the cla echoendoscope beyond the sigmoid required the use of an overtube or a guide wire previously placed with a colonoscope .
binmoeller et al . have reported the feasibility and safety of the front - view forward - array echoendoscope for evaluating right colon lesions .
thus , we used a front - view forward - array echoendoscope to safely evaluate the intrapelvic tumor and sample the tissue .
a correct diagnosis was obtained using this method . in summary , this is the first known case report diagnosing gossypiboma by eus - fna .
|
gossypiboma is a term used to describe a foreign mass within the body that is composed of a cotton matrix .
gossypiboma can cause infection or abscess formation from an early stage , or it can remain clinically silent for many years .
gossypiboma can be diagnosed using several methods including abdominal x - ray films , us of the abdomen , contrast - enhanced computed tomography of the abdomen , and during surgery .
however , the variable appearance of gossypiboma can lead to diagnostic misinterpretations . here , we report a successful diagnosis of gossypiboma using a novel method , that is , endoscopic ultrasound - fine - needle aspiration .
|
INTRODUCTION
CASE REPORT
DISCUSSION
|
a 55-year - old woman visited the emergency clinic of hanyang university seoul hospital with chest pain that had persisted for one month and had become aggravated two days earlier .
ten years before this admission , the patient had undergone double valve replacement with aortic and mitral mechanical valves due to aortic regurgitation and mitral steno - insufficiency .
echocardiography revealed normal motion and function of the mitral and aortic valve prostheses , and coronary angiography findings were also normal .
however , coronary computed tomographic angiography revealed three large lobulated aneurysms with calcified walls bulging from the base of the left ventricle ( fig .
the leak in the left ventricle was located in the sub - mitral valve prosthesis area .
based on this finding , we diagnosed the patient with a pseudoaneurysm of the left ventricle , and surgical treatment was planned .
arterial cannulation via the left femoral artery and venous cannulation via the left femoral vein and left pulmonary artery were performed .
three communicating aneurysmal sacs were observed in a single plane , identical to that observed in coronary computed tomographic angiography .
the neck of the first sac was located at the base of the left ventricle . under cardiopulmonary bypass and fibrillation ,
a defect measuring 15 mm in diameter was identified in the sub - mitral left ventricular wall with calcification but with no infection .
closure of the defect was performed with a supple peri - guard pericardium patch with apex processing ( synovis , st .
the walls of the aneurysmal sacs were closed , and the patient was weaned from cardiopulmonary bypass without difficulty .
the vital signs of the patient remained stable , and she was extubated six hours after the operation .
follow - up echocardiography was performed on postoperative day eight , and no abnormal findings , including cardiac wall motion , were noted .
no leakage at the closed aneurysmal neck was observed , and there was no evidence of recurrence of the pseudoaneurysm ( fig .
she has remained disease - free for six years postoperatively , with regular coumadin anticoagulation therapy due to the presence of the prosthetic aortic and mitral valves .
a left ventricular ( lv ) pseudoaneurysm , or false aneurysm , is defined as a contained rupture or perforation of the myocardium .
the rupture of the myocardium occurs rarely in clinical practice ; it is most often associated with myocardial infarction or cardiac surgery , such as mitral valve replacement ( mvr ) , and is usually fatal [ 24 ] . in 1980 , cobb et al .
roberts and morrow suggested that inadvertent invasion into the lv free wall is possible during excision of the mitral valve if there is poor visualization of the operating field when using the tips of the scissors .
other causes of complete or incomplete rupture of the lv free wall include an oversized prosthetic valve , excessive extirpation of calcium in the mitral annulus , myocardial erosion caused by the struts of the prosthetic valve , the untethering of the fibrous structures of the left ventricle during resection of mitral leaflets , an increase in lv contractility after aortic cross- clamping , enhanced lv wall stress with the support of inotropic agents , and other mechanical trauma between the free wall and the papillary muscles , such as rubber catheter wedging or metal pump suction during valve replacement .
the wall of a false aneurysm comprises the fibrous obliteration of the pericardial sac , resulting from adhesion between the parietal and visceral layer of the pericardium .
while a true aneurysm has nonrestrictive continuity with the lv cavity , a pseudoaneurysm has a defect in the myocardial continuity and a well - defined neck , representing a history of lv wall perforation .
thus , such pseudoaneurysms are more likely to undergo rapid enlargement and rupture than true aneurysms . therefore , surgical correction , including the resection of the aneurysmal sac and patch repair or primary closure of the aneurysmal neck , is indicated and recommended for pseudoaneurysms . in the present case , we diagnosed a lv pseudoaneurysm using coronary computed tomographic angiography .
myocardial infarction was excluded as a cause of the lv pseudoaneurysm because coronary angiography revealed normal findings and cardiac markers were in the normal range , although electrocardiography revealed myocardial ischemia in the inferior wall .
the cause of lv pseudoaneurysm in our patient was a late - term complication of mvr .
echocardiography revealed normal function of the mitral valve prosthesis ; therefore , mitral reoperation was not indicated . in such patients ,
if a repeat sternotomy for entering the operative field is considered , it may be difficult to approach the heart due to massive pericardial adhesions , and the possibility of injury or rupture of the pseudoaneurysm during dissection may be higher .
a direct approach to the operative field via a left lateral thoracotomy can avoid these complications and preserve the previously implanted prosthetic valves .
further , it has other advantages , such as making it easier to establish cardiopulmonary bypass and obviating the need to cross - clamp the aorta , in contrast to the sternotomy approach . in our operation , a fibrillator was used and the exposure of the operative field was excellent . in the operative field , we found three large lobulated pseudoaneurysms with calcified walls in a line , bulging from the base of the left ventricle .
the configuration of the pseudoaneurysms suggested a previous episode of lv rupture , which had developed over time . in patients with lv pseudoaneurysms , there is a well - defined approach to surgical correction .
the neck of the pseudoaneurysm is dissected , and the perforated site can be closed by patch repair or primarily . the mortality rate of this surgery is approximately 10% .
, we have reported a case of lv pseudoaneurysm that occurred as a late complication after mvr .
we performed a left lateral thoracotomy via the fifth intercostal space to approach the operative field directly and successfully repaired the sub - mitral lv pseudoaneurysm using a prosthetic patch .
a few cases of lv pseudoaneurysms have been previously reported ; however , no case reports have previously been presented of korean patients with an lv pseudoaneurysm following mvr who were treated with an operative technique employing a left lateral thoracotomy .
|
we present a case of left ventricular pseudoaneurysm , which is a very rare and fatal complication of cardiac procedures such as mitral valve replacement .
a 55-year - old woman presented to the department of thoracic and cardiovascular surgery at hanyang university seoul hospital with chest pain .
ten years prior , the patient had undergone double valve replacement due to aortic regurgitation and mitral steno - insufficiency .
surgical repair was successfully performed using a prosthetic pericardial patch via a left lateral thoracotomy .
|
CASE REPORT
DISCUSSION
|
data on the adult ( over 20 years ) population has been reported elsewhere ( 5 ) . the present analysis focused on albertan youth < 20 years of age .
flagged in the databases and were defined as any alberta inhabitant registered under the federal indian act and entitled to treaty status with the canadian government .
the status aboriginal identifier captures first nations and inuit peoples both on- and off - reserve with treaty status , but not mtis individuals or aboriginal individuals without treaty status who are included in the general population comparison group .
alberta law requires that all residents and dependants register with the alberta health care insurance plan , thus the alberta central stakeholder registry functioned as a denominator .
diabetes cases were identified by applying the national diabetes surveillance system ( ndss ) algorithm ( 13 ) , which has recently been validated for the determination of pediatric diabetes rates ( 14 ) .
the ndss methodology of identifying has been validated in non - aboriginal populations that include persons of varying geographical locations ( 15,16 ) , and also within an aboriginal community in alberta ( 17 ) .
the algorithm requires an individual to have either 2 physician visits or 1 hospitalization for diabetes ( icd-9 codes starting with 250 and icd-10 codes e10e14 ) within 2 years to be labelled as case of diabetes ( 13 ) .
in contrast to the ndss case definition , pregnant women that may have had gestational diabetes were not excluded in the current analysis due to the elevated risk of subsequent diabetes ( 18 ) . also , since the numbers of diabetes cases are small among this population , only crude , unadjusted rates are provided . for both populations ,
the denominators were based on the health insurance registry ( which provincial law requires all residents and dependants to register ) for the entire population as of june 30 in each year .
the first year in which an individual met the criteria for diabetes ( with no diabetes claims in the preceding 2 years ) was considered an incident year , and considered a prevalent case in subsequent years .
prevalence was determined using the following group - specific formula : total number of youth with diabetes in the calendar year / the total population for the calendar year .
incidence was determined using the following formula : total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases ) .
odds ratios ( or ) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . to examine trends over time , average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 ( rockville , md ) for crude values .
the aapc provides a relative summary measure of the trend over a pre - specified fixed interval .
tests of parallelism were then performed to determine if trends over time differed by group or sex .
the denominators were based on the health insurance registry ( which provincial law requires all residents and dependants to register ) for the entire population as of june 30 in each year . the first year in which an individual met the criteria for diabetes ( with no diabetes claims in the preceding 2 years )
prevalence was determined using the following group - specific formula : total number of youth with diabetes in the calendar year / the total population for the calendar year .
incidence was determined using the following formula : total number of youth with a diabetes incident date for in calendar year/(total population count for calendar year)(prevalent diabetes cases)+(incident diabetes cases ) .
odds ratios ( or ) and 95% confidence intervals were calculated and used to compare the likelihood of being a prevalent case and incident case of diabetes for the 2 populations in 2007 . or 95% confidence intervals that did not include the null value of 1.00 were considered statistically significant with a 5% error . to examine trends over time , average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared using joinpoint version 3.4.2 ( rockville ,
the aapc provides a relative summary measure of the trend over a pre - specified fixed interval .
tests of parallelism were then performed to determine if trends over time differed by group or sex .
based on alberta health and wellness administrative databases , 853,733 youth ( 51.2% male ) were living in the province as of 2007 , of which 50,930 were status aboriginal ( 51.2% male ) . among these youth
a total of 2,589 prevalent diabetes cases were apparent in 2007 , with 140 cases occurring amongst status aboriginal youth .
the 2007 crude prevalence rate of diabetes was 0.27 and 0.31% in the status aboriginal and general population youth populations respectively ( fig . 1 , table i ) .
incidence was 0.59 per 1,000 for status aboriginal and 0.49 per 1,000 for general population .
no discernible differences were seen in diabetes prevalence or incidence with respect to group or sex in 2007 ( fig . 1 ,
crude rates of diabetes among status aboriginal and general population youth in alberta , 19952007 ( adss ) . a = prevalence ; b = incidence .
crude diabetes prevalence and incidence among status aboriginal and general population youth , 2007 crude rates of both diabetes prevalence and incidence increased in status aboriginal and general population youth over the observation period ( fig . 1 , table ii ) .
however , prevalence grew to a greater extent among status aboriginal youth ( aapc 6.98 ) compared to those in the general population ( aapc 3.93 ; p < 0.01 ) , with status aboriginal males experiencing the largest increase in diabetes prevalence ( aapc 9.18 ) .
ethnic differences in diabetes incidence trends were only observed among the male population , which saw a significantly larger rise in incidence for status aboriginal ( aapc 11.65 ) compared to general population males ( aapc 4.62 ; p < 0.01 ) .
male youth , regardless of group , experienced greater increases in both prevalence and incidence over time than that of respective female youth ( table ii ) .
age - specific crude prevalence and incidence of diabetes by group are shown in fig .
2 . age specific crude rates of diabetes among status aboriginal and general population youth , 1995 and 2007 .
ethnicity comparisons of aapc ( average annual percent change ) in diabetes prevalence and incidence among status aboriginal and general population youth , 19952007 p < 0.05 for aapc ethnicity comparison .
paralleling adult populations , diabetes among youth is increasing worldwide ( 20 ) . while type 1 diabetes remains
the majority of cases in youth , type 2 diabetes ( a disease typically regarded as an adult phenomenon ) is becoming progressively more common in children and adolescents ( 20 ) .
we report that youth - onset diabetes is an increasing problem in alberta , especially among status aboriginals .
though our results are in line with international data showing increasing trends ( 20,21 ) , they contradict the recent ndss reports ( 12,22 ) , which found diabetes prevalence and incidence rates remained stable at 0.3 and 0.4 ( per 1,000 ) respectfully among general population youth between 20012006 .
it is possible that differences in ethnicities and/or socioeconomic parameters contribute to these differences . among indigenous populations
especially , including american indian / alaskan natives , australian aborigines , the maori of new zealand , and canadian aboriginals , youth - onset diabetes is increasingly being identified ( 14,2327 ) . in canada
, longitudinal trends of diabetes in aboriginal youth had previously only been explored in manitoba .
dean and colleagues ( 10 ) showed the crude type 2 diabetes incidence rate rose to 0.55/1,000 ( for ages 019 ) in 2001 . similarly ,
crude diabetes incidence increased from 0.22 per 1,000 to 0.59 per 1,000 over a 12 year time span in the current analysis .
the observed accelerated increase in diabetes among status aboriginal youth is consistent with reports of a decrease in the age of diagnosis of diabetes among aboriginal adults . in saskatchewan
type 2 diabetes incidence was highest among status aboriginal adults aged 4049 , while most new diabetes cases among the general population were in those > 70 years of age ( 4 ) .
also , as indicated by the first nations and inuit regional health survey , the majority ( 53% ) of those with diabetes in aboriginal communities were < 40 years of age ( 28 ) . compared to general population , diabetes grew approximately twice as much among status aboriginal youth .
ethnic comparisons have remained largely unexplored in youth , despite numerous studies documenting the divergence in diabetes epidemiology in adult populations . in the beginning of adss observation 1995 , incidence and prevalence of diabetes were distinctly higher among general population . by 2007
however , status aboriginal youth have caught up to the point where no detectable differences were apparent , likely ( as mentioned above ) due to type 2 diabetes . reinforcing this notion ,
a recent national surveillance study amongst practicing physicians found the incidence of type 2 diabetes among aboriginal children ( 0.23/1,000 ) was strikingly 46-fold higher than caucasian children ( 0.005/1,000 ) over a 24 month period ( 29 ) .
we observed an increased rise of diabetes among young status aboriginal males , confirming earlier studies .
sex ratios of prevalence and incidence ( female : male ) of approximately 4:1 and 2:1 in 1995 decreased to 1:1 and 1:1 respectively in 2007 .
additionally , the aapc in prevalence was highest amongst status aboriginal males ( aapc 9.18 ) compared to females ( aapc 5.70 ) . over a 15 year time period in manitoba , the early predominance of youth diabetes cases among aboriginal females ( 8-fold compared to males ) disappeared by 2001 ( 10 ) . from 1990 to 1998
, american indian and alaskan native young males also experienced relative increases in diabetes prevalence almost double that of their female counterparts ( 30 ) .
similarly , the gap in diabetes prevalence between adult status aboriginal females and males is also diminishing ( 4,5 ) .
reasons for the accelerated rise of diabetes among status aboriginal males have not been explored , but may be due in - part to escalating childhood overweight and obesity ( 31 ) with a possible gender - based lag in the obesity epidemic where it may have occurred earlier in time amongst females . in countries with universal healthcare , administrative data have become common and invaluable sources for the population - based surveillance of many chronic diseases .
ndss methodology has been validated in adults and youth , and is a standard for diabetes monitoring at the population level ( 16,18,32,33 ) .
for instance , guttmann et al . ( 33 ) recently found the ndss algorithm in youth ( 019 years of age ) in ontario was associated with 100% sensitivity and 94% specificity . using a similar algorithm ( 4
physician claims over a 2-year period ) , diabetes prevalence rose from 0.18 to 0.24 and incidence ( per 1,000 ) increased from 0.25 to 0.32 among ontarian youth between 1994 and 2003 ( 32 ) . in support , general population youth in the current study experienced nearly identical increases in prevalence and incidence between 1995 and 2003 : 0.19 to 0.25 and 0.22 to 0.32 ( per 1,000 ) respectively , with rates progressively increasing until the end of the study period in 2007 .
we are unable to distinguish the type of diabetes in our administrative data . however , our clinical experience suggest that the majority of the diabetes in aboriginal youth is type 2 diabetes , whereas in the non - aboriginal population it is heavily dominated by type 1 diabetes .
moreover , rates of type 1 diabetes are much lower among american indian and canadian aboriginal youth compared to the general population historically ( 34 ) .
future research aimed at describing the contribution of both type 1 and type 2 diabetes to the observed trends is warranted .
possible gestational diabetes cases were included and may have inflated rates in status aboriginal as gestational diabetes has been shown to be more common in adult aboriginal populations ( 35 ) .
it is possible that increased awareness and screening in youth may have contributed to the observed increases , however for various projects ( http//:www.braiddm.ca ) we have screened 799 youth at risk ( mostly aboriginal ) in rural alberta since 2001 and have found only 3 ( 0.4% ) of those had undiagnosed diabetes ( unpublished ) . by contrast , the rates of undiagnosed diabetes are approximately 4% in at risk adults .
thus , it appears that diabetes in youth is not often asymptomatic , and therefore is less likely to be detected by increased awareness and screening .
our results can not be generalized to non - registered aboriginal or mtis youth , whom could not be identified and were included in the general population group .
also , due to the small number of diabetes cases in youth , age - standardized prevalence and incidence rates were not calculated and only crude rates were reported .
lastly , amendments to the indian act in 1985 are possibly increasing the status aboriginal denominator ( bill c-31 ) and likewise some descendants of status aboriginal people are losing their status through the three generations rule ( 36 ) . taken together , prudence is needed when interpreting the observed epidemiological findings . in conclusion
, diabetes prevalence and incidence has increased among all alberta youth from 1995 to 2007 .
status aboriginal youth , males in particular , experienced a disproportionate growth in diabetes . if unabated , increasing diabetes in youth will likely only further perpetuate the diabetes epidemic in the status aboriginal population .
type 2 diabetes is typically the tip of the iceberg as it is often preceded by several co - morbidities ( obesity , hypertension , metabolic syndrome , pre - diabetes , etc ) , rendering its increased diagnosis in youth a potential public health crisis .
this work was supported in part by a team grant to the alliance for canadian health outcomes research in diabetes ( achord ; reference # : otg-88588 ) , sponsored by the canadian institutes for health research institute of nutrition , metabolism and diabetes .
this study is based in part on de - identified data provided by the alberta health and wellness .
|
objectivesto track and compare trends in diabetes rates from 1995 to 2007 for status aboriginal and general population youth.study designlongitudinal observational research study ( quantitative ) using provincial administrative data.methodsde-identified data was obtained from alberta health and wellness administrative databases for status aboriginal ( first nations and inuit people with treaty status ) and general population youth ( < 20 years ) .
diabetes cases were identified using the national diabetes surveillance system algorithm .
crude annual diabetes prevalence and incidence rates were calculated .
the likelihood of being a prevalent case and incident case of diabetes for the 2 populations was compared for the year 2007 .
average annual percent changes ( aapc ) in prevalence and incidence from 1995 to 2007 were determined and compared between the 2 groups to examine trends over time.resultswhile the prevalence of diabetes was higher in the general population in 1995 , by 2007 there were no between group differences , reflected in the significantly higher aapc of 6.98 for status aboriginal youth . status aboriginal males had a lower diabetes risk in 1995 compared with females , and experienced a greater increase in prevalence over the 13 years ( aapc 9.18 ) so that by 2007 their rates were equivalent to those of the females .
differences in diabetes incidence trends were only observed among male youth , where increases in incidence were greater for status aboriginal ( aapc 11.65 ) compared to general population males ( aapc 4.62 ) ( p = 0.03).conclusionyouth - onset diabetes is an increasing problem in alberta , especially among young status aboriginal males .
|
Methods
Statistical analysis
Results
Discussion
Conflict of interest and funding
|
a case control study was performed using the dutch pharmo record linkage system ( rls ) database ( www.pharmo.nl ) .
the database contains pharmacy dispensing data ( including dispensed drug , type of prescriber , dispensing date , amount dispensed , and written dosage instructions ) of about 1 million dutch residents , linked to a nationwide hospital discharge register .
diagnoses are coded according to the international classification of diseases , 9th revision ( icd-9 ) .
patients are included irrespective of health insurance or socioeconomic status and represent about 7% of the general population .
the pharmo rls database has a high level of completeness , as shown in several independent validation studies .
cases were defined as patients who had sustained their first hip fracture during the 10-year study period ( 1 january 1991 to 31 december 2002 , at least 18 years of age ) . up
to four controls were selected for each case , matched by year of birth , gender , and geographic location .
control patients were registered in the database and had no record for a hip fracture hospitalization .
, we restricted the study population to subjects who were at least 50 years of age at the index date .
history of primary ka before index date was determined using icd-9 surgical procedure code 81.54 .
time since onset ( recency ) of the ka was determined by calculating the time between the index date and the earliest hospital admission for the ka .
we created a proxy for unilateral / bilateral ka by stratifying ka patients into ( 1 ) subjects with one primary ka record before the index date and ( 2 ) those with multiple primary ka records before the index date . in a sensitivity analysis , we stratified patients who had undergone a ka to the region of the body in which oa was recorded .
we used icd-9 codes 715.6 ( oa of lower leg ) as well as 715.0715.5 and 715.7715.9 ( oa of other or unspecified regions ) to identify a history of oa .
in addition , time since onset of oa was calculated similarly to that of time since ka .
odds ratios ( ors ) for fracture risk were estimated using conditional logistic regression ( sas version 9.1.3 , phreg procedure ; sas institute , cary , nc ) .
the following risk factors were considered as potential confounders : use of benzodiazepines in the 3 months before the index date ; use of bronchodilators , inhaled corticosteroids , oral corticosteroids [ 12 , 13 ] , statins , antipsychotics , lithium , antidepressants , beta - blockers , opioids ( tramadol and stronger ) , antiepileptics , thiazide diuretics , renin angiotensin
aldosterone system inhibitors , acid suppressants , two or more dispensings of a nonsteroidal anti - inflammatory drug ( nsaid ) , disease - modifying antirheumatic drugs , organic nitrates , antidiabetic drugs , bisphosphonates , hormone - replacement therapy , calcium / vitamin d supplements , digoxin , and other antiarrhythmics within the 6 months before the index date . in addition , a diagnosis of anemia , mental disorders , impaired renal functioning , skin or subcutaneous disease , any serious injury within the year before the index date , or a history of malignant neoplasm , endocrine disorder , cardiovascular disease , obstructive airways disease , inflammatory bowel disease , musculoskeletal diseases ( excluding oa ) , and connective tissue diseases or rheumatoid arthritis ever before index date were considered as potential confounders .
parameters were included in the final regression model if they independently changed the beta coefficient for arthroplasty with > 5% in the logistic regression model . in a sensitivity analysis , we included use of bisphosphonates and hormone - replacement therapy within 6 months before index date in the final regression model , regardless of the change in beta coefficient for arthroplasty caused by these treatments . the longitudinal relationship between the risk of hip fracture and time since ka was visualized using a smoothing spline regression plot ( sas version 9.1.3 , gplot procedure ) .
a case control study was performed using the dutch pharmo record linkage system ( rls ) database ( www.pharmo.nl ) .
the database contains pharmacy dispensing data ( including dispensed drug , type of prescriber , dispensing date , amount dispensed , and written dosage instructions ) of about 1 million dutch residents , linked to a nationwide hospital discharge register .
diagnoses are coded according to the international classification of diseases , 9th revision ( icd-9 ) .
patients are included irrespective of health insurance or socioeconomic status and represent about 7% of the general population .
the pharmo rls database has a high level of completeness , as shown in several independent validation studies .
cases were defined as patients who had sustained their first hip fracture during the 10-year study period ( 1 january 1991 to 31 december 2002 , at least 18 years of age ) . up
to four controls were selected for each case , matched by year of birth , gender , and geographic location .
control patients were registered in the database and had no record for a hip fracture hospitalization .
, we restricted the study population to subjects who were at least 50 years of age at the index date .
history of primary ka before index date was determined using icd-9 surgical procedure code 81.54 .
time since onset ( recency ) of the ka was determined by calculating the time between the index date and the earliest hospital admission for the ka .
we created a proxy for unilateral / bilateral ka by stratifying ka patients into ( 1 ) subjects with one primary ka record before the index date and ( 2 ) those with multiple primary ka records before the index date . in a sensitivity analysis , we stratified patients who had undergone a ka to the region of the body in which oa was recorded .
we used icd-9 codes 715.6 ( oa of lower leg ) as well as 715.0715.5 and 715.7715.9 ( oa of other or unspecified regions ) to identify a history of oa .
in addition , time since onset of oa was calculated similarly to that of time since ka .
odds ratios ( ors ) for fracture risk were estimated using conditional logistic regression ( sas version 9.1.3 , phreg procedure ; sas institute , cary , nc ) .
the following risk factors were considered as potential confounders : use of benzodiazepines in the 3 months before the index date ; use of bronchodilators , inhaled corticosteroids , oral corticosteroids [ 12 , 13 ] , statins , antipsychotics , lithium , antidepressants , beta - blockers , opioids ( tramadol and stronger ) , antiepileptics , thiazide diuretics , renin angiotensin aldosterone system inhibitors , acid suppressants , two or more dispensings of a nonsteroidal anti - inflammatory drug ( nsaid ) , disease - modifying antirheumatic drugs , organic nitrates , antidiabetic drugs , bisphosphonates , hormone - replacement therapy , calcium / vitamin d supplements , digoxin , and other antiarrhythmics within the 6 months before the index date .
in addition , a diagnosis of anemia , mental disorders , impaired renal functioning , skin or subcutaneous disease , any serious injury within the year before the index date , or a history of malignant neoplasm , endocrine disorder , cardiovascular disease , obstructive airways disease , inflammatory bowel disease , musculoskeletal diseases ( excluding oa ) , and connective tissue diseases or rheumatoid arthritis ever before index date were considered as potential confounders .
parameters were included in the final regression model if they independently changed the beta coefficient for arthroplasty with > 5% in the logistic regression model . in a sensitivity analysis , we included use of bisphosphonates and hormone - replacement therapy within 6 months before index date in the final regression model , regardless of the change in beta coefficient for arthroplasty caused by these treatments .
the longitudinal relationship between the risk of hip fracture and time since ka was visualized using a smoothing spline regression plot ( sas version 9.1.3 , gplot procedure ) .
table 1 shows baseline characteristics of the fracture cases and controls . as expected ( due to matching ) , cases and controls had a similar age and gender distribution .
fracture cases had recently used more medication that has been associated with fracture , such as oral glucocorticoids and strong opioid analgesic . compared to controls ,
they had more often a history of comorbid conditions.table 1characteristics of hip fracture cases and controlscharacteristiccases ( % ) controls ( % ) ( n = 6,763)(n = 26,341)gender female4,929 ( 72.9)19,138 ( 72.7)age ( years ) 18701,641 ( 24.3)6,554 ( 24.9 ) 71802,144 ( 31.7)8,496 ( 32.3 ) > 802,978 ( 44.0)11,291 ( 42.9)use 6 months prior to index date oral glucocorticoids366 ( 5.4)918 ( 3.5 ) paracetamol882 ( 13.0)2,247 ( 8.5 ) > 1 nsaid929 ( 13.7)2,584 ( 9.8 ) opioids253 ( 3.7)455 ( 1.7 ) dmards115 ( 1.7)202 ( 0.8 ) antipsychotics412 ( 6.1)921 ( 3.5 ) calcium / vitamin d supplements362 ( 5.4)894 ( 3.4)hospitalization ever prior to index date osteoarthritis220 ( 3.3)773 ( 2.9 ) rheumatoid arthritis245 ( 3.6)731 ( 2.8 ) musculoskeletal / connective tissue disease ( excluding osteoarthritis)469 ( 6.9)1,328 ( 5.0 ) endocrine disorders199 ( 2.9)381 ( 1.4 ) obstructive airway disease266 ( 3.9)643 ( 2.4)nsaid nonsteroidal anti - inflammatory drug , dmard disease - modifying antirheumatic drug characteristics of hip fracture cases and controls nsaid nonsteroidal anti - inflammatory drug , dmard disease - modifying antirheumatic drug table 2 shows the relationship between time since ka and the risk of hip fracture . in the adjusted analysis , we found a 54% increased risk of hip fracture among patients with ka ( adjusted [ adj . ] or = 1.54 , 95% confidence interval [ ci ] 1.161.99 ) . in the sensitivity analysis ,
use of bisphosphonates and hormone - replacement therapy within 6 months before index date did not substantially change the increased risk ( adj . or = 1.54 , 95% ci 1.192.00 ) .
similarly , the increased risk was not changed when looking at subjects aged 50 years only ( 93.2% of the study population ; adj . or = 1.55 , 95% ci 1.192.01 ) .
there was a suggestion that the increased risk of hip fracture was greatest in the first few years after the first ka ( fig . 1 ) ,
no substantial differences were found with regard to the proxy for unilateral ( adj . or = 1.40 , 95% ci 1.011.95 ) and bilateral ( adj . or = 1.81 , 95% ci 1.202.74 ) ka ( p = 0.34 ) .
furthermore , timing of increased hip fracture risk following the most recent ka was comparable between the two groups ( data not shown).table 2risk of hip fracture with knee arthroplastycases ( % ) controls ( % ) crude or ( 95% ci)adj . or ( 95% ci)(n = 6,763)(n = 26,341)never knee arthroplasty6,674 ( 98.7)26,133 ( 99.2)1.00 referent1.00 referentever knee arthroplasty89 ( 1.3)208 ( 0.8)1.69 ( 1.322.18)*1.54 ( 1.192.00)*<2 years before index28 ( 0.4)69 ( 0.3)1.60 ( 1.032.49)*1.43 ( 0.892.29)25 years before index40 ( 0.6)79 ( 0.3)2.01 ( 1.372.96)*1.96 ( 1.312.92)*>5 years before index21 ( 0.3)60 ( 0.2)1.37 ( 0.832.26)1.08 ( 0.631.85)by number of primary ka records before index one ka record53 ( 0.8)135 ( 0.5)1.54 ( 1.112.12)1.40 ( 1.011.95 ) multiple ka records36 ( 0.5)73 ( 0.3)1.98 ( 1.332.96)1.81 ( 1.202.74)or odds ratio , adj adjusted , ci confidence interval * statistically significant differences compared to referentadjusted for use of benzodiazepines within 3 months prior , use of bronchodilators , antipsychotics , antidepressants , opioids , antiepileptics , disease - modifying antirheumatic drugs , calcium / vitamin d supplements , a history of anemia , skin or subcutaneous disease , or serious injuries 1 year prior , malignant neoplasms , endocrine disorders , cardiovascular disease , obstructive airway disease , inflammatory bowel disease , musculoskeletal / connective tissue disease ( excluding osteoarthritis ) , or rheumatoid arthritis ever before index datefig
. 1smoothed spline visualization of the relationship between time since first ka and adjusted risk of hip fracture .
as shown in table 2 risk of hip fracture with knee arthroplasty or odds ratio , adj adjusted , ci confidence interval * statistically significant differences compared to referent adjusted for use of benzodiazepines within 3 months prior , use of bronchodilators , antipsychotics , antidepressants , opioids , antiepileptics , disease - modifying antirheumatic drugs , calcium / vitamin d supplements , a history of anemia , skin or subcutaneous disease , or serious injuries 1 year prior , malignant neoplasms , endocrine disorders , cardiovascular disease , obstructive airway disease , inflammatory bowel disease , musculoskeletal / connective tissue disease ( excluding osteoarthritis ) , or rheumatoid arthritis ever before index date smoothed spline visualization of the relationship between time since first ka and adjusted risk of hip fracture .
adjusted for confounders as shown in table 2 table 3 shows that the increase in risk of hip fracture in patients with ka was highest in patients aged 1870 years ( adj . or = 2.76 , 95% ci 1.166.59 ) .
the increase in hip fracture risk rapidly decreased toward baseline levels with increasing age ( fig .
( 95% ci 1.152.57 ) , while the risk was no longer elevated in patients who were older than 80 years ( adj . or = 1.16 , 95% ci 0.771.75 ) .
the increase in risk of hip fracture tended to be higher in females ( adj . or = 1.62 , 95% ci 1.222.15 ) compared to males ( adj . or = 0.82 , 95% ci 0.332.03 ) , although this difference did not reach statistical significance.table 3risk of hip fracture with knee arthroplasty stratified by gender , age and medication usecases ( % ) controls ( % ) crude or ( 95% ci)adj . or ( 95% ci)(n = 6,763)(n = 26,341)never knee arthroplasty6,674 ( 98.7)26,133 ( 99.2)1.00 referent1.00 referentever knee arthroplasty89 ( 1.3)208 ( 0.8)1.69 ( 1.322.18)*1.54 ( 1.192.00)*by gender males7 ( 0.1)24 ( 0.1)1.17 ( 0.502.74)0.82 ( 0.332.03 ) females82 ( 1.2)184 ( 0.7)1.76 ( 1.352.29)*1.62 ( 1.222.15)*by age ( years ) 187013 ( 0.2)13 ( 0.0)4.18 ( 1.909.19)*2.76 ( 1.166.59 ) * 718043 ( 0.6)95 ( 0.4)1.82 ( 1.262.63)*1.72 ( 1.152.57 ) * > 8033 ( 0.5)100 ( 0.4)1.27 ( 0.851.89)1.16 ( 0.771.75)by use of pain relievers 6 months before yes43 ( 0.6)75 ( 0.3)2.25 ( 1.553.28)*1.93 ( 1.282.91 ) * no46 ( 0.7)133 ( 0.5)1.37 ( 0.971.92)1.33 ( 0.931.89)by use of oral corticosteroids 6 months before yes6 ( 0.1)12 ( 0.0)1.94 ( 0.735.18)1.41 ( 0.474.21 ) no83 ( 1.2)196 ( 0.7)1.68 ( 1.292.17)*1.56 ( 1.182.05)*by use of calcium / vitamin d supplements 6 months before yes7 ( 0.1)18 ( 0.1)1.77 ( 0.734.30)1.40 ( 0.553.58 ) no82 ( 1.2)190 ( 0.7)1.64 ( 1.252.15)*1.56 ( 1.182.06)*or odds ratio , adj adjusted , ci confidence interval*statistically significant differencesadjusted confounders as shown in table 2 compared to referent , except for the stratified covariate of interestopioids ( tramadol or stronger ) , paracetamol , or more than one nsaid prescriptionfig
. 2smoothed spline visualization of the association between first ka and adjusted risk of hip fracture , by age at the index date . dashed lines represent 95% confidence interval bands . adjusted for confounders as shown in table 2 risk of hip fracture with knee arthroplasty stratified by gender , age and medication use or odds ratio , adj adjusted , ci confidence interval * statistically significant differences adjusted confounders as shown in table 2 compared to referent , except for the stratified covariate of interest opioids ( tramadol or stronger ) , paracetamol , or more than one nsaid prescription smoothed spline visualization of the association between first ka and adjusted risk of hip fracture , by age at the index date .
as shown in table 2 ka patients who were dispensed pain relievers ( opioids [ tramadol or stronger ] , paracetamol , or more than one nsaid prescription ) 6 months before the index date did not have a significantly higher risk of hip fracture ( adj . or = 1.93 , 95% ci 1.282.91 ) compared to patients without a history of pain reliever use ( adj . or = 1.33 , 95% ci 0.931.89 ) 6 months before ( p = 0.17 ) ( table 3 ) .
similarly , patients who had used oral corticosteroids in the 6 months before ( adj . or = 1.41
, 95% ci 0.474.21 ) were at the same risk of hip fracture compared to patients without use of oral corticosteroids in the same period ( adj . or = 1.56 , 95% ci 1.182.05 ) .
table 4 shows that the association between ka and hip fracture did not substantially change when kas were restricted to patients with a history of oa . the proportion of lower leg oa in patients who had undergone ka was 85% .
ka patients with lower leg oa had the same risk of hip fracture ( adj . or = 1.45 , 95% ci 1.081.95 ) compared to ka patients without correction of lower leg oa ( adj . or = 1.54 , 95% ci 1.192.00).table 4risk of hip fracture with knee arthroplasty among osteoarthritis of lower legcases ( % ) controls ( % ) crude or ( 95% ci)adj
. or ( 95% ci)(n = 6,763)(n = 26,341)never knee arthroplasty6,674 ( 98.7)26,133 ( 99.2)1.00 referent1.00 referentever knee arthroplasty89 ( 1.3)208 ( 0.8)1.69 ( 1.322.18)*1.54 ( 1.192.00)*by osteoarthritis at any site never before index12 ( 0.2)21 ( 0.1)2.23 ( 1.104.55)*1.84 ( 0.883.85 ) ever before index77 ( 1.1)187 ( 0.7)1.63 ( 1.242.13)*1.48 ( 1.111.97)*by site of osteoarthritis lower leg73 ( 1.1)179 ( 0.7)1.61 ( 1.222.12)*1.45 ( 1.081.95 ) < 2 years before24 ( 0.4)61 ( 0.2)1.56 ( 0.972.50)1.39 ( 0.832.32 ) 25 years before32 ( 0.5)64 ( 0.2)1.98 ( 1.293.03)*2.00 ( 1.293.11 ) * > 5 years before17 ( 0.3)54 ( 0.2)1.24 ( 0.722.15)0.93 ( 0.511.69 ) other regions4 ( 0.1)8 ( 0.0)2.00 ( 0.606.64)2.17 ( 0.617.66)or odds ratio , adj adjusted , ci confidence interval * statistically significant differences compared to referentadjusted for confounders as shown in table 2 risk of hip fracture with knee arthroplasty among osteoarthritis of lower leg or odds ratio , adj adjusted , ci confidence interval * statistically significant differences compared to referent adjusted for confounders as shown in table 2
this study showed a 1.5-fold increased hip fracture risk in patients who had undergone a ka .
the risk of hip fracture was greatest in young patients ( 1870 years ) . with increasing age , we found a rapid decrease in strength of association , which was no longer elevated in patients aged 81 years and above .
the association tended to be greater during the first few years after surgery , but it did not reach statistical significance .
recent use of pain relievers or glucocorticoids did not alter the overall risk of hip fracture .
this is the second study that has evaluated risk of hip fracture in patients with a history of ka and is in line with the first study , which showed a 58% increased risk of hip fracture in british patients within the first year after their ka .
studies that investigated the association between oa ( the main indication for ka ) and risk of hip fracture have yielded conflicting findings .
some authors suggested a decreased fracture rate among patients with oa [ 57 ] , possibly due to higher bmd levels .
although data are controversial , patients with oa may have increased osteoblastic activity at the oa site , resulting into higher bmd levels and therefore lower fracture rates . on the other hand
, others reported an increased hip fracture risk , which is in line with our study results .
found an increased risk of both vertebral ( 2.0-fold ) and nonvertebral ( 1.5-fold ) fractures in patients with knee
oa . similarly , arden et al . demonstrated that patients with knee pain or a clinician diagnosis of knee oa have an increased risk of hip and nonvertebral fractures .
this is probably explained by an increased severity of falls since they could not detect an increased number of falls .
it should be noted , however , that data collection on falls is often incomplete .
this could explain the results of a different study that found an increased occurrence of falls among patients with lower limb oa .
furthermore , looking at differences in fracture types , arden et al . and vestergaard et al
. found a substantially higher increase in risk of hip fracture compared to other fractures ( such as distal forearm fractures ) .
this may suggest an important role for the nature of falls in patients with knee oa , as explained by arden et al . a us case
control study has shown that hip fractures tend to result from falling sideways or straight down ( low walking speed ) , whereas forearm fractures may be more likely to be the result of falling backward .
overall , our findings support the studies that found an increased hip fracture risk , indicating that the increased number and severity of falls may attenuate any potentially beneficial effects of higher bmd levels on fracture risk in these patients .
given this proposed mechanism , we would have expected hip fracture rates to decrease after the ka procedure as the surgery relieves pain and partially restores the biomechanical properties of the knee .
one study reported fewer falls within 1 year after the ka , while a recent danish study could not detect any decreases in hip fracture rates during that period . in line with the danish study and
the british study mentioned earlier , we found no obvious decrease in hip fracture risk shortly after the surgery .
a possible explanation for our findings is that patients become rapidly more active after their ka , due to effective knee pain relief .
this could also explain our observed effect modification by age : the youngest patients were at highest hip fracture risk .
compared with elderly patients , these patients may be more likely to increase their physical activity quickly after surgery .
in addition , residual knee pain and stiffness in the first months after surgery may be present in some ka patients and could further explain our observed increased hip fracture risk .
strengths of our study include its population - based setting and that it had a reasonable sample size and longitudinal data collection [ 10 , 1214 ] .
linkage with the dutch national hospitalization registry assured routinely collected ka surgeries and hip fractures .
limitations include the lack of data on physical activity , which could be an alternative explanation for our observed association between ka and hip fracture .
physical activity is significantly increased in ka patients within 9 months postoperatively , while a rapid increase could potentially initiate falls .
in addition , we did not have data on body mass index ( bmi ) , which could have underestimated our observed association between ka and hip fracture .
an increased bmi is a well - known risk factor for knee oa , while it is inversely associated with risk of hip fracture .
nevertheless , our findings are similar to the bmi adjusted results from the general practice research database ( gprd ) study .
similar to the british study , we could not differentiate between the sides of ka or the sides of hip fracture .
this information could be helpful in understanding the mechanism of the observed increased risk of hip fracture following ka .
local bone loss may be induced on the side of the replaced knee , possibly resulting in an increased fracture risk of the hip on the same side . the only feasible way to investigate
this is to link a dedicated joint registry to a hospital / general practitioner database , which has been planned for the uk national joint registry and the gprd .
furthermore , we did not have data on bmd or falling , which could have been useful for the assessment of causality and the underlying mechanism .
frail , unexposed subject bias may have occurred if ka patients had lower mortality rates compared to subjects who had not undergone ka ( due to clinical assessment of operative risk ) .
this was probably not the case : within our control subjects ( those without a hip fracture ) , proportions of cardiovascular hospitalizations were not lower in ka patients ( 6.7% ) compared to patients without a history of ka ( 4.9% ) .
unfortunately , we did not have data on other fracture types ( such as distal forearm fractures ) .
as our data source only keeps track of hospitalizations , fractures other than those of the hip would suffer from underrecording .
although oa diagnosis and ka surgery have not been validated in this data source , we expect high completeness for ka registration .
our hospitalization source was primarily designed to keep track of economic parameters ( e.g. , health - care cost ) . given the high cost of ka surgery , we would expect adequate recording of this procedure . in conclusion
, we showed that ka was associated with a 54% increased risk of hip fracture , which was not influenced by recent use of pain relievers or corticosteroids .
the increase in risk was highest among younger patients ( < 71 years ) , which may reflect a rapid increase in physical activity immediately after surgery .
risk assessment of hip fracture could therefore be considered in patients who are about to undergo a ka .
|
the majority of knee arthroplasties ( kas ) are performed in patients with osteoarthritis ( oa ) .
although bone mass may be increased in these patients , subjects with knee oa may have an increased risk of hip fracture , possibly due to an increased severity of falls .
however , in patients with kas , risk of hip fracture has not been studied extensively .
we evaluated the association between kas and hip fracture risk in a population - based case
control study using the dutch pharmo record linkage system ( 19912002 , n = 33,104 ) .
cases were patients with a first admission for hip fracture ; controls were matched by age , gender , and geographic location .
neither group had a previous history of fracture .
time since first ka was calculated .
analyses were adjusted for disease and drug history .
a 54% increased hip fracture risk was found in patients who underwent ka ( adjusted [ adj . ] or = 1.54 , 95% ci 1.192.00 ) .
we found a strong effect modification by age in these patients : the youngest patients ( aged 1870 years ) were at more increased risk for hip fracture ( adj . or = 2.76 , 95% ci 1.166.59 ) , while we could not detect a statistical increase in patients aged > 80 years .
furthermore , the association tended to be greater during the first few years after surgery , although it did not reach statistical significance .
we found that kas are associated with a 54% increased risk of hip fracture , in particular among adult patients aged < 71 years old .
fracture risk assessment could be considered in patients who are about to undergo a ka .
|
Methods
Settings and Study Design
Cases and Control Subjects
KA Definition
Statistical Analysis
Results
Discussion
|
digital radiograph or radiovisiography was first described in 1987 and was commercially introduced by trophy radiologie in collaboration with mouyen12 in 1993 .
three systems are employed for radiovisiography : the ccd sensor ( charge - coupled device ) , cmos sensor ( complementary metallic oxide sensor ) , and psps ( psps phosphor storage plate optical plate ) ; the latter includes the digora , digident , denoptix and den ortix systems11 .
however , some doubts remain in the use of radiovisiography based on the optical plate system as to the durability of the plate3 , image quality according to the processing delay1,2,14 , and storage conditions10 .
no study has yet investigated the influence of utilization of opaque cases on the quality of digital image .
this ex vivo study evaluated the quality of digital radiographic images obtained with the digora system according to the processing delay and maintenance of optical plates in either opaque ( supplied with the system ) or transparent protective plastic cases during this period .
the study was conducted on a dry human mandible fixed on an acrylic resin base .
a plastic tube was fixed frontally to the mandible to standardize the position of the x - ray source , thus standardizing the vertical and horizontal angles ( figure 1 ) .
a groove was prepared on this resin base to standardize the position of the optical plate .
radiographs were obtained from the mandibular molar region by utilization of optical plates with an x - ray unit ( gnatus model xr 6010 ; gnatus , ribeiro preto , sp , brazil ) set at 60 kvp and 10 ma , with open tube , total filtration of 1.5 mm al , exposure time of 0.3 s , and optical plate of the digora system ( soredex , orion corporation , finland ) .
two groups were constituted for x - ray exposure : in group a , the optical plate was inserted and sealed in the opaque plastic case supplied by the manufacturer and kept as such until processing ; in group b , the optical plate was stored in a transparent plastic case ( figure 2 ) .
the optical plates were processed in the digora scanner immediately and 5 , 60 and 120 min after x - ray exposure , and were transferred to the computer for analysis of digitized images .
analysis was performed directly on the computer screen by three examiners using the digora for windows software .
the images were analyzed as to brightness , contrast and resolution of tooth structures ( enamel , dentin , root and pulp cavity ) and periapical structures ( periodontal space , cortical bone , alveolar bone ) , which received scores 0 to 2 , as follows : 0- poor image quality ; 1- good image quality ; 2- excellent image quality .
data were submitted to the kruskal - wallis non - parametric test , and comparison of results between opaque and transparent cases were submitted by the mann - whitney test .
table 1 presents the scores assigned by examiners to the images , according to processing delays and utilization of opaque or transparent plastic cases .
there was statistically significant difference ( p<0.05 ) regarding the processing delays , namely immediate , 5 and 60 min with utilization of opaque case ( table 2 ) .
this difference was observed between the 120-min delay and the other periods ( table 3 ) .
no statistically significant difference ( p>0.05 ) was observed among processing delays with utilization of transparent case ( table 4 ) .
comparison of results between opaque and transparent cases did not reveal significant differences ( p>0.05 ) ( table 5 ) .
10.05882 ; chi - square with 3 degrees of freedom ; probability = 0.018072 ( significant ) u=5.00000000;exact probability = 0.150794 ; normal approximation : z=1.96396101 ; probability = 0.04953461 hc= 5.164999 ; chi - square with 3 degrees of freedom ; probability = 0 .
digital radiography was introduced to dental practice with a view to replace the conventional radiography due to the shorter exposure to radiation , achievement of high - quality images , possibility of adjustments with aid of software , and comparable reliability to conventional radiography , for both endodontic diagnosis and treatment3,57 ,
the main disadvantage of the optical plate system is the need of additional time for processing and possible loss of quality when the sensitized optical plate is exposed to light . some authors have emphasized that , after exposure to x - ray , the optical plates should be processed within 1 h in order to avoid loss of image quality1,2 .
other authors believe that the plates can be processed within 6 h if stored in appropriate cases10 . in the present study , a processing delay of 120 min caused a mild reduction in image quality , yet without compromising the quality of diagnosis ( table 1 ) .
it should be highlighted that the images were not altered as to the brightness and contrast provided by the system , being analyzed as produced on the computer screen .
together with the digora system , the manufacturer supplies opaque plastic cases that offer protection against contamination and the deleterious effect of light . images of optical plates maintained in the original opaque cases after exposure to x - ray maintained their image quality up to 120 min ( table 2 ) , with significant difference between the 120- min processing delay and the other periods ( table 3 ) .
when the optical plates were maintained in transparent cases , there was also a progressive reduction of image quality ( table 1 ) , which was more accentuated at 120 min ( table 4 ) , though without statistical difference among periods .
this is due to the fact that the optical plate protected with transparent cases is subjected to the continuous action of light , with progressive loss of image quality .
however , images obtained with optical plates protected with both opaque and transparent cases and with processing delays of up to 120 min provided good quality for diagnosis .
the findings of the present study revealed that the processing delay of 120 min for the digora system caused a reduction in image quality , yet without interfering with the quality of diagnosis .
the opaque case supplied by the system 's manufacturer provided better protection to the optical plate than the transparent case .
|
this ex vivo study evaluated the quality of digital radiographic images obtained with the photostimulable phosphor plate system ( digora ) according to the processing delay and maintenance of optical plates in either opaque ( supplied with the system ) or transparent protective plastic cases during this period .
five radiographs were obtained from the mandibular molar region of a dry human mandible using optical plates .
these plates were placed in the protective plastic cases before obtaining the radiographs and were processed immediately or after processing delays of 5 , 60 and 120 min , when the case was removed .
the results revealed a reduction in image quality when processing was delay 120 min compared to the other times .
the opaque case provided better protection to the sensor than the transparent case . in conclusion ,
a 120-min processing delay for the digora system caused a reduction in image quality , yet without interfering with the quality of diagnosis .
the opaque case supplied by the system 's manufacturer provided better protection to the optical plate than the transparent case .
|
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
|
complication following fracture of a central venous catheter can be catastrophic to both the patient and the attending doctor .
catheter fracture has been attributed to several factors namely prolong mechanical force acting on the catheter , and forceful removal or insertion of the catheter .
the tip of the catheter was notably missing , and an emergency chest radiograph confirmed our diagnosis of a retained fracture of central venous catheter .
the retained portion was removed by the interventional radiologist using an endovascular loop snare and delivered through a femoral vein venotomy performed by the surgeon .
endovascular approach to retrieval of retained fractured catheters has helped tremendously to reduce associated morbidity and the need for major surgery .
the role of surgery has become limited to instances of failed endovascular retrieval and in remote geographical locations devoid of such specialty .
werner forssman first reported the insertion of a central venous catheter ( cvc ) almost 86 years ago .
thirty years later , introduction and wide spread use of the seldinger technique made the procedure safer and easily reproducible ( 1 ) .
patients with end stage renal impairment are often the most common subset of patients whom require insertion of central venous catheters .
the catheter acts as a hemodialysis access point pending creation of a more permanent arterio - venous fistula ( avf ) .
it is also inserted in patients presenting with blocked avf , for the purpose of emergency hemodialysis ( 2 ) .
central venous catheter related complications can be classified into mechanical events , infection and venous thrombosis ( 3 ) .
since the first report of an embolised catheter fragment in 1954 , overall complication rates have remained low and are only reported to occur in less than 1% of cases .
prior to introduction of endovascular approach , retrieval of a fractured segment almost always required high risk open surgery such as thoracotomy ( 5 ) . since the late 70 's , combined endovascular and open approach has seen much success and gained popularity as first line treatment in the advent of a retained fractured central venous catheter ( 6 ) .
limitation of the use of this approach would be in rural health care settings devoid of access to an interventional radiologist .
we report our experience with a successful retrieval of a central venous catheter utilizing the combined endovascular and open approach .
a third year medical resident noticed an incomplete catheter tip post removal of a right subclavian central venous catheter in a 56 year old man diagnosed to have end - stage renal disease .
the patient was otherwise well and did not complain of chest pain or shortness of breath .
the catheter had been earlier used as a temporary access for hemodialysis pending maturation of a more permanent brachiocephalic arterio - venous fistula ( avf ) . during the visit to the nephrology outpatient clinic
, it was decided that the avf was ready for use and that the central venous catheter was no longer needed .
emergency chest radiograph was requested , and it confirmed the diagnosis of retained fractured central venous catheter ( figure 1 ) .
the case was discussed with the interventional radiologist , and a central venogram was performed to identify the exact location of the fractured segment and to plan for retrieval approach .
the venogram noted the fractured catheter segment to lie at the junction of the right brachioceohalic vein and subclavian vein ( figure 2 ) .
following discussion with the vascular surgeons interventional radiologists , decision was made to proceed with combined endovascular and open approach of retrieval . under image intensifier guidance ,
a 15 mm amplatz gooseneck snare ( ev3 inc , usa ) was inserted through the right femoral vein , and the fractured catheter fragment pulled - back all the way to the groin region .
the surgical team then externalized the retrieved fragment through a femoral vein venotomy , all the while ensuring good proximal and distal control ( figures 3 , 4 ) .
the venotomy site was then closed using non - absorbable sutures in the usual manner .
the patient was discharged the next day and continued with his hemodialysis through his matured brachiocephalic avf .
chest radiograph showing the right subclavian catheter prior to planned removal retained portion of catheter seen on venogram the tip of the retained part as being extracted from femoral vein via groin incision the fractured retained part of the catheter after being completely retrieved in one piece
central venous catheters are increasingly used worldwide in critically ill patients for administration of intravenous resuscitation fluids and medication , parenteral nutrition , chemotherapy agents , and monitoring of central venous pressure .
they have become ubiquitous in the intensive care unit and are available as single and multi - lumen variety . despite being a commonly performed procedure , it is not devoid of risks and complications .
mechanical complications include hemorrhage , pneumothorax , cardiac arrhythmias , malposition , catheter fracture and distant embolization of the fractured fragment into cardiac chambers or pulmonary artery .
venous thrombosis and central vein occlusion have also been noted to occur with long - term central vein catheter placement ( 3,4 )
patients with a fractured catheter may remain asymptomatic or experience shortness of breath , chest pain and even syncope .
diagnosis is often incidental and made in the presence of a radiopaque fragment on routine chest radiograph .
incomplete catheter tip noted during the process of catheter removal may also give rise to clinical suspicion that would warrant further radiological confirmation .
mechanism of catheter fracture has been largely attributed to mechanical shearing forces acting on the catheter over a prolonged period .
although it is a less popular approach , the supraclavicular technique is actually a safer option as it avoids many of the complications otherwise noted in the infraclavicular counter - part .
complications such as guide wire kinking , catheter compression and catheter fracture are less frequently seen in supraclavicularly placed catheters .
the reason for the apparent superiority has been studied and linked to the coaxial lie of the catheter itself within the vessel , and absence of need to maneuver through narrow musculo - osseous spaces ( 7 ) .
besides being fractured , the retained portion of the catheter may also dislodge and embolise distally .
mortality rate associated with intravascular foreign body embolization has been reported to range anywhere from 24% to 60% ( 3,8 ) .
retrieval of retained intravascular devices may be approached through the conventional open technique or percutaneous method .
to date , there are no consensus or clear guidelines for management of a fractured and retained central venous catheter . over the years
, endovascular approach to retrieval of such intravascular foreign bodies has been reported with much success ( 6 ) .
the lack of need for major open surgery has propelled its popularity due to the obvious reduction in overall morbidity and mortality .
patients who were subject to the endovascular approach were also able to assume normal activity of daily living sooner and experience early return to work . given such positive results , conventional surgical approaches have been limited to instances of failed endovascular approach or in rural areas devoid of access to an interventional radiologist .
percutaneous retrieval of intravascular foreign bodies was initially reserved for retained catheters and wire fragments . in recent years
, the spectrum of intravascular device and objects has broadened significantly to include items such as vena cava filters , embolization coils , and endovascular stents . a wide variety of percutaneous tools
they include snares , biopsy forceps , dormia baskets , and tip - deflecting wires ( 6 ) .
the amplatz gooseneck snare ( ev3 inc , usa ) used in our patient is a nitinol based micro snare that has gained tremendous popularity over the years . the snare 's super - elastic construction makes it less likely to be kinked or deformed during introduction into the vessel of choice .
the snare catheter also contains gold tungsten loops that provide excellent radiopacity for improved procedural precision ( 9 ) .
given the possible catastrophic complications following fracture of an intravascular catheter or device , it has become prudent to ensure that all cases of catheter fracture or retained catheter fragments are treated as an emergency situation that mandates prompt removal .
due to the superiority and good safety profile , percutaneous retrieval technique should be the treatment of choice ( 8) .
loop snares such as the amplatz gooseneck snare ( ev3 inc , usa ) should be used when and if available . in the advent of failure to retrieve the retained catheter using a loop snare ,
the likelihood of success with other percutaneous tools is low , and open surgery should be considered ( 8,9 ) .
this , however , does not rule out the need for surgery in case of failed endovascular therapy .
|
backgroundcomplication following fracture of a central venous catheter can be catastrophic to both the patient and the attending doctor .
catheter fracture has been attributed to several factors namely prolong mechanical force acting on the catheter , and forceful removal or insertion of the catheter.case detailsin the present case , the fracture was suspected during the process of removal .
the tip of the catheter was notably missing , and an emergency chest radiograph confirmed our diagnosis of a retained fracture of central venous catheter .
the retained portion was removed by the interventional radiologist using an endovascular loop snare and delivered through a femoral vein venotomy performed by the surgeon.conclusionendovascular approach to retrieval of retained fractured catheters has helped tremendously to reduce associated morbidity and the need for major surgery .
the role of surgery has become limited to instances of failed endovascular retrieval and in remote geographical locations devoid of such specialty .
|
Background
Case Details
Conclusion
Introduction
Case Report
Discussion
|
in 1959 ernst mayr ( figure 1 ) flung down the gauntlet at the feet of the three great population geneticists ra fisher , sewall wright and jbs haldane ( figure 2 ) : " but what , precisely , " he said , " has been the contribution of this mathematical school to the evolutionary theory , if i may be permitted to ask such a provocative question ? " his skepticism arose in part from the fact that the mathematical theory at the time had little to say about speciation , mayr 's major interest .
a particular focus was the simplification that he called " beanbag genetics " , in which " evolutionary genetics was essentially presented as an input or output of genes , as the adding of certain beans to a beanbag and the withdrawing of others . " .
photograph reproduced with permission from the archives of the ernst mayr library of the museum of comparative zoology , harvard university .
( a ) r a fisher ( 18901962 ) , ( b ) sewall wright ( 18891988 ) photograph reproduced with permission from the capitol times , and ( c ) j b s haldane ( 18921964 ) .
mayr was , however , criticizing textbook simplifications , rather than the actual work of the three pioneers .
far from treating gene frequency changes as analogous to the consequence of beans jostling at random in a bag , both fisher and wright considered gene interactions in detail .
fisher ( figure 2a ) showed that , despite interactions between genes , natural selection acts on the additive component of the genetic variance .
the beanbag criticism was particularly inappropriate for wright ( figure 2b ) , who specifically devised his ' shifting balance ' theory as a way for a population to go from one harmonious gene combination ( mayr would say " integrated genotype " ) to another when intermediates were disadvantageous . who was to answer mayr 's criticism ?
fisher was already dead , and in any case preferred attack to defense , and wright was too gentle though admittedly not always when mayr was involved : returning from italy where he had received the prestigious balzan prize in 1984 , wright told me that the value of the prize was considerably diminished when he discovered that mayr had won it the year before . in the event , however , it was haldane ( figure 2c ) who took up the challenge .
this was haldane at his best witty , spirited , informed , interesting and convincing .
but the larger question remains : what indeed has been the contribution of mathematical theory to evolution ?
mathematics is not central to evolution in the way it has been in theoretical physics .
solid advances have been made without using mathematics , much being due to mayr himself . and these continue .
yet , i shall argue that mathematical ideas have made important , and often essential , contributions , and still do .
many concepts that are now established were arrived at mathematically , although their origins have since been forgotten .
for example , the idea that polymorphisms become stabilized in populations because heterozygotes are at an advantage is now found in elementary textbooks , but fisher was the first to formulate it .
loss of heterozygosity with inbreeding is also textbook knowledge , but it was not clear until wright developed the theory and invented a simple algorithm for quantifying it . similarly , the idea that the impact of mutation on the population depends on the mutation rate rather than the magnitude of the mutant effect is now taken for granted , but that was not known until haldane showed it mathematically .
one final example is the inheritance of the abo blood groups , which was in doubt from the time of their discovery at the turn of the twentieth century until bernstein 's mathematical population analysis in 1924 .
all of these applications used only elementary methods , and they must have been known to mayr . often , concepts that were developed mathematically were later explained in intuitive , non - mathematical ways .
it 's a lot easier to find an intuitive explanation when you already know the answer .
his theory of " genetic revolutions " assumed that from a well integrated population , genetic drift in a small founder offshoot will sometimes produce a population with a new set of genotypes integrated in a new way .
intuitively , a small founder population seemed a particularly unlikely place to find a new favorable gene combination , and this was indeed shown to be the case in a very detailed mathematical analysis by barton and charlesworth .
if mayr had had more respect for mathematical population genetics , he never would have made what most theorists regard as the mistake of proposing that small founder populations are a likely source of major evolutionary changes by genetic drift .
partly this is due to skilled mathematicians entering the field and bringing new techniques with them ; especially noteworthy are stochastic processes .
an additional influence is the explosive growth of molecular data , which lend themselves to mathematical treatment . in the first half of the twentieth century
, population genetics and evolution had a beautiful theory , but there were very limited opportunities to apply it .
what are some of the newer developments in evolution that are owed to mathematical theory ? here are a few .
one striking result in the post - mayr period was motoo kimura 's neutral theory , independently developed in 1968 by him and by jack king and thomas jukes .
these writers shocked the biological world by arguing that the bulk of molecular evolution is due to selectively neutral mutations driven by the mutation process rather than selection .
i think it would please mayr that the general idea that the rate of evolution in the population is equal to the rate of mutation in a single individual can be derived by simple reasoning using school mathematics . yet , in order to apply the idea , we need to know how long a time period must be observed .
this depends on how long it takes for a lucky new mutant to increase in frequency and completely replace its predecessors .
when selection and migration are taken into account , the theory is much more complicated .
one contribution of the neutral theory has been to provide a rationale for a molecular clock .
essentially , all our estimates of evolution rates depend on the assumption that the molecular changes used in constructing the clock are mutation - driven .
fortunately , enough of the dna does not have an obvious function and can reasonably be supposed to be evolving by neutral kinetics , or near enough so that the neutral theory can be used in practice . and
a second important attribute of the neutral theory is that it supplies a natural null hypothesis for the study of selection . and
yet another outgrowth of the neutral theory is the view that much of the molecular polymorphism in natural populations is effectively neutral .
this is especially useful now that variation in the frequencies of single - nucleotide polymorphisms ( snps ) is easily observed .
the various measures that are used to quantify genetic variability are outgrowths of population genetics theory .
one striking result of such theory is the realization that all of the worldwide human population is descended from africa , and moreover from a small area within africa .
the evidence for this striking conclusion is that molecular variance is greater in african peoples than elsewhere .
the molecular clock can be used as one measure of the time taken during various human migrations and , of course , homo sapiens is not the only species that can be studied in this way .
a new favorable allele arises by mutation , spreads through the population and becomes fixed at a rate that is determined mainly by how favorable it is .
a consequence of this fixation is that neutral or weakly selected alleles linked to the locus are swept along with it . because of this , there is a region on either side of the selected locus that is deficient in genetic variability .
such regions of reduced variability are footprints of a selective sweep in the past and , remarkably , provide evidence for events that occurred long ago and which can no longer be observed . although the basic idea is simple and requires no mathematics , an assessment of how much the variability is reduced and the linkage distance over which the reduced variability occurs depend on mathematical theory .
an area of biology in which mathematics , and especially computers , have become absolutely essential is systematics , ernst mayr 's own field .
formerly , assessing species relationships and building phylogenetic trees based mainly on morphological differences was a matter of intuition and judgment .
a mammalian dna sequence supplies billions of bits of information , thus for the first time providing an opportunity for a procedure independent of personal judgments . in recent decades
the preferred procedures , such as fisher 's maximum likelihood , required a great deal of computation , and for a while this meant that large phylogenies were out of computer range .
it is now de rigueur to do statistical tests of significance of the tree structure and parts thereof . many of these involve permutation methods , which have the merit of requiring minimum assumptions .
they are computation - intensive , but with modern computers this is no longer an impediment .
one striking example from such studies , which came as a complete surprise to classical systematists , is the close relationship of the elephant to the shrew .
molecular analysis of dna sequences , using the newly developed theory , has shown that our closest relatives are chimpanzees . furthermore , that we and the chimpanzees are 99% identical at the dna level came as a surprise to many .
equally surprisingly , we share some 90% of our dna with mice , rabbits , dogs , horses and elephants .
these numbers are fully consistent with expectations based on mutation rates and the times involved . finally , there is now help available in the form of computer programs that can work out phylogenies and display the information graphically ( see ) .
these not only eliminate a lot of tedious work , but place advanced methods in the hands of relative novices .
finally , there has been a major theoretical advance , coalescent theory . instead of looking forward in time
any two alleles or homologous nucleotides are ultimately derived from a single one ; that is , looking backward , they coalesce .
one problem for which coalescent theory provided at least an approximate answer is the question of whether there was any mating between our ancestors and contemporary neanderthals .
small amounts of admixture are not ruled out , but coalescent theory has shown that any substantial intermating is very unlikely , as discussed by john wakeley .
my other examples have been relatively simple , but this one is n't , as is apparent from this discussion .
another example , also given by wakeley , is evidence for a selective sweep in drosophila simulans . until recently ,
mayr emphasized allopatric speciation and the prevailing model , due to dobzhansky and muller , prevailed .
recent mathematical studies support it and favor the view that speciation genes correspond to normal genes , selected for their effects within the species .
thus , hybrid incompatibility is a by - product of ordinary selection in geographically isolated populations .
there is no evidence that random drift plays an important part , so mayr 's ' genetic revolution ' and similar ideas have little support . yet
it is important to point out that , aside from this , mayr has usually been right .
the field of mathematical studies of speciation is barely started ; it will surely increase .
i have given only a few examples of the part that mathematical theory has played in evolution studies .
there are many more , but these , i hope , constitute a convincing sample of the importance of mathematics in population genetics and evolution .
i do not intend to imply that all evolutionary study need be mathematical and theory - driven .
for example , ' evo - devo ' studies , looking at changes in development during evolution , have produced exciting results while largely ignoring population genetics .
another non - mathematical example is horizontal gene transfer brought about by transposable elements , which is especially important in the evolution of microorganisms .
there is also abundant evidence for increases in genetic complexity by the accumulation of small duplications . and , as always , a lot of morphological and behavioral evolution is interesting in and of itself . yet
, my guess is that as these subjects become more quantitative , population genetic theory will play an increasing role .
the rise of molecular methods has led to an increase in the importance of mathematics in population genetics and evolution . the abundance of data that require mathematical analysis has greatly increased . at the time of mayr 's challenge
now the situation is reversed : data appear faster than existing theory can deal with them . that mathematics will play
i think these examples show not only that mathematical theory is helpful , but that it is often essential .
he might have had a change of mind , but i doubt it . knowing how much he enjoyed arguing , i suspect he would be quite critical of much that i have written .
unfortunately , although he lived to be 100 , he was not immortal and died in 2005 .
were he still alive , i would surely hear from him and whatever his opinions , he would not keep them to himself .
he would have enjoyed an argument , preferably over a glass of sherry . and so would i.
i am indebted to bret payseur for reading the manuscript and offering some very useful suggestions .
|
in 1959 ernst mayr challenged the relevance of mathematical models to evolutionary studies and was answered by jbs haldane in a witty and convincing essay .
fifty years on , i conclude that the importance of mathematics has in fact increased and will continue to do so .
|
None
Neutral theory, molecular clocks and selective sweep
Mathematics and the computation of family relationships
Coalescence and speciation
Looking to the future
Envoi
Acknowledgements
|
testosterone is essential for the normal growth and development of the prostate gland and is also a possible risk factor for prostate cancer .
huggins and hodges 's seminal work in the 1940s first demonstrated the hormone dependence of prostate cancer , consequently establishing testosterone as a key therapeutic target for managing prostate cancer .
androgen deprivation therapy to lower the serum testosterone level remains a standard treatment for advanced disease to the present day .
isbarn et al 's recent studies , however , show a result opposite that of huggins and hodges , implying that testosterone neither increases the risk of prostate cancer nor causes cancer recurrence in men who have been treated successfully for prostate cancer .
a recent study by morote et al showed that prostate cancer risk and tumor aggressiveness are unrelated to serum testosterone .
other recent epidemiologic studies also have found no association between testosterone and prostate cancer [ 6 - 10 ] . our main objective was to analyze the relationship between serum testosterone , prostate - specific antigen ( psa ) , and prostate cancer risk in high - risk patients .
the study population consisted of 120 men with a psa level of 10 ng / ml or higher .
the data were collected from january 2008 to january 2010 . to determine the relationship between testosterone , psa , and prostate cancer risk in a high - risk group , we limited our study population to men with a psa level of 10 ng / ml or higher .
we excluded from this analysis men who were taking medications known to lower psa , such as finasteride or dutasteride .
twelve cores were obtained and two extra biopsies were taken if hypoechoic or otherwise suspicious areas were noted on ultrasound .
serum testosterone levels were measured by using a solid - phase competitive chemiluminescent enzyme immunoassay with the advia centaur immunoassay system ( bayer health care , chicago , il , usa ) .
we classified the men as hypogonadal if their serum testosterone level was < 300 ng / dl , according to the criteria used by rhoden et al . for possible correlation between serum testosterone , psa , and prostate cancer , we included age , psa density ( psad ) , prostate volume , and gleason score for patients with prostate cancer .
statistical analysis was performed by using the student 's t - test , the pearson correlation test , simple linear regression , and binary logistic regression . odds ratios with 95% confidence intervals ( cis ) for psa , psad , serum testosterone , and age were determined to predict prostate cancer risk .
all data are presented as the mean2 sd to define and characterize quantitative variables and as percentages to characterize qualitative variables ; we considered a p - value<0.05 to be statistically significant .
out of 120 patients , the samples of 85 ( 70.1% ) patients were diagnosed as benign and those of 35 ( 29.2% ) patients were diagnosed as being prostate cancer .
there was no significant difference in the patient 's average age , psa , prostate volume , or testosterone between patients without and those with prostate cancer .
only psad differed significantly between the two groups ( t - test , p=0.012 ) ( table 1 ) .
hypogonadal and eugonadal men did not differ significantly in cancer detection rate ( 30.2% vs. 32.0% , respectively ) .
the testosterone level did not differ significantly between patients without and those with prostate cancer in either hypogonadal ( 248.1650.41 ng / dl vs. 251.3543.61 ng / dl , respectively ) or eugonadal ( 501.52137.06 ng / dl vs. 506.22126.40 ng / dl , respectively ) men ( t - test , p>0.05 ) .
testosterone was unrelated to age , psa , psad , or prostate volume in both men without and those with prostate cancer , and prostate cancer aggressiveness also was unrelated to serum testosterone in prostate cancer patients ( p>0.05 ) ( table 2 ) .
the binary logistic regression also confirmed that none of the variables used in this study was an independent predictor of prostate cancer risk for men with a psa level > 10 ng / ml ( p>0.05 ) ( table 3 ) .
further evaluation with simple linear regression confirmed that testosterone and psa did not show any correlation ( p>0.05 ) ( fig .
when we divided the patients into 11 groups according to their testosterone level , within a 150 ng / dl range , prostate cancer prevalence did not change as the testosterone level increased ( p>0.05 ) ( table 4 ) .
in men , the leydig cells in the testes produce approximately 90% of testosterone ; the adrenal glands produce the rest .
testosterone plays a key role in the development of male reproductive tissues such as the testes and prostate gland . under testosterone influence , the prostate gland experiences two main growth periods .
the first occurs early in puberty , when the prostate doubles in size . at around age 25 ,
when the prostate is growing , testosterone is converted into dihydrotestosterone ( dht ) , which is the androgen receptor 's major activator .
after dth binds to androgen receptors , it translocates to the nucleus , where it mediates the transcriptional activation of target genes .
these androgen - stimulated changes in gene expression promote cellular growth , which often results in benign prostatic hyperplasia in elderly men .
long - term cessation of the prostate 's exposure to androgen appears to protect against the development of cancer , but no dose - response relationship between testosterone level and cancer risk has been established .
the prostate cancer risk for men with a testosterone concentration in the normal range remains unclear .
recent chemopreventive trials with 5-alpha - reductase inhibitors show the role of testosterone in prostate cancer development .
finasteride reduced the risk of prostate cancer by 24.8% compared with a placebo in healthy men .
recently , the reduce study showed that dutasteride also reduced the risk of prostate cancer by 23% compared with a placebo in healthy men .
huggins and hodges first showed the effect of testosterone on prostate cancer patients in 1941 .
they showed the hormonal responsiveness of prostate cancer by reporting that markedly reducing the testosterone level by castration and estrogen treatment caused metastatic prostate cancer to regress , and injecting testosterone caused prostate cancer to grow . in 1995
wu et al found that the distribution of dihydrotestosterone - to - testosterone ratios parallels both the incidence of and mortality from prostate cancer .
the demonstration that androgen suppression effectively treats advanced prostate cancer , and the fact that elevated serum androgen levels might predispose people to prostate cancer , have attracted persistent interest .
however , a recent study refuted any connection between elevated testosterone levels and increased prostate cancer risk .
endogenous hormones and prostate cancer collaborative group et al meta - analyzed the serum concentrations of sex hormones from subjects in 18 prospective studies that included 3,886 men with incident prostate cancer and 6,438 control subjects .
they concluded that endogenous hormones , including testosterone , were not associated with prostate cancer .
morote et al recently conducted another study of 478 patients , all of whom were assessed by trus - guided prostate biopsy because of an abnormal digital rectal examination , psa > 4 ng / ml , or both .
they found no difference in serum testosterone level between patients with and those without prostate cancer and concluded that the serum testosterone level was not associated with prostate cancer risk or aggressiveness .
an animal study by morgentaler and traish showed that beyond a certain serum testosterone concentration , androgens have a limited ability to stimulate prostate cancer growth .
subsequent increases in serum testosterone levels beyond that concentration did not stimulate the prostate because the binding capacity of the intra - prostatic androgen receptors had been saturated .
our results are similar to those of the above studies even though we limited our study population to patients with psa > 10 ng / ml . we found that the serum testosterone level at the time of diagnosis was unrelated to psa and prostate cancer risk and aggressiveness in both hypogonadal and eugonadal patients .
ideas about the interaction between testosterone and prostate cancer have changed considerably over the past decade . like our study , most recent epidemiologic studies have found no association between testosterone and prostate cancer [ 6 - 10 ] .
a few studies published during the past decade contrast with our results , however , and show that low testosterone levels seem to be related to an increased risk of prostate cancer and tumor aggressiveness , such as a high gleason score [ 23 - 28 ] . in our study , we compared age , psa , prostate volume , and psad with testosterone .
only a known prostate cancer predictor , psad , showed a significant difference between patients with and those without prostate cancer , even in high - risk patients with a psa level of 10 ng / ml or higher .
patients without prostate cancer had relatively lower psa and larger prostates than did patients with prostate cancer .
however , our binary logistic regression showed that psad was unsuitable as an independent predictor of prostate cancer risk in men with a psa level of 10 ng / ml or higher .
we measured the serum testosterone level in the patients only once , before their biopsy was done .
second , we checked only their total testosterone level and were unable to check their free testosterone level .
, the present study found that the serum testosterone level at the time of diagnosis was unrelated to psa and prostate cancer risk and aggressiveness . because testosterone levels change with age and time , a prospective study with long - term testosterone monitoring is required to find a relationship between testosterone and prostate cancer .
our results show that the serum testosterone level at the time of diagnosis was unrelated to psa and prostate cancer risk and aggressiveness .
additional studies with long - term follow - up are needed to explain the possible mechanism and relationship between testosterone , prostate cancer , and psa .
|
purposetestosterone is essential for the prostate gland 's normal growth and development and is also a possible risk factor for prostate cancer .
this study 's aim was to determine the significance of serum testosterone for prostate - specific antigen ( psa ) elevation and prostate cancer prediction in high - risk men.materials and methodsthe study included 120 patients with psa > 10 ng / ml who underwent a transrectal - prostate biopsy .
serum testosterone , prostate volume , and psa density ( psad ) were checked in all patients .
patients were divided into two groups , patients with and those without prostate cancer ; and testosterone - related factors , prostate volume , psa , psad , age , prostate cancer prediction rate , and cancer aggressiveness were evaluated.resultsthirty-five patients ( 30.2% ) were confirmed as having prostate cancer . the average serum testosterone level in patients without and in those with prostate cancer was 452.25154.62 ng / dl and 458.10158.84 ng / dl , respectively ; average psa was 17.589.02 ng / ml and 18.626.53 ng / ml , respectively ; and average age was 69.027.52 years and 70.697.02 years , respectively ( p>0.05 ) . hypogonadal and eugonadal
patients showed no significant difference in cancer prevalence ( 30.3% vs. 32.0% , respectively ) .
the testosterone level did not differ significantly in patients with and those without prostate cancer in either hypogonadal or eugonadal men ( p>0.05 ) .
serum testosterone showed no correlation with psa , psad , or age in either group ( p>0.05 ) and was unrelated to prostate cancer risk or aggressiveness ( p>0.05).conclusionsin our study 's results , serum testosterone at the time of diagnosis was unrelated to psa elevation , prostate cancer risk , and aggressiveness .
|
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
|
amniotic band syndrome has many synonyms such as , congenital constriction rings , amniotic bands , limb body wall complex , adam complex
this syndrome comprises a variety of anatomical abnormalities among newborns , and is associated with intrauterine struggling by the fetus in the uterus , thereby leading to deformations , malformations , or ruptures .
amniotic band syndrome occurs in around 1:1,200 to 1:1,500 live newborns , but lack of correct diagnosis certainly underestimates the real occurrence rate .
the clinical manifestations of amniotic band syndrome are extremely variable , and their extent may range from a single abnormality , like a constriction ring , to multiple abnormalities .
almost all cases are sporadic , even though some rare evidences of familial cases have been described .
limb constrictions occur most frequently , ranging in severity from deformity of the distal part of the limb to partial or total amputation .
craniofacial abnormalities are the most serious type , because of the importance of the organs involved .
visceral abnormalities are a rare form of the syndrome , among which gastroschisis is the most frequent type .
generally the diagnosis is made by means of two - dimensional ultrasonography ( 2dus ) at the end of the first or the beginning of the second trimester of pregnancy .
three - dimensional ultrasonography ( 3dus ) in rendering mode allows reconstruction of the surface of the fetus , thus , enabling spatial analysis . in cases of fetal malformation ,
only three case reports describing the use of 3dus for diagnosing amniotic band syndrome exist in the literature . in two of them ,
3dus was used at the end of the first and the beginning of the second trimester , and only one case was in the third trimester .
we present a case of amniotic band syndrome diagnosed in the 34 week , in which 3dus enabled clear analysis of this malformation , as well as better understanding and better family counseling .
her prenatal care had begun late , and she had only undergone one ultrasound examination , in the 20 week , that had not shown any fetal abnormalities .
all the prenatal laboratory tests were normal , and the patient said that she had not been making any personal use of medicines or using any illicit drugs , and that there were no cases of malformations or chronic diseases like arterial hypertension and diabetes mellitus among members of her family .
2dus showed normal fetal growth , with a weight prediction of 2.242 g , amniotic fluid index ( afi ) of 140 , and anterior placenta .
there was an accessory lobe on the left lateral posterior wall , and a membrane ( amniotic band ) was present between this lobe and the main placental mass .
morphological analysis showed two bands adhering to the right forearm of the fetus , but with normal movements of this limb and its fingers [ figure 1 ] .
3dus was performed using a multifrequency volumetric transducer ( accuvix v20 ; samsung medison , seoul , korea ) and clearly showed the spatial relationship between the bands and the body of the fetus , thus proving that the bands had really adhered to the forearm , but without strangling it [ figure 2 ] .
the 3d image allowed the parents to gain a better understanding of the condition and enabled better counseling toward a good prognosis for the fetus .
the pregnant woman had her delivery scheduled as a caesarian section in the 38 week , and the postnatal phenotypic examination confirmed the diagnosis of amniotic band syndrome without injury to the forearm of the fetus .
the newborn underwent surgery to remove the bands on the third day of life and was subsequently discharged in a good general condition .
two - dimensional ultrasonography shows adherence of two amniotic bands to the right forearm of the fetus ( white arrows ) .
( a ) and ( b ) three - dimensional ultrasonography in rendering mode , shows amniotic bands adhering to the right forearm of the fetus ( white arrows ) , without constriction rings .
two theories have been formulated in attempts to explain the pathogenesis of amniotic band syndrome .
the streeter theory , which was accepted for a long time , takes the view that imperfect local embryonic development would indicate the abnormalities observed .
the theory put forward by torpin in 1965 is the one that is most accepted .
this author took the view that the problem begins because of rupturing of the amniotic sac , thereby separating the chorion and amnion , such that some of the amniotic liquid goes out into the chorionic cavity .
the fibrotic strings formed from the chorion would prevent normal development of some anatomical parts of the fetus , thus resulting in different kinds of abnormalities . according to higginbottom et al .
, three mechanisms would explain the role of the bands in originating the malformations : a ) interruption of the normal morphogenesis , for example , at the time of the facial fusion process ; b ) deformation due to compression of parts of the fetus ( oligohidramnios ) or interlacing of an anatomical part of the fetus by a band ; or c ) injury to a part of the fetus that had already formed , like in the cases with constriction rings around the limbs .
one of the possible causes of an amniotic lesion is maternal abdominal trauma ; other , less likely causes might be drug use or ammonites .
some cases of amniotic band syndrome in patients that underwent amniocentesis on the beginning of the 2 trimester and chorionic villus sample have been described .
rare descriptions of intrafamilial repetitions of constriction rings or congenital limb amputation give rise to the assumption that a genetic contribution could occur in some cases . in our case
, the patient did not have any antecedents , so we could assume that this case had a sporadic origin .
the late diagnosis was a consequence of prenatal care that had begun late and of an ultrasound scan performed in the 20 week that did not show any fetal abnormalities .
3dus enabled spatial analysis on the fetus proving that the bands had adhered to the surface of the right forearm of the fetus .
moreover , 3dus provides proof that the prognosis for the fetus is good when it does not show any strangling of the limb , and it allows better understanding and better parent counseling .
described a case of amniotic band syndrome diagnosed in the 14 week by 3dus , which was associated with severe fetus malformations , and in which the parents decided to terminate the pregnancy .
likewise , hata et al . described two cases of this syndrome diagnosed in the 13 and 14 weeks , in which 3dus made it possible to conduct analysis on the spatial relationship between the bands and the fetus , as well as enabling counseling for the parents .
the case of amniotic band syndrome with the latest diagnosis by 3dus ( and the only one in the third trimester until now ) was described by paladini et al .
these authors described a case in the 28 week , similar to ours , but in their case a constriction ring was observed on the forearm of the fetus and was confirmed after birth .
in summary , this is the second case description of late diagnosis of amniotic band syndrome by 3dus .
this method was shown to be important for proving that the bands had adhered to the limb of the fetus and showing that the prognosis is good when no constriction ring is demonstrated .
in addition , 3dus allowed better understanding of the pathology , better counseling for the parents , and a good prognosis for the newborn .
|
amniotic band syndrome is characterized by a build - up of bands and strings of fibrous tissue that adhere to the fetus and can compress parts of the fetus , thus causing malformations and even limb amputation while the fetus is still in the uterus .
the clinical manifestations are extremely variable and their extent may range from a single abnormality , like a constriction ring , to multiple abnormalities .
such abnormalities are generally diagnosed at the end of the first or the beginning of the second trimester using two - dimensional ultrasonography ( 2dus ) .
three - dimensional ultrasonography ( 3dus ) in rendering mode allows spatial analysis of the fetus and amniotic band , thus enabling better comprehension of this pathological condition and better counseling for the parents .
there has not previously been any evidence to show that 3dus would be useful in cases of late diagnosis ( third trimester ) of amniotic band syndrome . in the present case
, a primigravid woman underwent her second obstetric ultrasound scan in the 34th week , from which we observed two bands in contact with the right forearm , but with normal movement of this limb and its fingers .
3dus made it possible to see the spatial relationship of these bands to the fetal body , thereby confirming their adherence to the limb .
after the birth , the prenatal diagnosis of amniotic band syndrome without limb constriction was confirmed .
a surgical procedure was carried out on the third day after birth to excise the bands , and the newborn was then discharged in a good general condition .
|
INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION
|
mobile health , which is the use of mobile communication and computing technologies for medicine and public health , is rapidly expanding.1,2 a personal health record ( phr ) is defined as a health record created using mobile computing technologies in which health information and personal health data are maintained by the patient.3 models of phr vary across a large range .
one phr model utilizes patient - generated data about health and lifestyle that are recorded using a personal computer or web application and that help address specific health concerns.4,5 because a phr records health - related data generated by a patient , it is not only a repository of that patient s data but also a tool that facilitates interactions between the medical provider and patient via the provision of related health information.68 additionally , because technologies are designed to streamline the diagnosis and treatment processes , and big data analytics offer novel perspectives regarding the contribution of health data to health care , phrs provide the opportunity for a greater degree of interaction when managing health in one s daily life.9 several recent studies have used phrs to examine patient health and activity levels or to assess the management of patients through interventions outside medical institutions .
druss et al10 evaluated the effects of electronic phrs on the quality of medical care in 170 patients with serious mental illnesses who were treated at a community medical center and found that the use of phrs significantly improved the quality of medical care and increased the use of medical services by patients .
espie et al11 created a mobile health care application for the management of sleep disorders , which involved the development and psychometric validation of a brief scale ( the sleep condition indicator [ sci ] ) , to evaluate sleep disorders in everyday clinical practice .
these authors found that web - based cognitive behavioral therapy had a positive effect on the treatment of insomnia.12 park et al9 developed a phr based on teen - specific needs to promote better self - awareness and chronic disease management and determined that chronic psychological or physical states require constant attention because the symptoms can fluctuate spontaneously over time .
moreover , although patients often focus on immediate problems , the use of phrs may provide a tool that can gain the attention of individuals and remind them of these problems when not immediately apparent.9 taken together , these findings suggest that phrs constitute a useful tool to record the range of , and changes in , physical and psychological health states outside hospitals . a growing body of research has called attention to the influence of emotion on health and the possible management of the relationship between these two variables .
the instantiation of an affective state directly involves alterations in multiple physiological systems of the body , which leads to physiological responses that can directly influence physical health depending on the nature , frequency , and time course of the emotional state.13 physiological responses are meant to be adaptive in the short term but can lead to maladaptive outcomes in the long term if not correctly regulated.14 furthermore , recent evidence has sufficiently demonstrated the importance of comorbid relationships among emotional , psychological , and physical symptoms.1520 for example , a worldwide survey using a national representative sample identified an association between chronic pain and mental disorders.2123 similarly , there is a growing consensus that negative emotions influence the development of cardiovascular diseases24 and that chronic digestive disorders are closely linked with a variety of psychological disorders , including depression.25,26 taken together , these studies provide a clear indication that chronic physical symptoms are best understood in the context of psychological factors . in this respect ,
the use of phrs could provide a strategy for improving medical care for patients with comorbid psychological and physical illnesses . in the current study , a mobile application called mind mirror
was developed as a phr to evaluate the daily affective states and physical conditions of individual patients .
using participant - generated health records , detailed information on health was collected on a daily basis through the participants engagement and self - monitoring .
this mobile platform was developed in order to collect health records while maximizing the patient s self - assessment in terms of paying attention to their physical condition and feelings throughout the day .
a well - known example of self - assessment of daily experiences is the day reconstruction method ( drm ) , in which a participant reviews daily affective experiences and the subjective feelings.27,28 this was a 30-day observational study that aimed to characterize the dynamic relationship between the emotional state of patients and their physical states , including pain and fatigue , and to determine whether self - recorded tools such as phrs would be useful for the assessment of the relationship between an individual s emotional and physical conditions .
the inclusion criteria for the current study were as follows : participants were required to 1 ) be between 20 and 40 years of age , 2 ) have no history of any neuropsychological disorder or acute or chronic pain disorder , and 3 ) not be taking any type of medication . written informed consent was obtained prior to participation .
this study was conducted in accordance with the guidelines issued by the human subjects committee and approved by the institutional review board of kyung hee university in seoul , republic of korea .
the primary goals for the mind mirror mobile application were to assess the daily emotional and physical changes of patients and to provide a platform from which large - scale data could be collected and analyzed .
a novel user interface format was applied to measure the affective and physical states of a participant every 2 hours from 9:00 am to 9:00 pm for 30 days .
the natural starting point of research on the dynamic nature of these symptoms lies in the analysis of symptoms measured over different time points .
the analysis of time series data requires detailed records that reflect subtle changes in the emotional and physical conditions of individuals over time within a single day . in the current study
, participants completed the entries of their daily emotional and physical states using the mind mirror software ; they were instructed to complete the data input into the application at night prior to going to bed .
data regarding emotional valence , fatigue , and pain were entered by the participants over 30 days at seven time points each day between 9:00 am ( 09:00 ) and 9:00 pm ( 21:00 ) .
of the eligible participants ( n=21 ) , one participant was unable to finish the study due to loss of mobile phone ; thus , the final data analyses in the current study included 20 participants ( eight females , mean age = 24.7 years ) .
for the current study , an iphone operating system ( ios ) software application was developed using xcode 6.4 and the programming language
swift ; this application required ios 8.4 software development kit ( sdk ) or higher .
explicit methods were used to record the subjective ratings of the participants regarding their physical conditions and subjective valences of daily emotion .
the explicit method collects data on emotions and physical conditions that the participant cognitively feels by asking direct questions or presenting straightforward tasks regarding their daily condition.29 upon turning on the application , a title page with a logo , as well as a message that reads
once the participant has clicked on this message , the application begins its initial steps .
this application applied two main methods to measure explicit emotions or feelings that were presented as tasks for the participants to complete on a daily basis .
the first measure was designed to quantitatively visualize positive or negative affective states . on the screen , three auxiliary lines with the numbers 5 , 0 , and
the words pleasant and unpleasant were placed beside the numbers 5 and 5 to indicate positive and negative emotional valences , respectively .
circles that could be dragged were located on the middle line , and time points shown in units of 2 hours from 9:00 am to 9:00 pm were visible .
the participants moved the circles up and down on a vertical plane within the range of 5 to 5 to express their emotional states ( figure 1b ) .
the second measure depicted six basic emotions as described by ekman : anger , disgust , fear , joy , sadness , and surprise.13 the emotions were presented on each vertex of a hexagon , and the relative intensities of the six distinct emotions were defined as the circumplex model
. moreover , 10 small circles , which were depicted as drops of water , were presented at the center of the hexagon , and a single drop of water moved to attach to the corresponding emotion when the user touched any of the six emotions a single time .
each participant was asked to touch the emotions in order to distribute the 10 drops of water as a description of their overall emotional state for the day ; it was not necessary to use all 10 drops of water ( figure 1c ) .
the data from this measure were collected from the participants as well , but not used for the study analysis . the first step in determining a participant s physical state involved asking questions about the weather during each day using simple pictures . after choosing the weather ,
the participants were asked to complete their assessment of the length and quality of the previous night s sleep and to evaluate their digestive function throughout the day .
the last step involved assessing one chronic condition that the participant wanted to monitor for 30 days , which was reported prior to starting the study ; the personalized chronic symptoms ranged from eye irritation to dizziness . to assess the chronic symptom , each participant moved a button across a bar that was labeled at each end to indicate the worst and best conditions .
this system applied a variation of the visual analog scale ( vas ) in which performances in computer - based and web - based research have been validated.30 while categorical scales reach an ordinal - scale level , the vas extends the precision and discrimination of daily reports ( figure 1d ) .
the data from this measure were collected from the healthy participants and not used for the study analysis .
two main methods were applied to measure physical conditions according to time . like the emotional tasks , these were presented as tasks for the participants to complete on a daily basis and quantitatively described the positive or negative physical states of each participant .
the first task involved assessing one s overall pain and the second task involved assessing one s overall fatigue throughout the day . on the screen , three auxiliary lines with the numbers 10 and zero next to the top and bottom lines , respectively , were provided . the words extremely severe and none were placed besides the numbers 10 and zero to indicate the intensities of pain and fatigue .
circles that could be dragged were located on the middle line and time points shown in units of 2 hours from 9:00 am to 9:00 pm were visible .
the participants moved the circles up and down on a vertical plane within the range of 100 to express their physical states throughout the day ; the same procedure was applied for both pain and fatigue ( figure 1e and f ) . because this study focused on the dynamic relationship between emotional and physical states , records with a more frequent temporal resolution ( units of 2 hours ) were analyzed ; thus , a time series analysis of the data of 20 participants was conducted to determine the correlations between the emotional and physical states .
the collected data of the participants during the 30-day study were placed in a property list ( .plist ) file format that was converted to a comma separated values ( .csv ) file for the data analyses .
there were three different categories of time series data emotional valence , pain , and fatigue which illustrated the experiences and emotional states of the participants on a daily basis .
an interpolation process was applied to complete the data for time points between 11:00 pm and 7:00 am when the participants were assumed to be asleep . using this preprocessing procedure , five time points for a single night , or 150 time points in total ,
were filled in to complete the data for the 30-day study period . to fill in the empty time points , the function
in the forecast package of r ( http://www.r-project.org ) , which assumes a linear relationship , was used to complete the data set . following the preprocessing procedure ,
three sets of time series data for each of the 20 participants were available for data analysis .
a logarithmic transformation was applied to each of the emotional , pain , and fatigue series to ensure the normality and homogeneity of variance of the residuals .
cross - correlations between the emotional valence and pain , emotional valence and fatigue , as well as fatigue and pain time series were analyzed by participant , and the correlation coefficients were averaged to visualize the correlation analysis .
r software ( version 3.2.3 , wooden christmas tree ) and r studio ( version 0.99.892 ) were used for the data analyses . to investigate the possible relationships between emotional valence and physical states using the data of the 20 participants ,
a regression model was applied with consideration given for the random effects of the participants . when selecting a regression model to determine whether there were associations among emotion , fatigue , and pain , it was assumed that there were concurrent dynamics between emotion and the body based on cross - correlation results .
accordingly , a mixed model for multilevel data with a combination of between - subject and within - subject factors was used .
while some methods for time series analyses account for random effects in multilevel data,31 many other methods examine the concurrent changes between multiple time series without lagged effects .
thus , a single model was selected to analyze the three series to determine whether any associations existed .
the multilevel regression model , or the mlm,32,33 allows for the estimation of hierarchically structured longitudinal data on the individual and group levels . for the current data , a mixed model with participants as the random effect without temporal dislocation was applied because cross - correlation analyses of the series unanimously showed the highest correlation at lag 0 .
based on the regression beta coefficients , the dynamic structure between emotional valence , pain , and fatigue was visualized in a network ; the green line indicates a positive relationship , whereas the red line indicates a negative relationship .
the inclusion criteria for the current study were as follows : participants were required to 1 ) be between 20 and 40 years of age , 2 ) have no history of any neuropsychological disorder or acute or chronic pain disorder , and 3 ) not be taking any type of medication . written informed consent was obtained prior to participation .
this study was conducted in accordance with the guidelines issued by the human subjects committee and approved by the institutional review board of kyung hee university in seoul , republic of korea .
the primary goals for the mind mirror mobile application were to assess the daily emotional and physical changes of patients and to provide a platform from which large - scale data could be collected and analyzed .
a novel user interface format was applied to measure the affective and physical states of a participant every 2 hours from 9:00 am to 9:00 pm for 30 days .
the natural starting point of research on the dynamic nature of these symptoms lies in the analysis of symptoms measured over different time points .
the analysis of time series data requires detailed records that reflect subtle changes in the emotional and physical conditions of individuals over time within a single day . in the current study
, participants completed the entries of their daily emotional and physical states using the mind mirror software ; they were instructed to complete the data input into the application at night prior to going to bed .
data regarding emotional valence , fatigue , and pain were entered by the participants over 30 days at seven time points each day between 9:00 am ( 09:00 ) and 9:00 pm ( 21:00 ) . of the eligible participants ( n=21 ) , one participant was unable to finish the study due to loss of mobile phone ; thus , the final data analyses in the current study included 20 participants ( eight females , mean age = 24.7 years ) .
for the current study , an iphone operating system ( ios ) software application was developed using xcode 6.4 and the programming language
swift ; this application required ios 8.4 software development kit ( sdk ) or higher .
explicit methods were used to record the subjective ratings of the participants regarding their physical conditions and subjective valences of daily emotion .
the explicit method collects data on emotions and physical conditions that the participant cognitively feels by asking direct questions or presenting straightforward tasks regarding their daily condition.29 upon turning on the application , a title page with a logo , as well as a message that reads
once the participant has clicked on this message , the application begins its initial steps .
for the current study , an iphone operating system ( ios ) software application was developed using xcode 6.4 and the programming language
swift ; this application required ios 8.4 software development kit ( sdk ) or higher .
explicit methods were used to record the subjective ratings of the participants regarding their physical conditions and subjective valences of daily emotion .
the explicit method collects data on emotions and physical conditions that the participant cognitively feels by asking direct questions or presenting straightforward tasks regarding their daily condition.29 upon turning on the application , a title page with a logo , as well as a message that reads
once the participant has clicked on this message , the application begins its initial steps .
this application applied two main methods to measure explicit emotions or feelings that were presented as tasks for the participants to complete on a daily basis .
the first measure was designed to quantitatively visualize positive or negative affective states . on the screen , three auxiliary lines with the numbers 5 , 0 , and
the words pleasant and unpleasant were placed beside the numbers 5 and 5 to indicate positive and negative emotional valences , respectively .
circles that could be dragged were located on the middle line , and time points shown in units of 2 hours from 9:00 am to 9:00 pm were visible .
the participants moved the circles up and down on a vertical plane within the range of 5 to 5 to express their emotional states ( figure 1b ) .
the second measure depicted six basic emotions as described by ekman : anger , disgust , fear , joy , sadness , and surprise.13 the emotions were presented on each vertex of a hexagon , and the relative intensities of the six distinct emotions were defined as the circumplex model .
moreover , 10 small circles , which were depicted as drops of water , were presented at the center of the hexagon , and a single drop of water moved to attach to the corresponding emotion when the user touched any of the six emotions a single time .
each participant was asked to touch the emotions in order to distribute the 10 drops of water as a description of their overall emotional state for the day ; it was not necessary to use all 10 drops of water ( figure 1c ) .
the data from this measure were collected from the participants as well , but not used for the study analysis . the first step in determining a participant s physical state involved asking questions about the weather during each day using simple pictures . after choosing the weather ,
the participants were asked to complete their assessment of the length and quality of the previous night s sleep and to evaluate their digestive function throughout the day .
the last step involved assessing one chronic condition that the participant wanted to monitor for 30 days , which was reported prior to starting the study ; the personalized chronic symptoms ranged from eye irritation to dizziness . to assess the chronic symptom , each participant moved a button across a bar that was labeled at each end to indicate the worst and best conditions .
this system applied a variation of the visual analog scale ( vas ) in which performances in computer - based and web - based research have been validated.30 while categorical scales reach an ordinal - scale level , the vas extends the precision and discrimination of daily reports ( figure 1d ) .
the data from this measure were collected from the healthy participants and not used for the study analysis .
two main methods were applied to measure physical conditions according to time . like the emotional tasks , these were presented as tasks for the participants to complete on a daily basis and quantitatively described the positive or negative physical states of each participant .
the first task involved assessing one s overall pain and the second task involved assessing one s overall fatigue throughout the day . on the screen , three auxiliary lines with the numbers 10 and zero next to the top and bottom lines , respectively , were provided . the words extremely severe and none were placed besides the numbers 10 and zero to indicate the intensities of pain and fatigue .
circles that could be dragged were located on the middle line and time points shown in units of 2 hours from 9:00 am to 9:00 pm were visible .
the participants moved the circles up and down on a vertical plane within the range of 100 to express their physical states throughout the day ; the same procedure was applied for both pain and fatigue ( figure 1e and f ) .
this application applied two main methods to measure explicit emotions or feelings that were presented as tasks for the participants to complete on a daily basis .
the first measure was designed to quantitatively visualize positive or negative affective states . on the screen , three auxiliary lines with the numbers 5 , 0 , and
the words pleasant and unpleasant were placed beside the numbers 5 and 5 to indicate positive and negative emotional valences , respectively .
circles that could be dragged were located on the middle line , and time points shown in units of 2 hours from 9:00 am to 9:00 pm were visible .
the participants moved the circles up and down on a vertical plane within the range of 5 to 5 to express their emotional states ( figure 1b ) .
the second measure depicted six basic emotions as described by ekman : anger , disgust , fear , joy , sadness , and surprise.13 the emotions were presented on each vertex of a hexagon , and the relative intensities of the six distinct emotions were defined as the circumplex model .
moreover , 10 small circles , which were depicted as drops of water , were presented at the center of the hexagon , and a single drop of water moved to attach to the corresponding emotion when the user touched any of the six emotions a single time .
each participant was asked to touch the emotions in order to distribute the 10 drops of water as a description of their overall emotional state for the day ; it was not necessary to use all 10 drops of water ( figure 1c ) .
the data from this measure were collected from the participants as well , but not used for the study analysis .
the first step in determining a participant s physical state involved asking questions about the weather during each day using simple pictures . after choosing the weather , the participants were asked to complete their assessment of the length and quality of the previous night s sleep and to evaluate their digestive function throughout the day .
the last step involved assessing one chronic condition that the participant wanted to monitor for 30 days , which was reported prior to starting the study ; the personalized chronic symptoms ranged from eye irritation to dizziness . to assess the chronic symptom , each participant moved a button across a bar that was labeled at each end to indicate the worst and best conditions .
this system applied a variation of the visual analog scale ( vas ) in which performances in computer - based and web - based research have been validated.30 while categorical scales reach an ordinal - scale level , the vas extends the precision and discrimination of daily reports ( figure 1d ) .
the data from this measure were collected from the healthy participants and not used for the study analysis .
two main methods were applied to measure physical conditions according to time . like the emotional tasks , these were presented as tasks for the participants to complete on a daily basis and quantitatively described the positive or negative physical states of each participant .
the first task involved assessing one s overall pain and the second task involved assessing one s overall fatigue throughout the day . on the screen , three auxiliary lines with the numbers 10 and zero next to the top and bottom lines , respectively , were provided . the words extremely severe and none were placed besides the numbers 10 and zero to indicate the intensities of pain and fatigue .
circles that could be dragged were located on the middle line and time points shown in units of 2 hours from 9:00 am to 9:00 pm were visible .
the participants moved the circles up and down on a vertical plane within the range of 100 to express their physical states throughout the day ; the same procedure was applied for both pain and fatigue ( figure 1e and f ) .
because this study focused on the dynamic relationship between emotional and physical states , records with a more frequent temporal resolution ( units of 2 hours ) were analyzed ; thus , a time series analysis of the data of 20 participants was conducted to determine the correlations between the emotional and physical states .
the collected data of the participants during the 30-day study were placed in a property list ( .plist ) file format that was converted to a comma separated values ( .csv ) file for the data analyses .
there were three different categories of time series data emotional valence , pain , and fatigue which illustrated the experiences and emotional states of the participants on a daily basis .
an interpolation process was applied to complete the data for time points between 11:00 pm and 7:00 am when the participants were assumed to be asleep . using this preprocessing procedure , five time points for a single night , or 150 time points in total ,
were filled in to complete the data for the 30-day study period . to fill in the empty time points , the function
na.interp in the forecast package of r ( http://www.r-project.org ) , which assumes a linear relationship , was used to complete the data set . following the preprocessing procedure ,
three sets of time series data for each of the 20 participants were available for data analysis .
a logarithmic transformation was applied to each of the emotional , pain , and fatigue series to ensure the normality and homogeneity of variance of the residuals .
cross - correlations between the emotional valence and pain , emotional valence and fatigue , as well as fatigue and pain time series were analyzed by participant , and the correlation coefficients were averaged to visualize the correlation analysis .
r software ( version 3.2.3 , wooden christmas tree ) and r studio ( version 0.99.892 ) were used for the data analyses .
to investigate the possible relationships between emotional valence and physical states using the data of the 20 participants , a regression model was applied with consideration given for the random effects of the participants . when selecting a regression model to determine
whether there were associations among emotion , fatigue , and pain , it was assumed that there were concurrent dynamics between emotion and the body based on cross - correlation results .
accordingly , a mixed model for multilevel data with a combination of between - subject and within - subject factors was used .
while some methods for time series analyses account for random effects in multilevel data,31 many other methods examine the concurrent changes between multiple time series without lagged effects .
thus , a single model was selected to analyze the three series to determine whether any associations existed .
the multilevel regression model , or the mlm,32,33 allows for the estimation of hierarchically structured longitudinal data on the individual and group levels .
for the current data , a mixed model with participants as the random effect without temporal dislocation was applied because cross - correlation analyses of the series unanimously showed the highest correlation at lag 0 . based on the regression beta coefficients , the dynamic structure between emotional valence , pain , and fatigue was visualized in a network ; the green line indicates a positive relationship , whereas the red line indicates a negative relationship .
fuller test analyzing the three time series with no lagged differences indicated that the logarithmic emotion series , pain series , and fatigue series were each stationary time series .
thus , no temporal dislocation was required to meet the stationarity requirement for the regression analysis .
the correlation analysis revealed that there were negative correlations between emotional valence and fatigue , as well as emotional valence and pain , while pain and fatigue were positively correlated ; all three relationships showed the highest correlations at lag 0 .
figure 2 depicts the plots of the mean cross - correlation values among the participants between the emotional valence and fatigue , emotional valence and pain , and pain and fatigue series .
the cross - correlation was the strongest at lag 0 ; at lag 0 , the mean of the participants cross - correlation coefficient ( ccf ) values were as follows : emotion
fatigue = 0.289 ( p<0.001 ) ; emotion pain = 0.208 ( p=0.007 ) ; and pain
table 1 summarizes the dynamic interactions among the three series ( ) , as well as the akaike information criterion ( aic ) , bayesian information criterion ( bic ) , and log likelihood of each model .
the mlm revealed that fatigue was negatively associated with emotional valence ( = 0.233 , p<0.001 ) and positively associated with pain ( = 0.398 , p<0.001 ) .
additionally , pain was significantly associated with fatigue ( = 0.250 , p<0.001 ) and emotional valence was significantly more negative as fatigue increased ( = 0.150 , p<0.001 ) .
emotional valence also exhibited a negative change as pain increased ( = 0.022 , p=0.063 ) and pain increased as emotion changed negatively ( = 0.021 , p=0.063 ) , but these results were not statistically significant .
the dynamic network structure between emotional valence , pain , and fatigue is presented in figure 3 .
fuller test analyzing the three time series with no lagged differences indicated that the logarithmic emotion series , pain series , and fatigue series were each stationary time series .
thus , no temporal dislocation was required to meet the stationarity requirement for the regression analysis .
the correlation analysis revealed that there were negative correlations between emotional valence and fatigue , as well as emotional valence and pain , while pain and fatigue were positively correlated ; all three relationships showed the highest correlations at lag 0 .
figure 2 depicts the plots of the mean cross - correlation values among the participants between the emotional valence and fatigue , emotional valence and pain , and pain and fatigue series .
the cross - correlation was the strongest at lag 0 ; at lag 0 , the mean of the participants cross - correlation coefficient ( ccf ) values were as follows : emotion
fatigue = 0.289 ( p<0.001 ) ; emotion pain = 0.208 ( p=0.007 ) ; and pain fatigue = 0.321 , ( p<0.001 ) .
table 1 summarizes the dynamic interactions among the three series ( ) , as well as the akaike information criterion ( aic ) , bayesian information criterion ( bic ) , and log likelihood of each model . the mlm revealed that fatigue was negatively associated with emotional valence ( = 0.233 , p<0.001 ) and positively associated with pain ( = 0.398 , p<0.001 ) .
additionally , pain was significantly associated with fatigue ( = 0.250 , p<0.001 ) and emotional valence was significantly more negative as fatigue increased ( = 0.150 , p<0.001 ) .
emotional valence also exhibited a negative change as pain increased ( = 0.022 , p=0.063 ) and pain increased as emotion changed negatively ( = 0.021 , p=0.063 ) , but these results were not statistically significant .
the dynamic network structure between emotional valence , pain , and fatigue is presented in figure 3 .
the current study developed a phr called mind mirror for the recording of daily emotions , pain , and fatigue . by using explicit methods to retrospectively record changes in emotional valence , pain , and fatigue every 2 hours on a daily basis
, this mobile platform enabled the collection of a set of individualized time series data that illustrated emotional and physical changes in individual patients .
the analyses revealed that there were dynamic relationships between daily emotional and physical states in healthy participants . at lag 0 , emotion and fatigue
were negatively correlated , while fatigue and pain were positively correlated . in the dynamic structural network produced by the mlm , the body , or the physical states of pain and fatigue , instantly interacted with emotional valence .
additionally , the overall physical states of fatigue and pain seemed to positively interact with each other , which may have interactively facilitated either the improvement or worsening of a condition .
the drm is one way of self - assessment on the daily affective experiences.27,28 while self - assessments of the recent affective experiences such as drm have been studied to contribute to subjective well - being , our study focused on not only the emotional states but also the participant s physical symptoms that may affect their daily experiences . by doing so , this mobile platform aimed to provide a tool for assessing daily situations , as well as a daily record of psychological and physical states .
the current findings suggest that emotional valence and fatigue directly influence one another and that emotional valence and pain influence one another through the mediating symptom of fatigue , even in healthy individuals .
these findings agree with those of previous studies that identified a relationship between negative emotions and physical states.2024,3538 for example , there are strong genetic links between chronic widespread musculoskeletal pain and fatigue as well as between chronic widespread pain and depression.34 other studies have identified a close relationship between emotional valence and one s physical condition .
accordingly , chronic pain is associated with mental disorders , and negative emotions are known to influence the development of cardiovascular diseases.21,22,24,35 pain disorders without a definite cause , including fibromyalgia ( in which the most common symptoms are pain , fatigue , and depression ) , affect patients in such a way that treatments help to relieve symptoms but not to eliminate the cause of the disorders.34,36 diseases such as cancer are associated with a variety of emotional symptoms , including anxiety and depression , as well as physical symptoms , including pain and fatigue.20,37 on the other hand , although there is evidence that mood influences pain disorders , the influence of a negative emotional state on one s physical state has also been shown to be either selective , general , or unclear.23,38 a 30-day observational study demonstrated that emotions affect healthy individuals and that this influence is instantly evident even in the absence of awareness of a patient.24 in this respect , phrs may provide a possible strategy for improving medical care for patients with comorbid psychological and physical illnesses.39 thus , the findings of the current study can be applied to health management by medical professionals and institutions for the purpose of analyzing daily emotions that can lead to diseases as well as for predicting possible changes in daily emotions .
participant - generated health data , such as those produced by the current study , hold potential for the self - monitoring and daily measurement of health conditions , which will aid in the investigation of possible relationships between an individual s emotions and chronic physical symptoms outside the hospital . in a more general sense ,
phrs point to the promise of health technologies for managing health and preventing the occurrence or worsening of various disorders among members of the general population who have access to mobile technology .
moreover , a variety of diverse information can be obtained using this format , depending on the target population , including inpatients within medical facilities , outpatients who make regular visits , and healthy individuals who are yet to receive medical checkups . reduced gaps in health - related information may aid in the diagnoses of individuals who do not have a clear cause for their symptoms .
as these technologies are developed and distributed in the near future , it will be essential to ensure that they are available for and tested in patients with psychological and/or psychosomatic symptoms .
this study found marked and direct , but not significant , relationships between pain and emotion , which may be interpreted from two different perspectives .
first , this study was conducted with a limited number of participants and , thus , further investigations are needed to compare these findings with those of other studies in order to establish generalizability with other age groups and health settings with a larger population .
second , this study was conducted using only a subset of participants with regular emotional and physical health statuses and , thus , they may have had different relationships between their emotional and physical states relative to chronic disease patients .
for instance , mental disorders , including depression , are known to be comorbid with chronic pain.40 thus , further research is needed to examine the benefits of this application and other types of novel technologies in different patient groups .
furthermore , the mobile application - based phr used in the current study required that the participants enter all data and access their records using smartphones , which may have limited the population eligible for this intervention .
third , the retrospective assessment of the pain , fatigue , and emotional valence by the participants , as indicated in our study , may introduce recall bias . while the strategy to use this mobile platform minimized any missing values during the trial , the recall bias may have consequences for the data quality .
one way of avoiding this problem , as used by other mobile platforms , is to prompt the participants several times during the day .
finally , the current analyses did not include data collected from the circumplex model of categorical emotion or other items , including sleep or other chronic symptoms , because it was focused on the temporal dynamics of the relationships between the emotional and physical states of patients .
it would be interesting to investigate the interplay between different types of emotions and physical symptoms in the future .
future studies in which a large sample size and the analysis of a combination of other items are utilized when an application - based phr is widely used worldwide should be conducted .
the current 30-day observational study examined relationships among pain , fatigue , and emotional valence and provided evidence that the physical condition and emotional state of healthy participants are interrelated .
positive changes in emotional valence were associated with improvements in physical condition via decreases in pain and fatigue , while negative changes in emotional valence were associated with the aggravation of pain and fatigue . by measuring differences in daily emotions ,
the current study provided basic information about both emotional and physical health in daily life using a mobile platform that recorded emotional and physical changes throughout the day on a daily basis .
these findings also suggest that further data collection and analyses will contribute to the ability to predict an individual s emotional and physical health conditions , which would aid in the management of an individual s health on a daily basis .
|
objectivesrecently , there has been increasing interest in preventing and managing diseases both inside and outside medical institutions , and these concerns have supported the development of the individual personal health record ( phr ) .
thus , the current study created a mobile platform called mind mirror to evaluate psychological and physical conditions and investigated whether phrs would be a useful tool for assessment of the dynamic relationship between the emotional and physical conditions of an individual.methodsmind mirror was used to collect 30 days of observational data about emotional valence and the physical states of pain and fatigue from 20 healthy participants , and these data were used to analyze the dynamic relationship between emotional and physical conditions .
additionally , based on the cross - correlations between these three parameters , a multilevel multivariate regression model ( mixed linear model [ mlm ] ) was implemented.resultsthe strongest cross - correlation between emotional and physical conditions was at lag 0 , which implies that emotion and body condition changed concurrently . in the mlm ,
emotional valence was negatively associated with fatigue ( = 0.233 , p<0.001 ) , fatigue was positively associated with pain ( = 0.250 , p<0.001 ) , and pain was positively associated with fatigue ( = 0.398 , p<0.001).conclusionour study showed that emotional valence and one s physical condition negatively influenced one another , while fatigue and pain positively affected each other .
these findings suggest that the mind and body interact instantaneously , in addition to providing a possible solution for the recording and management of health using a phr on a daily basis .
|
Introduction
Methods
Participants
Study design and procedures
Development of software for Mind Mirror
Measurements of emotional and physical states using PHRs
Measurement of emotional states
Measurement of physical states
Data processing and analysis
Mixed linear model (MLM)
Results
Correlations between emotional and physical states
Multilevel regression model of the emotional and physical states
Discussion
Conclusion
|
rheumatoid arthritis ( ra ) is a chronic , progressive , inflammatory autoimmune disease in which the body 's immune system mistakenly attacks the joint .
the disease produces an inflammatory infiltrate of immune cells as well as a series of destructive events such as synovial hyperplasia , pannus setting , bone and cartilage erosion , and joint destruction .
it results in swelling and pain in the joints and around them [ 1 , 2 ] . in ra ,
activation of innate immunity in early disease , followed by the appearance of adaptive immune responses ultimately leads to a destructive phase .
the pathophysiology of ra implies the existence of t and b cells , various immune modulators ( cytokines and effector cells ) , and signalling pathways .
the complex interaction of immune modulators causes joint damage starting at the synovial membrane and covering most structures .
this disequilibrium between pro- and anti - inflammatory cytokine activities facilitates the induction of autoimmunity , chronic inflammation , and joint damage .
it is less known , though , how cytokines are organized within a hierarchical regulatory network and which cytokines may qualify as best targets for clinical intervention a priori [ 35 ] .
ra pathogenesis is caused by b cells not only through antigen presentation , but also through the production of antibodies , autoantibodies , and cytokines [ 2 , 5 ] .
there are no disease - specific diagnostic features in ra and patients can have a wide range of manifestations .
the diagnosis of ra is given by a combination of symptoms , signs , serologic tests , and radiologic findings , as established by the american college of rheumatology .
since early inflammatory arthritis is a clinically heterogeneous disease , cytokine networks are known to play a critical role in the pathogenesis of rheumatoid arthritis ; a panel of pro- and anti - inflammatory cytokines and associated autoantibodies were measured to identify the biologically based subsets of early rheumatoid arthritis ( era ) .
it requires assessment , not only of the current clinical picture but also of the potential for change .
therefore , identifying the early rheumatoid arthritis ( ra ) is a crucial step in controlling the progress of the disease .
. early diagnosis of rheumatoid arthritis ( ra ) is essential , because there is strong evidence that early treatment with one or more disease - modifying antirheumatic drugs improves the evolution [ 10 , 11 ] .
when dmards therapy is introduced early in patients , function and radiological outcome in the long term are better than cases when it is delayed .
our research objectives were to assess the concentrations of il-13 and il-17 in serum of patients with era , the investigation of correlation between the concentrations of these cytokines and disease activity score and the concentration of some autoantibodies in relation to the control group , and the evaluation of the utility of il-13 and il-17 concentration measurements as markers of disease activity .
we accomplished a study which included 30 patients diagnosed with early rheumatoid arthritis , gender ratio 6 m/24 f , and mean age 56.22 years ; in parallel , we investigated a control group that included 28 persons unaffected by early rheumatoid arthritis or other diseases . controls were matched for sex , age at the time point of blood sampling , and area of residence ( rural or urban ) .
early ra patients fulfilled the american college of rheumatology ( acr ) 1987 revised criteria for the classification of ra .
they were all investigated , diagnosed , and included in the studied group , following the revised classification criteria of the american college of rheumatology , in 2010 .
all patients accomplished the inclusion criteria for early rheumatoid arthritis ( two or more swollen joints dating more than 2 weeks , but less than 12 months from onset ) .
we excluded , from the start of the study , patients with other autoimmune diseases , those who received treatment with dmards , glucocorticoids , or / and vitamins , the women during pregnancy , and persons with diabetes mellitus or metabolic syndrome . the study cohort comprised patients firstly evaluated for early arthritis because we aimed to investigate era patients to find a better and faster way of discrimination between affected and unaffected cases . based on the das28 results ,
the 30 era patients were subdivided into three groups : mild ( 2.6 < das28 3.2 ) , moderate ( 3.2 < das28 5.1 ) , and severe ( 5.1 < das28 ) .
serum samples were collected from 30 patients and from 28 controls ( healthy persons ) and analyzed for concentrations of il-13 and il-17 , anticyclic citrullinated peptide antibodies ( anti - ccp ) , rheumatoid factor igm isotype ( igm - rf ) , anti - cardiolipin igg isotype ( igg - acl ) , anti - ra33 , erythrocytes sedimentation rate ( esr ) , and c - reactive protein ( crp ) .
blood samples were obtained from all subjects into tubes without additives by venous puncture in a fasting state in the morning .
peripheral venous blood was collected into separator vacutainers and allowed to clot for 30 minutes at room temperature .
the test tubes were centrifuged at 3.000 g for 10 minutes , and serum samples were further divided into aliquots and stored at 80c , until assessment . before testing , frozen probes were brought to room temperature , avoiding freezing - unfreezing cycles .
the analysis of serum parameters was based on a quantitative sandwich elisa , according to the manufacturer 's instructions .
igg anti - ccp 3.1 and igg - acl autoantibodies were determined by elisa , using quanta lite-inova diagnostics kits , usa ( autoantibodies seropositivity was defined as a titer > 20
the investigation of serum igm - rf concentrations was achieved using elisa - aescu germany kits ( positive > 15
u / l ) and of anti - ra33 antibodies using elisa - kit human , wiesbaden , germany ( positive results > 25 u / ml ) . for hscrp dosage , drg elisa international inc .
serum concentrations of il-13 and il-17 were measured in patients with early , untreated inflammatory arthritis and in control persons , using elisa techniques with invitrogen corporation kits ( camarillo , ca , usa ) .
the values obtained were expressed in pg / ml . in looking for a method of measuring serum cytokine ,
concentrations were taken into account studies that show good stability for samples stored at 70c until dosage .
all the procedures were followed in accordance with the ethical standards of the institutional responsible committees for human studies and with the helsinki declaration of 1975 , as revised in 2008 . for realisation of this study
, we obtained the approval of the committee of ethics and academic and scientific deontology of the university of medicine and pharmacy from craiova number 76/2014 .
patients ' data , management system , and data processing were performed using microsoft excel and data analysis module ; statistical analysis was done using graphpad prism 5 .
the significance of differences between groups was examined with a mann - whitney u test or kruskal - wallis , when multiple comparisons were made .
correlation analysis between the concentration of il-13 and il-17 and the degree of disease activity ( das28 ) , as well as the concentration of some autoantibodies , crp and esr , were conducted with a pearson 's test .
the diagnostic values of studied markers were evaluated using receiver operating characteristic ( roc ) curves analysis .
the performance was expressed as the area under the roc curve ( auc , area under roc curve ) together with 95% confidence interval ( 95% ci ) and p statistics for the difference between calculated auc and auc = 0.5 ( weak discriminative marker ) . cut - off values corresponding to the highest accuracy were determined and for various threshold values investigated at each marker ,
we calculated the sensitivity ( sn ) , specificity ( sp ) , and youden index ( sensitivity + specificity 1 ) .
among the 30 patients , initially diagnosed with era , 80% were female ( sex ratio : 24 female/6 male ) , with age , mean stdev 55.77 10.87 years . in controls
there was no significant difference in age between the two groups ( table 1 ) . in our study
, we found that both il-13 ( 18.20 pg / ml , 95% ci : 16.4719.92 ) and il-17 ( 17.87 pg / ml , 95% ci : 12.9922.75 ) concentrations in the serum of patients suffering from era were higher than those in the control group ( 4.80 pg / ml , 95% ci : 3.895.71 , p < 0.0001 , and 4.20 pg / ml , 95% ci : 3.365.05 , p < 0.0001 , resp . ) .
we also found differences in serum concentrations of il-13 and il-17 in subgroups of era disease patients with different clinical activity stages .
the concentrations of il-13 in the severe and moderate groups were statistically higher than in the mild and control groups ( p < 0.05 ) .
there were no statistical differences between severe and moderate groups . also , in the case of il-17 serum concentrations increased proportionally with the disease activity of era , the highest concentrations were in patients with severe activity disease ( figure 2 ) .
statistically significant differences were observed between both the moderate and the severe groups and a mild group ( p < 0.05 ) , as well as between the group with moderate disease activity and the control group ( p < 0.001 ) . in the studied cohort of patients , we observe statistically significant differences in the concentrations of crp and the levels of esr between patients with era and the control group ( crp / control group , p < 0.0001 ; esr / control group , p < 0.0001 ) ( table 1 ) . analyzing the relationship between serum levels of crp and esr and different disease activity stages , we observed only statistically significant differences between severe and moderate group ( p < 0.0379 ) ( table 2 ) .
our study showed different profiles of igg anti - ccp and igm - rf concentrations in serum of patients suffering from era in different clinical activity stages .
igg anti - ccp and igm - rf concentrations were increasing along with the disease activity . in both cases , there were statistically significant differences between severe groups and moderate and mild groups ( igg anti - ccp : severe versus moderate group , p = 0.0011 , severe versus mild group , p = 0.0030 ; igm - rf : severe versus moderate group , p = 0.0014 , severe versus mild group , p = 0.0039 ) .
there was a weak , statistically not significant correlation between il-17 and igm - rf ( r = 0.320 , p = 0.085 ) .
there was a significant positive correlation between the concentrations of il-17 and crp ( r = 0.366 , p = 0.047 ) and a significant negative correlation between the concentrations of il-13 and crp ( r = 0.334 , p = 0.041 ) .
disease activity score , das28 , was strongly positively correlated with levels of esr ( r = 0.967 , p 0.001 ) and weakly positively correlated with concentrations of anti - ra33 autoantibodies ( r = 0.404 , p = 0.027 ) .
concentrations of anti - ccp autoantibodies positively correlated fairly well with crp and igm - rf , and concentrations of anti - ra33 positive correlated with levels of esr . comparing
the roc curves for the studied parameters in the patients with era indicated that il-13 has a higher diagnostic utility than il-17 , crp , esr , igm - rf , and anti - ccp as markers of disease activity ( table 4 ) .
roc analysis revealed that il-13 concentration indicated era presence with 100% accuracy using the concentration of 10.73 pg / ml as an optimal cut - off value for discrimination between patients with era and controls ( 95% ci : 0.9621.000 , p < 0.0001 ) .
the likelihood ratios of positive and negative results obtained on the basis of optimal threshold values specific for era were as follows : lr(+ ) = 28.00 and lr( ) = 1.12 with sensitivity and specificity equal to 100 and 100% , respectively ; youden index was 1.00 ( figure 3 ) . in case of il-17 ,
the calculated cut - off value for discrimination between patients with era and controls was 9.40 pg / ml and using this value the diagnostic accuracy of il-17 was 90.2% ( 95% ci : 0.8090.995 , p < 0.0001 ) .
the likelihood ratios of positive and negative results obtained on the basis of optimal threshold values specific for era were as follows : lr(+ ) = 24.27 and lr( ) = 1.14 with sensitivity and specificity equal to 86.67 and 100% , respectively ; youden index was 0.866 . in the studied cohort of patients , crp and esr
have discriminative power towards diagnosis of era ( sensitivities for both crp and esr were found to be lower in comparison to the il-13 ; diagnostic accuracy of crp was 96.9 and 94.8% , resp .
we also noticed that although they have a specificity less than il-13 and crp , autoantibodies igm - rf and anti - ccp have a good tendency to discriminate patients with era from healthy ones ( diagnostic accuracy 98.1 and 94.7% , resp . ) .
chronic inflammatory and autoimmune diseases are the result of an interplay between genetic factors and environmental ones that culminate in the phenotypes of the established disease .
owing to the prevalence and accessibility of joint samples for laboratory investigation , ra has been a suitable model for the study of numerous inflammatory and immune - mediated conditions .
the formulation of a definition for early ra was difficult , but the majority of the rheumatologists use the term early for symptoms shorter than three months . there was a tendency to accept the involvement of fewer affected joints [ 9 , 16 ] .
changing from health to established disease in ra is generally clearly understood.early rheumatoid arthritis ( ra ) and very early ra are major targets of research and clinical practice [ 15 , 17 ] .
we found a predominance ( 80% ) of women in patients affected by era , finding which are congruent with the results of other studies having identified a female predominance in ra [ 9 , 15 , 18 ] .
both pro- and anti - inflammatory cytokines were found elevated in ra patients over controls claiming opinion that cytokine networks play critical rolls in the pathogenesis of rheumatoid arthritis [ 4 , 7 , 1928 ] .
the present study reveals that levels of serum il-13 and il-17 cytokines were significantly higher in era patients than in age- and sex - matched healthy persons .
our results are related to the cell - mediated immune response intervention in disease onset .
a broad range of inflammatory processes that are involved in the pathophysiology of rheumatoid arthritis are regulated by cytokines .
the imbalance between pro- and anti - inflammatory cytokine activities favours the induction of autoimmunity , chronic inflammation , and thereby joint damage [ 5 , 27 ] .
some cytokines , such as tumour necrosis factor- ( tnf- ) alpha , il-17 , and ( il)-1 , function by promoting inflammatory responses and by inducing cartilage degradation .
others such as il-4 , il-10 , and il-13 are mainly anti - inflammatory molecules [ 18 , 20 , 25 ] .
even if present in rheumatoid joints , in progressive ra anti - inflammatory cytokine levels are too low to neutralize the deleterious effects of proinflammatory cytokines .
the suppression of both the secretion and action of il-17 by il-13 is of potential clinical importance [ 1922 ] .
il-13 is a protein , secreted by activated t cells , that modulates b cell function in vitro and plays an important part in their proliferation and differentiation ; the high local il-13 levels were observed in patients with ra , correlated with b lymphocyte proliferation .
interleukin 13 induces interleukin-4-independent igg4 and ige synthesis and cd23 expression by human b cells .
some researchers support a role for il-13 as an in vivo antiangiogenic factor and provide a rationale for its use in ra to control pathologic neovascularization .
treatment with th2 cytokines ( il-4 , il-10 , and il-13 ) was tested in many animal models of arthritis based on the th1 bias of t cells , showing considerable promise . in patients suffering from ra , disruptions in self - tolerance lead to abnormalities such as recognition of citrullinated antigens by t and b cells .
the proportion of lymphocyte differentiation in ra is skewed towards the th1 phenotype , to the detriment of the th2 , th17 , and t regulator ( treg ) ones .
imbalances appear in the main cytokine systems including il-1 , tnf , il-6 , il-18 , il-15 , il-33 , il-22 , and il-13 .
however , the destruction of the joint in ra is caused not only by these cytokine imbalances but also by matrix production dysregulation responsible for cartilage damage .
il-17 levels fall after administration of anti - inflammatory cytokines such as il-4 or il-13 .
il-13 levels were significantly higher in patients with early ra ( p < 0.001 ) than in reference group , suggesting the different pathogenic mechanisms involved in joint inflammation .
serum il-13 values increased in ra have been reported in many works [ 20 , 22 , 28 ] . lower interleukin 13 levels were communicated in patients with arthritis by some investigators as barra and contributors and woods et al . .
assume that the production of rf and antinuclear antibodies by b cells could depend on different cytokines action . in their study , il-13 serum levels correlated with those of rf in ra patients and they suggested that il-13 may be involved in the pathogenesis of autoimmune rheumatic diseases , with a relevant role on rf production .
we found that detection of il-13 in era patients was not affected by rheumatoid factor igm ( r = 0.206 , p = 0.274 ) , a fact revealed by other researchers too .
interleukin 13 inhibits the production of proinflammatory cytokines , chemokines , and hematopoietic growth factors by activated human monocytes .
the increase of biologically active il-13 in ra supports the hypothesis that il-13 regulates immune cell ( including dendritic cell ) activity and indicates how the varied anatomical distribution of cytokines may play a role in the ra disease process .
the differential regulation of circulating il-13 and m - csf levels by tnf antagonists further implies discrete roles in the tnf - cytokine network in ra [ 33 , 34 ] .
. showed that il-13 was present in 27 out of 28 serum samples from patients with ra , indicating that this cytokine is constantly present in rheumatoid joints .
detected increased levels of the th2 cytokines il-4 and il-13 in synovial samples from early ra patients .
il-13 causes b cell proliferation and differentiation , including ige production , and the expression of certain adhesion molecules on endothelial cells .
all these biological properties of il-13 are shared with il-4 , but in contrast to il-4 , il-13 does not act on t cells . besides the increased values of il-13 , we detected circulating il-17 levels significantly higher in patients with era ( p < 0.001 ) when compared to those in the reference group .
strong correlations of serum il-17a levels with anti - ccp were found by rou et al . .
th17 with decreased circulating levels in era seems to be a marker of anti - ccp seropositivity .
considering the complexity and heterogeneous nature of ra , it is unlikely that only cytokines investigation may provide sufficient discrimination ; predicting the era is better with a combination of biomarkers .
il-17 has several sources : th17 cells , which are a subset of cd4+helper t cells , mast cells , nk cells , and t cells ; all of them contributing to the pathogenesis of inflammatory arthritis [ 39 , 40 ] . in a recent study comparing individuals before the onset of symptoms ( defined as prepatients ) and after the onset of ra with matched control subjects , kokkonen et al
. showed that il-17 was present at its highest concentrations in prepatients , and the level had decreased within 7.7 months following the onset of disease .
il-17a was detected at higher levels in early disease compared with late , established disease .
reported il-17a levels significantly higher in synovial fluid ( sf ) and serum from era patients compared to osteoarthritis ( oa ) .
in other previous studies , there were higher serum and sf il-17a levels in ra patients as compared to healthy controls , which suggests that the cytokine is mainly produced locally in the inflamed joint .
other results sustain that despite the significant increases in th17 and il-17 cd4 + t cells in the blood of ra patients , these did not correlate with esr , crp , or das28 , suspecting that the presence of il-17 producing cd4 + t cells in the blood from patients with established ra is of limited use as a biomarker to indicate disease activity . in our study , there were significant correlations ( r = 0.366 , p = 0.047 ) found between il-17 and crp serum levels , but not with esr or disease activity score , supporting partially arguments of these investigators . in another work ,
leipe et al . demonstrated that th17 cells play an important role in inflammation in human autoimmune arthritides , both at the onset and in established disease .
they claim that the levels of il-17 are connected to the systemic disease activity at both the onset and the progression of ra . taken together
, these data suggest that il-17 may be a key activator of t cell - driven inflammation and thus may contribute to the pathogenesis of ra [ 46 , 47 ] .
it can be asserted that il-17 represents a member of the proinflammatory cytokine family produced by ra synovium and inhibited by some th2 cytokines . in this way , il-17 contributes to the active , proinflammatory pattern characteristic to ra , whose production and function are regulated by il-4 and il-13 .
reduction of synovial inflammation may be protective through a direct effect on il-17 , inducing proinflammatory effects .
thus , il-17 appears to represent a target for treatments of ra [ 48 , 49 ] .
overexpression of il-17 has been shown to be associated with a number of pathological conditions .
since il-17 was found at high levels in the synovial fluid around the affected cartilage in patients with inflammatory arthritis , it is assumed that this determines the direct effect on articular cartilage .
il-17 functions as a direct and potent inducer of matrix breakdown and an inhibitor of matrix synthesis in articular cartilage explants .
it was observed that individuals in whom ra later developed had significantly increased concentrations of several cytokines , closer to the onset of symptoms , and that there exists a relationship between th1 , th2 , treg , and th17-cytokines and the presence of anti - ccp antibodies .
sensitivity , though , was not increased by the combination of anti - ccp antibodies and these cytokines [ 18 , 41 , 51 , 52 ] . because we know the statistical limits of the present study , the relatively small number of patients , we propose in the future longitudinal studies with regular serum analysis to determine more precise roles of il-17 , il-13 , and autoantibodies in ra pathogenesis .
with il-13 and il-17 serum concentrations increasing proportionally with the disease activity of era , the highest concentrations were in patients with severe activity disease . based on the results of this study , we can conclude that the presence of higher il-13 , il-17 , igm - fr , and anti - ccp serum levels in patients , compared to those of controls , confirms that these markers , found with high specificity , might be involved in the pathogenesis of era . il-13 and il-17 might be of better usefulness in the prediction of era activity status than igm - rf and anti - ccp .
investigation of the association between cytokine profile and autoantibodies status may lead to prognostic and treatment decisions in era patients .
the basis for the new therapies in patients with ra is represented by inhibiting the action of proinflammatory cytokines by using specific cytokine inhibitors or anti - inflammatory cytokines .
the combination of il-17 and anti - ccp autoantibodies may have the potential as biomarkers in early ra , especially for their clinical utility .
|
aims . in the present study , we aimed to assess the concentrations of il-13 and il-17 in serum of patients with early rheumatoid arthritis ( era ) , the investigation of correlation between the concentrations of these cytokines and disease activity score , and the concentration of some autoantibodies and the evaluation of the utility of il-13 and -17 concentration measurements as markers of disease activity .
materials and methods .
serum samples were collected from 30 patients and from 28 controls and analysed parameters .
results .
the serum concentrations of il-13 , il-17 , anti - ccp , and igm - rf were statistically significantly higher in patients with era , compared to the controls .
il-13 concentrations in the severe and moderate groups with era were statistically higher than in the mild and control groups .
also , in the case of il-17 , serum concentrations increased proportionally with the disease activity of era .
we observe that concentrations of il-13 and -17 did not correlate with autoantibodies .
il-17 concentration significantly positively correlated with crp , while il-13 concentration significantly negatively correlated with crp .
disease activity score , das28 , was strongly positively correlated with levels of esr and weakly positively correlated with concentrations of anti - ra33 autoantibodies .
il-13 has a higher diagnostic utility than il-17 , crp , esr , igm - rf , and anti - ccp as markers of disease activity .
conclusions .
the presence of higher il-13 and il-17 serum levels in patients , compared with those of controls , confirms that these markers , found with high specificity , might be involved in the pathogenesis of era . il-13 and il-17 might be of better usefulness in the prediction of era activity status than igm - rf and anti - ccp .
|
1. Background and Aims
2. Materials and Methods
3. Results
4. Discussions
5. Conclusions
|
atherosclerosis is the leading cause of peripheral artery disease ( pad ) and coronary artery disease ( cad ) .
some previous studies have reported that pad is a coronary heart disease risk equivalent.1)2 ) another study , however , reported that risk factors for pad are different to those for cad.3 ) risk factors for atherosclerosis are hypertension ( ht ) , diabetes mellitus ( dm ) , hypercholesterolemia { high total cholesterol ( tc ) levels , high low density lipoprotein - cholesterol ( ldl - c ) levels , and/or low high density lipoprotein - cholesterol ( hdl - c ) levels } , smoking , older age , obesity , metabolic syndrome ( mets)4 ) and chronic kidney disease ( ckd).5 ) risk factors for pad may vary depending on the affected arteries.6 ) although the prevalence of pad and cad is known to increase with the ageing population,7 ) few studies have compared risk factors for pad to cad solely in the korean population . therefore , our objective in the present study was to analyze and compare risk factors for pad , cad , and for normal controls under the hypothesis that risk factors for pad and cad are different from those for normal controls .
we reviewed the records of patients diagnosed with pad and cad at the cardiac and vascular center from november 1994 to november 2004 as well as those of healthy subjects ( normal control group ; control ) who underwent health examinations of digestive organs during the same period at the health promotion center of samsung medical center .
we excluded patients with cardiovascular disease ( cvd ) , cerebrovascular accident , or lung cancer from the normal control group .
the enrolled subjects consisted of 1 ) patients with pad ( n=415 ) who had over 50% peripheral artery occlusion confirmed by lower extremity computed tomography angiography , 2 ) patients with cad ( n=3686 ) including those with stable angina , unstable angina , and acute myocardial infarction confirmed by cardiac catheterization , and 3 ) control ( n=3835 ) . in addition , self reported information on the absence of cad and pad was used in control .
this study was approved by the samsung medical center institutional review board ; informed consent was waived for this retrospective study .
subjects were defined as having ht if they were taking an anti - ht drug , had been clinically diagnosed with ht , or had either a systolic blood pressure ( sbp ) 40 mm hg or a diastolic blood pressure ( dbp ) 90 mm hg . subjects who met one of the following requirements were defined as having dm : on an oral hyperglycemic agent , using insulin , clinical diagnosis of diabetes , or a fasting glucose level > 126 mg / dl .
subjects were defined to have hypercholesterolemia if they met one of the following requirements : diagnosis of hypercholesterolemia or a medication history of hypercholesterolemia or tc > 200 mg / dl or ldl - c > 130 mg / dl . the following body mass index ( bmi ) categories
were recognized : normal ( 18.5bmi<23 ) , overweight ( 23bmi<25 ) and obese ( bmi25 ) .
there was no statistical meaning of adding an underweight category because the number of underweight patients was two in the pad group , thus we included them into the bmi normal group in pad .
a patient who had smoked within a year prior to the study was defined as a smoker .
the estimated glomerular filtration rate ( egfr ) , which was used as an indicator of kidney function , was calculated using the modification of diet renal disease study formula : egfr ( ml / min/1.73 m)=186.3{serum creatinine ( cr)}(age)(0.742 if women)(1.21 if african - americans ) .
the national kidney foundation kidney disease outcome quality initiative defined ckd as an egfr < 60 ml / min/1.73 m. patients with mets were classified into two groups based on the modifications suggested by the national cholesterol education program adult treatment panel iii.1 ) diagnosis of mets in this study was based on the presence of three or more of the following symptoms : 1 ) bmi 25 ( bmi categories for asia of international obesity taskforce ) , 2 ) triglyceride ( tg ) levels 150 mg / dl , 3 ) hdl - c levels < 40 mg / dl for men and < 50 mg / dl for women , 4 ) ht with sbp 130 mm hg , dbp 85 mm hg , or undergoing active antihypertensive drug therapy , and 5 ) fasting blood sugar ( fbs ) 100 mg / dl or active use of oral hypoglycemic agents or insulin .
a number of pad patients had had cad , however , cad patients were not diagnosed with pad in our data .
patients with pad were divided into two groups based on the absence or presence of coexisting cad .
pad subjects were also classified into two groups based on the modified recommendations of haltmayer et al.8 ) according to the affected arteries of the lower limb : 1 ) aortoiliac ( ai ) disease including occlusion or > 50% stenosis in the abdominal aorta and common and external iliac arteries , 2 ) femoropopliteal ( fp ) disease including occlusion or > 50% stenosis in the common , superficial and deep femoral , popliteal , and infrapopliteal arteries .
the fp and ai groups were compared with the cad group , because the coronary arteries are similar to the fp arteries in size .
general characteristics of pad , cad , and control subjects were analyzed by one - way analysis of variance with the bonferroni method in multiple comparisons testing for continuous variables .
the -test was used to compare categorical variables . to analyze and compare risk factors between pad subjects with coexisting cad and those with no coexisting cad and between pad subjects with ai and fp , we employed student 's t - test for continuous variables and the -test for categorical variables .
simple logistic regression analysis and multinomial logistic regression analysis were carried out to determine the association among cardiovascular risk factors in pad , cad , and control subjects .
i , age , gender , ht , dm , hypercholesterolemia , obesity grade ( obese ) , smoking status and ckd were adjusted . model ii considered age , gender , smoking status , ckd , and mets .
we reviewed the records of patients diagnosed with pad and cad at the cardiac and vascular center from november 1994 to november 2004 as well as those of healthy subjects ( normal control group ; control ) who underwent health examinations of digestive organs during the same period at the health promotion center of samsung medical center .
we excluded patients with cardiovascular disease ( cvd ) , cerebrovascular accident , or lung cancer from the normal control group .
the enrolled subjects consisted of 1 ) patients with pad ( n=415 ) who had over 50% peripheral artery occlusion confirmed by lower extremity computed tomography angiography , 2 ) patients with cad ( n=3686 ) including those with stable angina , unstable angina , and acute myocardial infarction confirmed by cardiac catheterization , and 3 ) control ( n=3835 ) . in addition , self reported information on the absence of cad and pad was used in control .
this study was approved by the samsung medical center institutional review board ; informed consent was waived for this retrospective study .
subjects were defined as having ht if they were taking an anti - ht drug , had been clinically diagnosed with ht , or had either a systolic blood pressure ( sbp ) 40 mm hg or a diastolic blood pressure ( dbp ) 90 mm hg . subjects who met one of the following requirements were defined as having dm : on an oral hyperglycemic agent , using insulin , clinical diagnosis of diabetes , or a fasting glucose level > 126 mg / dl .
subjects were defined to have hypercholesterolemia if they met one of the following requirements : diagnosis of hypercholesterolemia or a medication history of hypercholesterolemia or tc > 200 mg / dl or ldl - c > 130 mg / dl .
the following body mass index ( bmi ) categories were recognized : normal ( 18.5bmi<23 ) , overweight ( 23bmi<25 ) and obese ( bmi25 ) .
there was no statistical meaning of adding an underweight category because the number of underweight patients was two in the pad group , thus we included them into the bmi normal group in pad .
a patient who had smoked within a year prior to the study was defined as a smoker .
the estimated glomerular filtration rate ( egfr ) , which was used as an indicator of kidney function , was calculated using the modification of diet renal disease study formula : egfr ( ml / min/1.73 m)=186.3{serum creatinine ( cr)}(age)(0.742 if women)(1.21 if african - americans ) .
the national kidney foundation kidney disease outcome quality initiative defined ckd as an egfr < 60 ml / min/1.73 m. patients with mets were classified into two groups based on the modifications suggested by the national cholesterol education program adult treatment panel iii.1 ) diagnosis of mets in this study was based on the presence of three or more of the following symptoms : 1 ) bmi 25 ( bmi categories for asia of international obesity taskforce ) , 2 ) triglyceride ( tg ) levels 150 mg / dl , 3 ) hdl - c levels < 40 mg / dl for men and < 50 mg / dl for women , 4 ) ht with sbp 130 mm hg , dbp 85 mm hg , or undergoing active antihypertensive drug therapy , and 5 ) fasting blood sugar ( fbs ) 100 mg / dl or active use of oral hypoglycemic agents or insulin .
a number of pad patients had had cad , however , cad patients were not diagnosed with pad in our data .
patients with pad were divided into two groups based on the absence or presence of coexisting cad .
pad subjects were also classified into two groups based on the modified recommendations of haltmayer et al.8 ) according to the affected arteries of the lower limb : 1 ) aortoiliac ( ai ) disease including occlusion or > 50% stenosis in the abdominal aorta and common and external iliac arteries , 2 ) femoropopliteal ( fp ) disease including occlusion or > 50% stenosis in the common , superficial and deep femoral , popliteal , and infrapopliteal arteries . the fp and ai groups were compared with the cad group , because the coronary arteries are similar to the fp arteries in size .
subjects were defined as having ht if they were taking an anti - ht drug , had been clinically diagnosed with ht , or had either a systolic blood pressure ( sbp ) 40 mm hg or a diastolic blood pressure ( dbp ) 90 mm hg . subjects who met one of the following requirements were defined as having dm : on an oral hyperglycemic agent , using insulin , clinical diagnosis of diabetes , or a fasting glucose level > 126 mg / dl .
subjects were defined to have hypercholesterolemia if they met one of the following requirements : diagnosis of hypercholesterolemia or a medication history of hypercholesterolemia or tc > 200 mg / dl or ldl - c > 130 mg / dl .
the following body mass index ( bmi ) categories were recognized : normal ( 18.5bmi<23 ) , overweight ( 23bmi<25 ) and obese ( bmi25 ) .
there was no statistical meaning of adding an underweight category because the number of underweight patients was two in the pad group , thus we included them into the bmi normal group in pad .
a patient who had smoked within a year prior to the study was defined as a smoker .
the estimated glomerular filtration rate ( egfr ) , which was used as an indicator of kidney function , was calculated using the modification of diet renal disease study formula : egfr ( ml / min/1.73 m)=186.3{serum creatinine ( cr)}(age)(0.742 if women)(1.21 if african - americans ) .
the national kidney foundation kidney disease outcome quality initiative defined ckd as an egfr < 60 ml / min/1.73 m. patients with mets were classified into two groups based on the modifications suggested by the national cholesterol education program adult treatment panel iii.1 ) diagnosis of mets in this study was based on the presence of three or more of the following symptoms : 1 ) bmi 25 ( bmi categories for asia of international obesity taskforce ) , 2 ) triglyceride ( tg ) levels 150 mg / dl , 3 ) hdl - c levels < 40 mg / dl for men and < 50 mg / dl for women , 4 ) ht with sbp 130 mm hg , dbp 85 mm hg , or undergoing active antihypertensive drug therapy , and 5 ) fasting blood sugar ( fbs ) 100 mg / dl or active use of oral hypoglycemic agents or insulin .
a number of pad patients had had cad , however , cad patients were not diagnosed with pad in our data .
patients with pad were divided into two groups based on the absence or presence of coexisting cad .
pad subjects were also classified into two groups based on the modified recommendations of haltmayer et al.8 ) according to the affected arteries of the lower limb : 1 ) aortoiliac ( ai ) disease including occlusion or > 50% stenosis in the abdominal aorta and common and external iliac arteries , 2 ) femoropopliteal ( fp ) disease including occlusion or > 50% stenosis in the common , superficial and deep femoral , popliteal , and infrapopliteal arteries . the fp and ai groups were compared with the cad group , because the coronary arteries are similar to the fp arteries in size .
general characteristics of pad , cad , and control subjects were analyzed by one - way analysis of variance with the bonferroni method in multiple comparisons testing for continuous variables .
the -test was used to compare categorical variables . to analyze and compare risk factors between pad subjects with coexisting cad and those with no coexisting cad and between pad subjects with ai and fp , we employed student 's t - test for continuous variables and the -test for categorical variables . simple logistic regression analysis and multinomial logistic regression analysis were carried out to determine the association among cardiovascular risk factors in pad , cad , and control subjects .
i , age , gender , ht , dm , hypercholesterolemia , obesity grade ( obese ) , smoking status and ckd were adjusted . model ii considered age , gender , smoking status , ckd , and mets .
the mean age of pad subjects was 64.4 ( 9.3 ) years , while the mean age of cad subjects was 61.2 ( 9.9 ) years , and that of control subjects was 59.9 ( 9.1 ) years ( p<0.001 ) .
the proportion of males in the subject groups was as follows : 90.6% for pad , 71.4% for cad , and 75.5% for control ( p<0.001 ) .
more pad subjects than cad and control subjects had ht , dm , and ckd ( p<0.001 ) , while more cad subjects were smokers , had hypercholesterolemia , and were obese than pad and control subjects ( p<0.001 ) . among the components of mets , more pad subjects had high blood pressure and high fbs ( p<0.01 ) than patients in the other two groups , while more cad subjects had low hdl - c levels and were obese than pad and control subjects ( p<0.01 ) .
tc , tg , ldl - c , hdl - c , fbs , and cr were significantly different among the three groups ( p<0.001 ) .
the results after bonferroni correction for multiple comparisons are shown in table 1 . among pad subjects , the proportion of ai subjects with coexisting cad was 58.4% , while the proportion of pad patients with no coexisting cad was 56.8% { p = nonsignificant ( ns)}. the proportion of coexisting cad was 33.6% in pad patients with ai and 32.2% in pad patients with fp ( p = ns ) ( table 2 ) . in model i , the adjusted odds ratios ( ors ) for ht { or 6.43 , 95% confidence interval ( ci ) 4.92 - 8.39 } , dm ( or 7.71 ,
95% ci 6.05 - 9.84 ) , hypercholesterolemia ( or 1.50 , 95% ci 1.18 - 1.90 ) , smoking ( or 10.3 , 95% ci 7.89 - 13.4 ) , and ckd ( or 1.54 , 95% ci 1.14 - 2.04 ) were significantly higher in subjects with pad compared to those in normal controls .
however , the ors for hdl - c ( or 0.92 , 95% ci 0.91 - 0.93 ) , being overweight ( or 0.51 , 95% ci 0.38 - 0.67 ) , and being obese ( or 0.32 , 95% ci 0.24 - 0.43 ) were significantly lower in pad subjects compared to those in normal controls .
the ors for ht ( or 2.89 , 95% ci 2.57 - 3.25 ) , dm ( or 4.90 , 95% ci 4.28 - 5.61 ) , smoking ( or 3.76 , 95% ci 3.28 - 4.30 ) , being overweight ( or 1.21 , 95% ci 1.04 - 1.41 ) , being obese ( or 1.74 , 95% ci 1.51 - 2.01 ) and ckd ( or 1.46 , 95% ci 1.24 - 1.72 ) were significantly higher in cad subjects than those in normal controls
. however , the ors for hypercholesterolemia ( or 0.67 , 95% ci 0.60 - 0.76 ) and hdl - c ( or 0.93 , 95% ci 0.92 - 0.94 ) were significantly lower in the cad group than those in the control group . in model ii , the ors for smoking ( or 9.38 , 95% ci 7.35 - 11.9 ) , ckd ( or 2.14 , 95%
ci 1.63 - 2.82 ) , and mets ( or 3.61 , 95% ci 2.89 - 4.51 ) were higher in pad subjects than those in normal controls . comparing cad subjects with normal control group subjects , the ors for smoking ( or 3.36 , 95% ci 2.99 - 3.79 ) , ckd ( or 1.80 ,
95% ci 1.56 - 2.09 ) , and mets ( or 3.66 , 95% ci 3.31 - 4.05 ) were significantly higher in cad subjects ( table 3 ) .
we analyzed the association between cardiovascular risk factors in 1 ) pad patients with or without coexisting cad , and in the normal control group and 2 ) the pad affected site ; ai or fp , and normal control group .
the ors in pad patients with or without coexisting cad group and ai or fp group for ht , dm , smoking , hdl - c , and obese grade ( model i ) and smoking , mets , and ckd ( model ii ) were similar to pad subjects ( table 4 and 5 ) .
the overall findings of this study revealed that ht , dm , hypercholesterolemia , obesity , smoking , ckd and mets are risk factors for pad and cad . however ,
these findings are consistent with those of previous studies that reported that risk factors for pad are similar to those for cad . for instance , ht,9 ) dm,10 ) hypercholesterolemia,11 ) smoking,12 ) ckd,13 ) and mets4 ) are known risk factors for cad .
ht,14 ) dm,15 ) hypercholesterolemia,16 ) smoking,17 ) ckd,5 ) and mets4 ) are risk factors for pad . in our study , the mean tc , ldl - c , hdl - c values were higher in the normal control group than those in the pad or cad group . the results from a japanese male worker
adult study based on the national health and nutrition examination surveys20 ) are consistent with the high tc , ldl - c , hdl - c values that we found in our normal control group .
obesity is one of the major risk factor for cvd , including pad.21 ) obesity is also associated with high mortality related to chronic disease.22 ) however , patients with cad or pad have an inverse correlation between bmi and cardiovascular mortality after adjustment for confounding variables in the factores de riesgo y enfermedad arterial registry.23 ) the obesity paradox24 ) is that obese patients receive better treatment and care , because they are perceived to be at high risk .
however , our pad obesity results can not be explained by the obesity paradox , because pad subjects had occlusion or > 50% stenosis in the peripheral artery confirmed by lower extremity computed tomography angiography .
this corresponds to an ankle - brachial index < 0.9 and the consequent development of intermittent claudication , gangrene , and pain .
our pad subjects who visited tertiary medical services already had progressed pad with a limited radius of action , immobilization , muscle atrophy , and depression requiring surgery or intervention .
the factors outlined above may explain why the obesity results were inconsistent between pad and cad subjects in our analysis .
the ors for the risk factors for the absence or presence of coexisting cad in pad and cad , with the exception of obesity , were similar among the groups .
a study on the association of cardiovascular risk factors with patterns of lower limb atherosclerosis in 2659 patients who underwent angioplasty revealed that dm predicted pad compared to no dm or current smoking status.25 ) an italian study also showed that preexisting cad with pad was associated with risk factors for pad.26 ) in our study , 33% of subjects with pad had coexisting cad .
this result is consistent with previous studies that reported 21% of pad subjects showed myocardial infarction and 26% of pad subjects had angina.27 ) a comparison of the ors for ai and fp according to the affected site in pad and cad revealed similar risks as those reported in a turkish study ( ankara),28 ) and two u.s.a - based studies ( san diego29 ) and southern california30 ) ) .
first , the study was conducted retrospectively at a single center , which may have caused selection bias .
we were also not able to eliminate the possibility of information bias when collecting medical records from the medical charts of the subjects and laboratory results .
second , the mean age of the subjects in the normal control group was younger than that of the subjects in the pad and cad groups . to minimize the effect of age , we conducted age - adjusted analysis .
a further limitation of our study is that we could not consider symptoms of patients with pad , physical activity , nutrition , socioeconomic position , waist circumference , or health behavior variables due to limited data .
further cardiovascular cohort studies considering these variables are therefore required to verify the risk factors for atherosclerosis .
we found significantly different ors for risk factors , namely age , gender , ht , dm , hypercholesterolemia , hdl - c , obesity , smoking , ckd , and mets , in the pad and cad groups compared to those in the control group .
interestingly , the ors for obesity were inconsistent between pad and cad subjects . in other words ,
obesity grade was showed opposite trends . however , in both diseases , cardiovascular risk factors were found to be risk factors . in conclusion , there appears to be no differences in risk factors for pad and cad in the korean population .
we found significantly different ors for risk factors , namely age , gender , ht , dm , hypercholesterolemia , hdl - c , obesity , smoking , ckd , and mets , in the pad and cad groups compared to those in the control group .
interestingly , the ors for obesity were inconsistent between pad and cad subjects . in other words ,
obesity grade was showed opposite trends . however , in both diseases , cardiovascular risk factors were found to be risk factors . in conclusion , there appears to be no differences in risk factors for pad and cad in the korean population .
|
background and objectivesthe objective of this study was to analyze and compare risk factors for peripheral artery disease ( pad ) and coronary artery disease ( cad).subjects and methodsthe sample included 7936 korean patients aged 20 years who were hospitalized from 1994 to 2004 .
of the 7936 subjects , pad ( n=415 ) , cad ( n=3686 ) , and normal controls ( control ) ( n=3835 ) were examined at the health promotion center , samsung medical center.resultsthe mean age ( years ) of pad subjects was 64.4 ( 9.3 ) , while cad subjects was 61.2 ( 9.9 ) , and control subjects was 59.9 ( 9.1 ) ( p<0.01 ) .
the proportion of males was 90.6% for pad , 71.4% for cad , and 75.5% for control subjects ( p<0.01 ) .
the adjusted odds ratios ( ors ) for hypertension , diabetes mellitus , hypercholesterolemia , smoking , metabolic syndrome and chronic kidney disease were significantly higher in subjects with pad or cad compared to those in control .
however , the ors for high density lipoprotein , being overweight , and being obese were significantly lower in pad subjects compared to those in control.conclusionwe found that cardiovascular risk factors were in fact risk factors for both pad and cad .
|
Introduction
Subjects and Methods
Study population and design
Diagnostic criteria
Cardiovascular risk factors
Statistical analysis
Results
Discussion
Conclusions
- Supplement -
|
neurological soft signs ( nss ) have long been considered one of the functional features ( tsuang and faraone , 1999 ; tsuang et al . , 1991 ) and endophenotypes ( chan and gottesman , 2008 ; chan et al . , 2010a ) of schizophrenia spectrum disorders .
however , most of the studies of nss have been limited to the use of clinical ratings . instead
, recent findings from both structural and functional imaging studies have shown that nss may be associated with specific brain alterations .
specifically , in individuals with psychosis , more nss have been associated with smaller volumes of the inferior frontal lobes ( thomann et al . , 2009a ) , the pre - central gyrus ( heuser et al . , 2011 ; mouchet - mages et al . , 2011
) , the global cortical sulci ( dazzan et al . , 2004 ; gay et al . ,
2013 ) as well as the cerebellum ( ho et al . , 2004 ; thomann et al .
, nss may be responsible for some of the observed clinical manifestations in schizophrenia ( keshavan et al .
, 2003 ; mouchet - mages et al . , 2011 ; schroder et al . , 1999 ) .
a recent meta - analysis of structural and functional imaging findings suggests that nss are associated with volume reductions of the pre - central gyrus , the cerebellum , the inferior frontal gyrus and the thalamus .
furthermore , the same meta - analysis also suggests that functional imaging studies support an association between nss and altered neural activation in the inferior frontal gyrus , the bilateral putamen , the cerebellum and the superior temporal gyrus in patients with schizophrenia ( zhao et al . ,
, these findings support the presence of a dysfunction of neural circuitry that underlies the presence of these minor neurological abnormalities , which are already present at illness onset . on the other hand , empirical findings
have also shown that non - psychotic first - degree relatives of schizophrenia patients exhibit a higher prevalence of nss compared to healthy controls ( egan et al .
a meta - analysis comparing the prevalence of nss between patients with schizophrenia , non - psychotic first - degree relatives and healthy controls indicated a mean effect size of 0.8 and 0.97 for patients and their non - psychotic first - degree relatives and for non - psychotic first - degree relatives and controls respectively ( chan et al . , 2010b ) .
these results are consistent with the argument that nss are familial in nature and segregate with the illness .
however , no studies had examined the brain correlates of nss in healthy relatives of patients with schizophrenia .
furthermore , all the aforementioned neuroimaging studies in schizophrenia patients used conventional subtraction analysis and did not examine the underlying connectivity between the brain regions identified . given that some nss , such as the fist
palm ( fep ) task ( first described by luria , see heuser et al . , 2011 ) , involve the functional integration or regulation of areas involved in motor sequencing rather than being directly activated , a connectivity approach is more appropriate to identify the specific network involved .
only one study ( rao et al . , 2008 ) adopted a psychophysiological interaction ( ppi ) ( friston et al . , 1997 ) method to re - analyze their previous findings on the fep task in healthy volunteers ( chan et al . , 2006 ) .
this new analysis showed enhanced functional connectivities between the left- and right sensorimotor cortices ( smc ) and the right inferior and middle prefrontal cortices during fep task performance compared to a simple palming task .
these findings suggest a regulatory role of the prefrontal cortex on fep task execution . in this study
, we examined whether prefrontal regions are involved in the integration or regulation of neural activity underlying motor sequencing in patients with first - episode schizophrenia and their non - psychotic first degree relatives ( fdr ) .
more specifically , we aimed to identify any frontal regions where coupling in these areas and the sensorimotor cortex ( smc ) significantly differed between the complex fep task and a simple control motor task using ppi analysis .
we hypothesized that patients with first - episode schizophrenia and their non - psychotic fdrs would show a reduced dysfunction of the prefrontal cortex and smc while performing the fep task .
thirteen right - handed first - episode schizophrenia patients were recruited from the mental health center , peking university , beijing , for the study .
participants were recruited to the study if they met the following inclusion criteria : a ) diagnosis of schizophrenia ascertained by experienced psychiatrists according to the dsm - iv ( apa , 1994 ) ; b ) aged 1840 years ; and c ) illness duration within 2 years .
the exclusion criteria were : a ) a history of neurological disorders ; b ) a lifetime history of substance abuse and c ) an estimated iq lower than 70 .
current symptom severity was assessed with the positive and negative syndrome scale ( kay et al . , 1987 ) .
fourteen non - psychotic fdrs of the patients taking part in the study were also invited to participate .
potential participants were excluded if they a ) met the dsm - iv criteria for substance abuse ; b ) were suffering from any clinically unstable medical disorder ; c ) had a history of head injury ( past or present ) ; and d ) had an estimated iq lower than 70 .
fourteen healthy controls matched with the patients in age , gender and handedness were recruited from local universities through advertisements .
the institutional review board of the institute of psychology , the chinese academy of sciences , approved the study protocol .
behavioral neurological soft signs were examined with the abridged version of the cambridge neurological inventory ( cni ) ( chan et al . , 2009 ) .
this abridged version offers instructions for eliciting and rating a comprehensive range of nss in motor coordination , sensory integration and disinhibition .
rao et al . , 2008 ) ( for the earliest description , please see luria , 1966 ; in heuser et al . , 2011 ) . in brief , the task consisted of three right - hand motor tasks which varied in complexity . in the simple palm tapping ( pt ) task
, participants were required to repeat only one right palm tapping in the prone position . in the intermediate complex pronation / supination ( ps ) task , participants were required to perform right palm tapping in the prone and supine positions alternatively . in the complex fep task , participants were required to successivley place their right hand in a fist resting position vertically ( fist ) , a palm resting position vertically ( edge ) , and a palm resting position horizontally ( palm ) . a resting condition ( a ) without any hand movement when participants were asked to focus on the screen
was used as the control baseline of the pt task , and the pt task was in turn used as the control baseline of the ps and fep tasks .
participants were asked to practice the motor actions correctly at a constant rate before entering the scanner . during scanning , their performance was monitored by the experimenter through the window of the scanner room . in the formal imaging task
, participants were instructed to perform the three tasks at a similar pace throughout the entire experiment .
pt and ps tasks were executed in 1 s and the fep task was executed in 1.5 s. the experimenter monitored the task performance outside the scanner room to ensure that the participants performed the correct hand movements .
first , a resting condition lasted for 20 s and then 6 s of counting backward reminded the participants to get ready for their hand movement . after backward counting , the pt or ps or fep task lasted for 40 s. one of these three hand movements would be presented on the screen .
participants were required to conduct the hand movements according to the demonstration on the screen .
the sequence of the three hand movement tasks was optimized and counterbalanced within the three runs .
the functional imaging data was originally acquired in a ge 3 t sigma scanner ( general electric , waukesha , wi , usa ) with a standard ge birdcage - type rf coil using a standard t2 * -weighted epi sequence .
the epi parameters were : tr = 2 s te = 60 ms , fov = 24 24 cm , matrix = 64 64 , flip angle = 60 , 22 axial slices ( 5 mm thick/1.2 mm sp , from superior to inferior ) .
the spatial resolution for the functional images was 3.75 3.75 6.2 mm .
high - resolution anatomical images were also obtained using the standard t1-weighted sequence ( 66 axial slices , 2.0 mm thick / interleaved , fov = 24 24 cm , matrix = 256 256 ) .
images were analyzed with statistical parametric mapping software ( spm8 , wellcome department of imaging neuroscience , london , uk ) implemented in matlab 2009b ( mathworks inc .
, sherborn , ma , usa ) . three dummy scans in the beginning of the experiment were removed automatically from the dataset .
images were registered to the first icbm 152 , which was based on 152 brains and was created by the montreal neurological institute ( mni ) with a 2 2 2 mm resolution . in the final step of pre - processing , the images were spatially smoothed by an isotropic gaussian kernel with fwhm of 8 mm .
conventional analyses were first conducted at the individual - level using voxel - wise general linear modeling ( glm ) and four t - contrasts were defined between the tasks and the corresponding baselines , i.e. , pt vs. rest , ps vs. rest , fep vs. rest and fep vs. pt .
data from the contrasts of the first level model in the healthy control group , the schizophrenia group and the fdr group were entered into this model . a threshold of alphasim corrected p < 0.01 and cluster size larger than 15 voxels were used to identify the activations associated with each contrast . furthermore , we used a regression model to find the regions with group difference .
we set up an f contrast to find the regions with main effect of group difference .
signal change percentage was retrieved from the rois with group difference for post - hoc analysis .
ppi analysis was used to estimate functional integration during task execution under different motor complexity conditions .
the left smc was determined a - priori as the reference region for the ppi analysis because the motor tasks in the present study were only completed with the right hand .
this region was defined by using a sphere with a radius of 8 mm and a center at the peak activation in the left smc activation ( mni coordinates = [ 36 28 52 ] , from the conventional analysis of the contrast of pt vs. rest ) .
we performed voxel - wise ppi analysis at individual - level for the left smc to see if any other brain areas connected to the smc showed a significant increase in functional coupling ( the slope of regression ) during the fep task compared with a control task ( e.g. , the pt or ps task ) .
for each participant , the activation time course signal in the reference region ( i.e. , the first eigenvariate time series , adjusted by effect of interest ) was extracted from the conventional glm and entered into the ppi analysis as the first regressor representing the physiological variable .
a second regressor representing the motor tasks with different complexity was entered into the ppi analysis as the psychological variable .
the psychophysiological interaction between task complexity and activation signal in the reference region was designated as the regressor of interest in the ppi analysis .
thereafter we performed group - level random effect analysis on the individual results using one sample t - tests for the contrast fep vs. pt .
group - level paired t - tests were conducted for the contrast fep vs. ps .
areas of significant activation were identified at a threshold of uncorrected p < 0.001 and cluster size larger than 15 voxels .
there was no significant difference between patients with schizophrenia , their fdrs and healthy controls in the three subscales and total score of cni .
conventional activation of the pt , ps and fep tasks in the three groups , after co - varying for age , is shown in table 3 .
the left frontal parietal region was significantly activated when participants performed the pt , ps and fep tasks with their right hand . moreover , in the pt rest contrast , activation in the left frontal parietal region , together with the left medial frontal region , were lowest in patients with schizophrenia , intermediate in the fdrs and highest in healthy controls ( scz < rel < hc ) .
we further used a regression model and conducted a post - hoc analysis to identify the regions that were different across groups both linearly and non - linearly . in the pt rest contrast , we confirmed that the left frontal precentral gyrus ( 30 34 64 , k = 100 , f(2,37 ) = 20.23 ) was linearly activated in all three groups ( scz < rel < hc ) .
the left medial frontal region ( hc = rel > scz , 8 18 64 , k = 136 , f(2,37 ) = 15.59 ) and the left middle temporal gyrus ( hc > scz = rel , 48 68 10 , k = 124 , f(2,37 ) = 11.7 ) were non - linearly activated in all three groups .
the activations associated with task complexity were examined in the fep ps and the fep pt contrasts . in the fep
pt contrast , the bilateral middle frontal regions were activated in the healthy control group .
the left middle frontal region was activated in the fdr group , but there was no frontal region activation in first episode schizophrenia patients .
2 illustrates the frontal activation in both healthy controls and fdrs . in the fep
ps contrast , the left middle frontal region was also activated in the fdr group , but not in the healthy control group or the schizophrenia group .
the ppi analysis identified right frontal regions in which the activity showed significant activation coupling to activity in the left smc during performance of the more complex fep task relative to the simple pt or ps tasks in the healthy control group , but not in the schizophrenia group .
in this study , we report for the first time the neural activation and connectivity elicited by execution of the fep task in patients with first episode schizophrenia and their healthy first degree relatives .
our main finding is that patients with first episode schizophrenia do not show , in comparison with healthy controls and their fdrs , activation of the left middle frontal gyrus in the execution of the fep versus a simpler motor task like the pt .
this provides evidence of a frontal dysfunction in these patients when performing a complex motor task .
the right inferior frontal gyrus was found to modulate the activation of the sensorimotor cortex with the increase in motor complexity in healthy controls .
a frontal dysfunction was also demonstrated by our second main finding , suggesting that with increase in task difficulty , patients with first episode schizophrenia do not show functional connectivity between the sensorimotor cortex and the right frontal gyrus , in contrast to healthy controls .
the presence of a prefrontal dysfunction in schizophrenia is supported by extensive evidence from both structural and functional neuroimaging studies .
prefrontal areas have been consistently reported as reduced in volume in patients with schizophrenia ( chan et al . , 2011 ; dazzan et al . ,
furthermore , fmri studies have shown altered activation of frontal areas during executive and working memory tasks , particularly with increasing capacity demand ( barch et al .
, 2012 ; callicott et al . , 2000 ; perry et al . , 2001 ) . at a functional level
, patients with schizophrenia also show an excess of signs reflecting frontal release , such as abnormalities in eye movements and short - term memory deficits , which again point to a frontal cortical alteration ( hyde et al . , 2007 ) .
in this study , we tested the activation of frontal areas during a complex motor task , the fep task , which has long been considered in neurological and neurocognitive studies as indicative of frontal lobe lesions ( luria , 1966 ) .
two previous fmri studies have reported that in healthy individuals , the execution of this task did not induce activation of prefrontal areas , but induced activation of other parts of the cortex , including the sensorimotor areas ( chan et al . , 2006 ; umetsu et al . , 2002 ) .
a subsequent study , using the same ppi approach that we used in this study , showed that this finding could reflect an indirect involvement of the prefrontal cortex , which could exert an integrative and regulatory function on neural circuitry involved in complex motor sequences .
we found here that in healthy individuals , the execution of the fep task , compared to a simpler motor act , was indeed associated with both direct activation of the prefrontal cortex , and greater coupling of prefrontal areas and sensorimotor cortex .
interestingly , a recent meta - analysis of the brain connectome suggests that the frontal lobe represents a hub ( a region highly interconnected with other brain regions and valuable for integrative information processing and adaptive behavior ) particularly affected in patients with schizophrenia ( crossley et al . , 2014 ) . there is a paucity of studies on brain activation during complex motor tasks in these patients .
however , neuroimaging studies that examined gross motor coordination signs such as finger - to - thumb operation tests in these patients have reported reduced activation in the smc and the supplementary motor area ( sma ) in comparison to healthy controls ( schrder et al . ,
a later similar study used pronation / supination tests and found significant reduced activation in the smc in patients with schizophrenia compared to healthy controls ( schroder et al . , 1999 ) .
the lack of activation we found with a task like the fep , which requires fine motor coordination and requires executive processes like inhibition , planning and updating , could be interpreted as part of the hypofrontality hypothesis of schizophrenia reflecting a reduced function of the prefrontal cortex ( callicott et al . , 2000 ) .
response relationship of the left frontal parietal activation across patients , their fdrs and healthy controls . here , in the pt vs. rest
contrast , activation was lowest in the schizophrenia group , intermediate in the fdr group and highest in the healthy control group . although these differences were highlighted with a motor sequence less complex than the fep , the finding suggests that at least part of the pathophysiological substrate that underlies motor difficulties in psychosis may be linked to a genetic susceptibility to schizophrenia .
indeed , deficits in motor skills have been reported extensively in individuals at risk of schizophrenia because of either genetic loading or prodromal features ( blanchard et al . , 2010 ;
first , the clinical sample size was relatively small . however , the merit of the present sample is that it included both first - episode schizophrenia patients and their non - psychotic siblings .
secondly , the use of ppi analysis may not fully detect the connectivity changes related to the performance of the fep task .
however , ppi is an appropriate method to test the relationship between two simple mental activities ( friston et al . , 1997 ) .
other more sophisticated approaches , such as dynamic casual modeling , may help identify further subtle connectivity changes in the performance of the fep task .
thirdly , the use of the screen to synchronize the motor actions might induce the activation of mirror neurons .
however , the contrasts between any two of the three motor actions ( fep , pt , and ps ) should minimize this effect by subtraction methods .
finally , we should also consider the validity of our task , since previous subtraction analyses of the fep task did not elicit activation of the frontal cortex .
however , the fact that the left frontal parietal region was significantly activated when participants performed the pt , ps and fep tasks with their right hand suggests that this hand movement imaging task was valid . notwithstanding these limitations
, our findings suggest that the fep task may be a useful endophenotype of schizophrenia , as it fulfills the criteria of being familial and shows a dose
previous study had demonstrated that patients with schizophrenia showed aberrant brain activation in the premotor area after a week of motor training ( kodama et al .
, 2001 ) . given this temporal stability of nss , the fact that this is an imaging endophenotype rather than a simple behavioral rating arguably enhances its precision .
this task is also simpler to carry out when compared to a full nss scale .
we believe that our findings add valuable information to the understanding of the origin and underlying neural mechanism of motor coordination signs .
future studies should recruit a larger sample of first - episode , preferably medication - nave schizophrenia patients for further validation of our findings .
|
neurological soft signs have been considered one of the promising neurological endophenotypes for schizophrenia . however ,
most previous studies have employed clinical rating data only . the present study aimed to examine the neurobiological basis of one of the typical motor coordination signs , the fist edge
palm ( fep ) task , in patients with first - episode schizophrenia and their non - psychotic first degree relatives .
thirteen patients with first - episode schizophrenia , 14 non - psychotic first - degree relatives and 14 healthy controls were recruited .
all of them were instructed to perform the fep task in a 3 t ge machine .
psychophysiological interaction ( ppi ) analysis was used to evaluate the functional connectivity between the sensorimotor cortex and frontal regions when participants performed the fep task compared to simple motor tasks . in the contrast of palm - tapping ( pt ) vs. rest , activation of the left frontal
parietal region was lowest in the schizophrenia group , intermediate in the relative group and highest in the healthy control group .
in the contrast of fep vs. pt , patients with schizophrenia did not show areas of significant activation , while relatives and healthy controls showed significant activation of the left middle frontal gyrus .
moreover , with the increase in task complexity , significant functional connectivity was observed between the sensorimotor cortex and the right frontal gyrus in healthy controls but not in patients with first episode schizophrenia .
these findings suggest that activity of the left frontal parietal and frontal regions may be neurofunctional correlates of neurological soft signs , which in turn may be a potential endophenotype of schizophrenia . moreover , the right frontal gyrus may play a specific role in the execution of the fep task in schizophrenia spectrum disorders .
|
Introduction
Method
Results
Discussion
|
these studies were approved by institutional review boards at the national institutes of health ( nih ) and washington university . a 28-year - old woman with job syndrome
pathologic examination of a bronchoalveolar lavage ( bal ) sample showed scattered cells , primarily columnar bronchial cells , with cytomorphologic changes reminiscent of bk polyomavirus ( bkpyv)infected decoy cells .
the cells stained positive with pab416 , a monoclonal antibody against the sv40 large t antigen .
the patient had bkpyv viremia ( 8.1 10 copies / ml ) and viruria ( 6.9 10 copies / ml ) .
the bal sample was weakly positive for bkpyv by pcr ( < 250 copies / ml ) and negative for jc polyomavirus .
nonenveloped virions were purified from the bal sample by using ultracentrifugation with optiprep ( # d1556 ; sigma - aldrich , st .
dna was extracted from the virion preparation and subjected to random - primed rolling circle amplification ( rca ) and restriction enzyme digestion , which yielded 2 strong bands .
the bands were cloned and identified as wupyv by using sanger sequencing . the complete genomic sequence of the isolate , designated j1 ( genbank accession no .
kj643309 ) , was confirmed by using miseq analysis ( illumnina , san diego , ca , usa ) of the rca product .
a second wupyv variant with 2-nt polymorphisms and a single base insertion was also detected in the rca product .
we developed an immunohistochemical ( ihc ) assay to detect the wupyv viral protein 1 ( wu - vp1 ) by using an igg2b designated nn - ab06 .
recombinant histidine - tagged wu - vp1 protein was generated by expressing wu - vp1 ( genbank accession no .
abq09289 ) in escherichia coli from a gateway pdest17 plasmid ( life technologies , carlsbad , ca , usa ) and purifying the protein by using an affinity ni - nta column ( pierce biotechnology , rockford , il , usa ) . after generation of hybridomas , we identified clones producing antibody against wu - vp1 by elisa and immunoblot .
clones that cross - reacted with ki polyomavirus vp1 ( ki - vp1 ) were identified by elisa with glutathione s - transferase tagged ki - vp1 ( 2 ) and eliminated . to generate positive control cells for ihc assay optimization
, we transfected 293 t cells with plasmid pdest26-wu - vp1 ( life technologies ) .
a subset of cells was fixed in 10% neutral - buffered formalin and embedded in paraffin .
ihc testing was performed by deparaffinizing slides in xylene and rehydrating them in a series of ethanol solutions . after treating slides with 3% hydrogen peroxide , antigen
was retrieved in citrate buffer , ph 6.0 ( 10 mmol / l citric acid , 0.05% tween 20 ) in a pressure cooker ( pc625 ; nesco , two rivers , wi , usa ) for 3 min on the high setting .
slides were blocked in 1.5% normal horse serum ( # s-200 ; vector laboratories , burlingame , ca , usa ) and incubated with nn - ab06 , then with biotinylated anti - mouse igg ( ba-2000 ; vector laboratories ) .
biotin complex kit ( # pk-6100 ; vector laboratories ) and ( 3,3-diaminobenzidine ) ( # sk-4100 ; vector laboratories ) , we counterstained tissues with hematoxylin .
a serial section of the same cell block stained with an isotype - matched antibody ( # 557351 , mouse igg2b : bd biosciences , san jose , ca , usa ) ( figure 1 , panel b ) and mock transfected cells stained with nn - ab06 ( figure 1 , panel c ) showed negative results .
western blotting was performed as an independent means of evaluating specificity of nn - ab06 ( 11 ) .
nn - ab06 reacted with wu - vp1 protein lysate but not with ki - vp1 lysate , which is the most closely related virus to wupyv .
wu polyomavirus antigen in bronchoalveolar lavage specimens from lungs transplanted into a recipient ( 28-year - old woman ) with job syndrome .
immunohistochemical analysis of 293 t cells transfected with pdest26-wu virus protein 1 and stained as follows .
d ) bronchoalveolar lavage specimen stained with nn - ab06 showing prominent dark staining of cells with enlarged nuclei and a ground glass appearance characteristic of viral cytopathic changes ( arrows ) .
original magnifications 400 in panels a c and 600 in panels d and e. we applied the wu - vp1 ihc assay to formalin - fixed , paraffin - embedded sections of the bal sample .
prominent dark staining of cells with enlarged nuclei and a ground glass appearance characteristic of viral cytopathic changes were observed ( figure 1 , panel d ) .
staining was not seen in serial sections stained with the isotype antibody ( figure 1 , panel e ) or with no antibodies .
many wupyv - positive cells were cuboidal to columnar and showed other morphologic features consistent with respiratory epithelial cells . to determine their etiology , we developed a double immunofluorescence ( dif ) assay with a polyclonal antibody against wu - vp1 ( 2 ) , designated nn - ab01 , and a monoclonal antibody against cytokeratins ( # m3515 ; dako , carpinteria , ca , usa ) .
deparaffinization and antigen retrieval were accomplished as noted above , and sections were blocked in superblock t20 ( # 37516 ; thermo scientific , waltham , ma , usa ) . to validate the assay
, we performed immunofluorescence analysis with nn - ab01 on positive control 293 t cells expressing wu - vp1 .
several wu - vp1-positive cells were observed ( figure 2 , panel a ) ; a serial section stained with preimmune serum at the same dilution showed a negative result ( figure 2 , panel b ) .
wu polyomavirus antigen in respiratory epithelial cells from lungs transplanted into a recipient ( 28-year - old woman ) with job syndrome .
immunofluorescence of 293 t cells transfected with pdest26-wu virus protein 1 and stained with a ) wu virus protein 1 polyclonal antibody ( nn - ab01 ) or b ) preimmune serum .
c ) double immunofluorescence with nn - ab01 ( red ) and a monoclonal antibody against cytokeratin ( green ) showing a double - positive cell from the bronchoalveolar lavage specimen .
d ) bronchoalveolar lavage specimen with multiple wu virus protein 1/cytokeratin double - positive cells .
original magnifications 100 in panels a and b , 600 in panel c , and 400 in panel d. for dif , bal sections were incubated first with the primary antibodies and then with fluorescently labeled secondary antibodies ( # a10042 anti - rabbit-568 and # a10042 anti - mouse-488 ; life technologies ) .
we observed cells positive for wu - vp1 and cytokeratin ( figure 2 , panels c , d ) , which identified these cells as epithelial cells .
of the 136 wu - vp1-positive cells , 77 ( 57% ) were also cytokeratin positive .
a serial section of the bal sample stained with an isotype - matched antibody to the cytokeratin antibody ( # 555746 mouse igg1 ; bd biosciences ) and preimmune rabbit serum was negative .
we hypothesized that the remaining 43% of wu - vp1-positive , cytokeratin - negative cells might be macrophages .
however , a dif assay using nn - ab01 and an antibody against cd68 ( # m0814 ; dako ) , a macrophage marker , showed wu - vp1 and cd68 single - positive cells but no double - positive cells .
in addition , a stain with nn - ab01 and an antibody against cd45 ( # m3515292 ; dako ) , a marker for hematopoietic cells , also showed negative results .
before this study , to our knowledge , no specific cell type had been identified as susceptible to wupyv infection .
the presence of nuclease - resistant viral dna from the optiprep gradient and detection of wu - vp1 , which is believed to be expressed concomitantly with dna replication ( 12 ) , suggests that the cells were infected by wupyv and that the wupyv life cycle reached at least the stage of late gene expression .
the clinical role of infection by wupyv is uncertain , given that the patient was not experiencing any recognizable symptoms .
although we attempted to identify a second population of wu - vp1-positive , cytokeratin - negative cells , the etiologic features of these cells remains uncertain .
our patient had job syndrome , a primary immunodeficiency not previously associated with polyomavirus susceptibility .
it is possible that immunosuppressant medications , which include prednisone and tacrolimus , altered susceptibility to virus infection . other human polyomaviruses are believed to exclusively cause disease in immunocompromised hosts
this case suggests that immunosuppression might also play a role in wupyv infection and expands our understanding of wupyv biology .
|
we detected wu polyomavirus ( wupyv ) in a bronchoalveolar lavage sample from lungs transplanted into a recipient with job syndrome by using immunoassays specific for the wupyv viral protein 1 .
co - staining for an epithelial cell marker identified most wupyv viral protein 1positive cells as respiratory epithelial cells .
|
The Study
Conclusions
|
toxoplasma gondii is an obligate intracellular parasite that infects a variety of mammals and birds , causing toxoplasmosis .
t. gondii is an important foodborne parasite that is primarily transmitted from animals to humans through the consumption of infected meat . in some countries ,
pork is the most common meat consumed , and several ethnic groups consume raw pork .
toxoplasmosis is a source of significant economic loss for swine farmers because of gross lesions in infected animals , which result in the carcass being condemned at the time of slaughter , the expense associated with treatment , and weight loss associated with clinical toxoplasmosis .
the development of effective diagnostic reagents or vaccines is very important for worldwide public health and economic repercussions of t. gondii infection .
the life cycle of t. gondii is relatively complex , and its antigenic component can change in specificity or makeup during different development stages ; therefore , the newly synthesized multiepitope antigen is one of the most promising antigens for the development of effective diagnostic reagents or vaccines [ 4 - 9 ] .
however , the study of epitope - based vaccines and diagnostic reagents is highly dependent on the accurate identification of b - cell epitopes and t - cell epitopes .
therefore , the identification of protein epitopes will be very important for diagnostic purposes and for the development of peptide vaccines [ 10 - 12 ] . among dense granule antigens ( gras ) ,
gra6 was also demonstrated to be useful for designing novel and alternative diagnostic methods for toxoplasmosis or vaccines [ 13 - 17 ] .
the gra6 gene does not contain any introns and is a single copy in the genome of t. gondii .
gra6 localized in the dense granules and in the parasitophorous vacuole closely associated to the network .
a total of 51 t. gondii - positive sera samples previously collected from pigs ( n=32 ) experimentally infected with the gansu jingtai strain ( isolated from a pig with acute toxoplasmosis ) in our laboratory were evaluated in this study .
the experimental protocol was approved by the ethical committee of the lanzhou veterinary research institute , chinese academy of agricultural sciences , china .
twelve pig serum samples were collected at the time of presentation of clinical symptoms ( g1 ) , 18 follow - up samples were collected on days 14 to 35 after the onset of symptoms ( g2 ) , and 21 samples were collected on days 60 to 120 after the onset of symptoms ( g3 ) .
the presence of toxoplasma igm and igg antibodies was determined by t. gondii lysate antigen - elisa .
t. gondii dna was obtained from gansu jingtai strain tachyzoites using the universal genomic dna extraction kit ( takara biotechnology co. , ltd , dalian , china ) , and the gra6 sequence was amplified using the primers 5-gcgaattcatggcacacggtggcatct-3 and 5-atgcggccgcttaaaaatcaaactcattc-3 .
the pcr amplification was performed using the takara taqtm kit according to the manufacturer s instructions .
the sample was subjected to an initial denaturation ( 94c for 5 min ) , 35 cycles of denaturation ( 94c for 1 min ) , annealing ( 60c for 30 sec ) and elongation ( 72c for 1 min ) , and a final extension at 72c for 10 min .
the pcr - generated fragment was purified and cloned into the pmd-18 t vector ( takara biotechnology ) .
the recombinant plasmid was used to transform escherichia coli jm 109 competent cells , and the recombinant cells were selected on lb plates with ampicillin ( 100 mg / l ) , x - gal ( 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ; 70 mg / l ) , and iptg ( isopropyl -d - thiogalactopyranoside ; 80 m ) at 37c for 24 hr ( ampicillin , x - gal and iptg were from takara biotechnology ) .
positive colonies were inoculated into lb liquid medium containing ampicillin ( 100 mg / l ) and incubated at 37c for 16 hr .
the positive colonies identified by pcr were sequenced by takara biotechnology . to analyze the gra6 b cell epitopes ,
the deduced amino acid sequence of gra6 was analyzed using the protean subroutine in the dnastar software package .
this subroutine uses the garnier - robson and chou - fasman algorithms for predicting the alpha , beta , and turn regions , the garnier - robson algorithm for predicting the coil regions , the kyte - doolittle algorithm for predicting hydrophilicity , the karplus - schultz algorithm for predicting flexibility , the emini algorithm for predicting surface probability , and the jameson - wolf algorithm for predicting antigenicity .
based on this analysis , the peptides with good hydrophilicity , high accessibility , high flexibility , and strong antigenicity were selected as the antigen epitopes .
enzyme - linked immunoassays specific for each peptide were performed as described by cardona et al . with minor modifications .
the microplates were coated with 100 l ( 10 g / ml ) of each peptide diluted in carbonate buffer , ph 9.6 ( na2co3 : 0.159 g/100 ml ; nahco3 : 0.293 g/100 ml ) .
the plates were incubated for 1 hr at 37c , then for 48 hr at 4c , and 1 hr at 37c .
non - specific ligand sites were blocked with 100 l 2% casein phosphate buffer for 1 hr at 37c . the plates were washed and incubated with 100 l serum diluted to 1:100 in 5% casein phosphate buffer for 1 hr at 40c .
after washing , 100 l rabbit anti - pig peroxidase - conjugated igg ( sigma ) diluted to 1:4,000 in 6% casein phosphate buffer was added for 20 min at 37c .
after the washes , the horseradish peroxidase activity was detected using tmb for 30 min at 37c and stopped with a 5% h2so4 solution .
a positive cut - off point was determined by estimating the mean average absorbance of 10 negative controls plus 2 sds . to determine the specificity of the anti - peptide antibody , elisa using irrelevant peptides from the bl21 of the orf virus previously synthesized by our laboratory ( sequence : vdvqskdkdadelre )
was also performed as described above . as controls , elisa using excretory / secretory antigen ( esa ) and recombinant gra6 from the rh strain of t. gondii previously expressed by our laboratory
briefly , non - specific ligand sites were blocked with 100 l 5% bsa phosphate buffer .
the rabbit anti - pig peroxidase - conjugated igg diluted 1:8,000 in pbs was used as the secondary antibody .
a total of 51 t. gondii - positive sera samples previously collected from pigs ( n=32 ) experimentally infected with the gansu jingtai strain ( isolated from a pig with acute toxoplasmosis ) in our laboratory were evaluated in this study .
the experimental protocol was approved by the ethical committee of the lanzhou veterinary research institute , chinese academy of agricultural sciences , china .
twelve pig serum samples were collected at the time of presentation of clinical symptoms ( g1 ) , 18 follow - up samples were collected on days 14 to 35 after the onset of symptoms ( g2 ) , and 21 samples were collected on days 60 to 120 after the onset of symptoms ( g3 ) .
the presence of toxoplasma igm and igg antibodies was determined by t. gondii lysate antigen - elisa .
t. gondii dna was obtained from gansu jingtai strain tachyzoites using the universal genomic dna extraction kit ( takara biotechnology co. , ltd , dalian , china ) , and the gra6 sequence was amplified using the primers 5-gcgaattcatggcacacggtggcatct-3 and 5-atgcggccgcttaaaaatcaaactcattc-3 .
the pcr amplification was performed using the takara taqtm kit according to the manufacturer s instructions .
the sample was subjected to an initial denaturation ( 94c for 5 min ) , 35 cycles of denaturation ( 94c for 1 min ) , annealing ( 60c for 30 sec ) and elongation ( 72c for 1 min ) , and a final extension at 72c for 10 min .
the pcr - generated fragment was purified and cloned into the pmd-18 t vector ( takara biotechnology ) .
the recombinant plasmid was used to transform escherichia coli jm 109 competent cells , and the recombinant cells were selected on lb plates with ampicillin ( 100 mg / l ) , x - gal ( 5-bromo-4-chloro-3-indolyl--d - galactopyranoside ; 70 mg / l ) , and iptg ( isopropyl -d - thiogalactopyranoside ; 80 m ) at 37c for 24 hr ( ampicillin , x - gal and iptg were from takara biotechnology ) .
positive colonies were inoculated into lb liquid medium containing ampicillin ( 100 mg / l ) and incubated at 37c for 16 hr .
to analyze the gra6 b cell epitopes , the deduced amino acid sequence of gra6 was analyzed using the protean subroutine in the dnastar software package .
this subroutine uses the garnier - robson and chou - fasman algorithms for predicting the alpha , beta , and turn regions , the garnier - robson algorithm for predicting the coil regions , the kyte - doolittle algorithm for predicting hydrophilicity , the karplus - schultz algorithm for predicting flexibility , the emini algorithm for predicting surface probability , and the jameson - wolf algorithm for predicting antigenicity .
based on this analysis , the peptides with good hydrophilicity , high accessibility , high flexibility , and strong antigenicity were selected as the antigen epitopes .
enzyme - linked immunoassays specific for each peptide were performed as described by cardona et al . with minor modifications .
the microplates were coated with 100 l ( 10 g / ml ) of each peptide diluted in carbonate buffer , ph 9.6 ( na2co3 : 0.159 g/100 ml ; nahco3 : 0.293 g/100 ml ) .
the plates were incubated for 1 hr at 37c , then for 48 hr at 4c , and 1 hr at 37c .
non - specific ligand sites were blocked with 100 l 2% casein phosphate buffer for 1 hr at 37c .
the plates were washed and incubated with 100 l serum diluted to 1:100 in 5% casein phosphate buffer for 1 hr at 40c .
after washing , 100 l rabbit anti - pig peroxidase - conjugated igg ( sigma ) diluted to 1:4,000 in 6% casein phosphate buffer was added for 20 min at 37c .
after the washes , the horseradish peroxidase activity was detected using tmb for 30 min at 37c and stopped with a 5% h2so4 solution .
a positive cut - off point was determined by estimating the mean average absorbance of 10 negative controls plus 2 sds . to determine the specificity of the anti - peptide antibody , elisa using irrelevant peptides from the bl21 of the orf virus previously synthesized by our laboratory ( sequence : vdvqskdkdadelre )
was also performed as described above . as controls , elisa using excretory / secretory antigen ( esa ) and recombinant gra6 from the rh strain of t. gondii previously expressed by our laboratory
briefly , non - specific ligand sites were blocked with 100 l 5% bsa phosphate buffer .
the rabbit anti - pig peroxidase - conjugated igg diluted 1:8,000 in pbs was used as the secondary antibody .
the secondary structure of gra6 was predicted by the garnier - robson and chou - fasman algorithms based on the sequence of the gra6 gene .
a flexibility plot , hydrophilicity plot , surface probability plot , and antigenic index for gra6 were obtained using the karplus - schulz , kyte - doolittle , emini , and jameson - wolf algorithms , respectively ( fig .
1 ) . based on the results obtained with these methods , potential b cell epitopes on gra6 were predicted , including 1 - 20 aa , 44 - 63 aa , 54 - 73 aa , 64 - 83 aa , 74 - 93 aa , 84 - 103 aa , 172 - 191 aa , 182 - 201 aa , 192 - 211 aa , and 202 - 221 aa .
all of the 10 predicted epitope peptides were evaluated by elisa using pig sera from various time points after infection .
the results of elisa for 3 peptides , p2 , p10 , and p11 , are shown in fig .
no significant differences were found between the mean absorbances of the 3 groups ( g1 , g2 , and g3 ) as determined by anova .
furthermore , no significant differences were found between the mean absorbances of the 3 peptides , p2 , p7 , and p9 .
the other 7 peptides were recognized by selection of sera from various time points after infection ( fig .
the number of positive samples / tested for each peptide was as follows : p1:8/51 , p3:25/51 , p4:18/51 , p5:9/51 , p6:27/51 , p8:20/51 , and p10:31/51 . to determine the specificity of the anti - peptide antibody , elisa using an irrelevant peptide
4a ) . to compare the serological reactivity of the peptides with esa and recombinant gra6 , elisa using esa and recombinant gra6
the adoption of immunoinformatics methods for the prediction of antigenic epitopes has become an indispensable tool for epitope localization .
in addition , such techniques are economical and effective and can substantially reduce experimental costs .
bioinformatics has been widely used in the analysis of protein epitopes [ 10 - 12 ] . in the present study ,
the secondary structure of gra6 was predicted by the garnier - robson and chou - fasman algorithms based on the sequence of the gra6 gene .
a flexibility plot , hydrophilicity plot , surface probability plot , and antigenic index for gra6 were obtained using the karplus - schulz , kyte - doolittle , emini , and jameson - wolf algorithms , respectively .
the existence of flexible regions , such as coil and turn region , provides powerful evidence for epitope identification . in the past ,
several experimental techniques were developed for mapping antibody interacting residues on an antigen , including the identification of interacting residues from the structure of antibody - antigen complexes .
many researchers have applied this technique to study epitopes [ 6,10 - 12,26,29 ] . using this technique and bioinformatics tools
, we found that many regions of gra6 , particularly the regions represented by peptides p2 , p7 , and p9 , are involved in the pig antibody response , and strong reactivity with the t. gondii - infected pig sera was observed .
the reactivity of these epitopes does not seem to be dependent upon the time of infection .
the identification of b cell epitopes is important for understanding antigenic structure and parasite - antibody interactions at the molecular level and may assist in the design of vaccines and diagnostic reagents .
conformational epitope selection relies on the determination of the tertiary structure of an antigen to identify residues that interact with antibodies .
the experimental techniques required to determine the tertiary structure of the antigen , such as crystallography , are expensive and time - consuming , and the mapping of conformational epitopes has been severely hampered .
the majority of methods and databases have focused on the identification of linear epitopes . in the present study
, linear epitopes were analyzed using synthetic peptide techniques and bioinformatics tools , and 3 of the 10 predicted epitope peptides were confirmed by synthetic peptide techniques .
the use of a molecular biology method in combination with a bioinformatics method is a useful method to screen for linear epitopes .
we have precisely located the epitopes of t. gondii gra6 using pig sera collected at different time points after infection .
the identification of specific epitopes targeted by the host antibody response is important both for understanding the natural response to infection and for the development of epitope - based vaccines and diagnostic methods .
there are more linear and conformational b cell epitopes than previously predicted ; therefore , the number of identified epitopes should also increase with further studies .
|
the study of antigenic epitopes from toxoplasma gondii has not only enhanced our understanding of the structure and function of antigens , the reactions between antigens and antibodies , and many other aspects of immunology , but it also plays a significant role in the development of new diagnostic reagents and vaccines . in the present study , t. gondii gra6 epitopes were identified using bioinformatics tools and a synthetic peptide technique .
the potential b cell epitopes of gra6 predicted by bioinformatics tools concentrated upon 3 regions of gra6 , 1 - 20 aa , 44 - 103 aa , and 172 - 221 aa .
ten shorter peptides from the 3 regions were synthesized and assessed by elisa using pig sera from different time points after infection .
three of the 10 peptides ( amino acids 44 - 63 , 172 - 191 , and 192 - 211 ) tested were recognized by all sera and determined to be immunodominant b - cell epitopes of gra6 .
the results indicated that we precisely and accurately located the t. gondii gra6 epitopes using pig sera collected at different time points after infection .
the identified epitopes may be very useful for further studies of epitope - based vaccines and diagnostic reagents .
|
INTRODUCTION
MATERIALS AND METHODS
Serum samples
Amplification, cloning, and sequencing of the GRA6 gene
Prediction of the epitopes
ELISA analysis
RESULTS
DISCUSSION
|
while the recent strategies of lung - protective ventilation and proper fluid restriction therapy have reduced mortality in patients with acute lung injury ( ali ) and acute respiratory distress syndrome ( ards ) , the mortality rate remains high .
although many pharmacological therapies have been attempted to treat ali / ards , no specific therapy has demonstrated a clear effect so far .
accordingly , mesenchymal stem cells ( mscs ) have been introduced as a possible therapy .
mscs are multipotent adult stem cells that have the ability to differentiate into many different cell lineages and the capacity for self - renewal ( 1 ) .
in early research on the use of mscs to treat ali / ards , the main mechanism of action of these cells seemed likely to involve the replacement of injured lung epithelium . however , subsequent studies have suggested that the most important therapeutic effect probably comes from the paracrine properties of mscs ( 2 , 3 ) .
thus far , the therapeutic potentials of mscs have primarily been supported by preclinical evidence and the need for clinical trials has been suggested ( 4 ) . as there is a lack of clinical data , we here introduce a patient who was treated with mscs in the course of ards and subsequent pulmonary fibrosis .
a 59-yr - old man with a history of pulmonary tuberculosis ( tb ) and no significant family history was diagnosed with idiopathic thrombocytopenia in june 2008 .
, he developed a cough , sputum , and rhinorrhea and five days later displayed fever and dyspnea .
his chest radiography and computed tomography ( ct ) scan showed multifocal patchy ground - glass opacities ( ggos ) in both lungs with underlying emphysema with large bullae in the left lower lobe and focal irregular nodular lesions in the right upper lobe that were presumed to be tb sequelae .
after five days , he was transferred to our hospital , a tertiary referral center , and admitted to the medical intensive care unit . at admission , he had tachypnea with a fever of 39. he had progressive bilateral diffuse infiltrations on his chest radiography .
his initial sao2 level was 75% and his pao2/fio2 ( p / f ) ratio was 166 mmhg .
we started a course of methylprednisolone at a dose of 40 mg twice a day to treat a possible pneumocystis jiroveci pneumonia in conjunction with a continuing regimen of empirical antibiotics and an antiviral agent .
however , no specific pathogen was identified in any specimen from this patient , including bronchoalveolar lavage , blood or sputum .
then , his chest radiography was stationary and his p / f ratio began to gradually improve . on hospital day ( hd ) 7 , he underwent tracheostomy to enable early weaning from mv .
however , he then developed hospital - acquired pneumonia and hydropneumothorax due to a ruptured bullae with bronchopulmonary fistula ( bpf ) .
he was conscious during this time but he could not communicate with the medical team or even his family members .
on hd 87 , he had a follow - up high - resolution ct , which showed aggravation of diffuse ggos and interlobular septal thickening in both lungs .
this indicated the progression of pulmonary fibrosis as a post - ards sequelae . after discussing the possibility of administering mscs with his family , we commenced with a trial of this therapy .
the umbilical cord blood ( ucb)-derived mscs ( ucb - mscs ) were prepared as follows .
ucb - mscs were produced at the good manufacturing practice facility of medipost co. , ltd .
quality control and quality assurance for the production of these cells were performed according to the standards of the korea food and drug administration .
flow cytometry analysis of expressed surface antigens showed that these cells were uniformly positive for cd29 , cd44 , cd73 , cd105 , and cd166 and negative for the hematopoietic lineage markers cd34 , cd45 , cd14 , and hla - dr .
the final ucb - mscs preparations used in the infusion were harvested from cell culture passage 6 and suspended at a final density of 7.510/1.5 ml in normal saline . on hd 114 , the patient underwent the intratracheal administration of ucb - mscs at a target dose of 110/kg . before the procedure , the ventilator was set to the pressure - controlled ventilation ( pcv ) mode of 26 cmh2o of inspiratory pressure ( ip ) and 0.45 of fio2 .
there were no peri - procedural complications . the next morning ( 16 hr later )
, he was able to communicate effectively with our medical team , which had not been possible in the previous few months .
after 24 hr , we changed the pcv to a pressure support mode with 18 cmh2o of support pressure with 0.4 of fio2 .
after 48 hr , pcv was applied again with 22 cmh2o with 0.35 of fio2 .
the dynamic compliance of his lung improved from a pre - procedure value of 22.7 to 26.5 , 27.3 , and 27.9 ml / cmh2o after 24 , 48 , and 72 hr , respectively .
his p / f ratio subsequently increased from pre - procedural 191 to 328 mmhg on day 1 after the procedure and to 334 mmhg on day 3 ( fig .
1 ) . a follow - up chest radiography showed a slight decrease in bilateral lung infiltrates ( fig .
2 ) . on day 3 , however , he suddenly experienced six generalized tonic - clonic seizures during the course of the day with a fever near to 39. he had no focal lateralizing signs . a brain ct was performed and showed no hemorrhage .
we discussed the case with neurological specialists and determined that the current use of carbapenem was the most probable cause of his seizure .
the weaning trial was restarted for our patient and the ip was reduced to 12 cmh2o with 0.3 of fio2 during the following week .
however , he could not be weaned because his repeated infection by multidrug - resistant pathogens was not controlled .
eventually , he suffered septic shock due to empyema and died on hd 231 or day 118 after msc administration .
our current case involved a patient who developed ards due to pneumonia . he subsequently had post - ards pulmonary fibrosis . weaning from mv was difficult as he did not improve for a long - period ( four months ) .
although he failed to survive , his immediate improvement after cell infusion is worthy of mention . his mental status ,
his lung compliance , p / f ratio and his chest radiography all showed improvement over the course of at least three days .
we speculate that these clinical , physiological and radiological improvements might be related to the paracrine properties of mscs , with immunomodulation , alveolar fluid clearance and regulation of lung protein permeability known as potential mechanisms ( 2 ) .
the main reasons for our failure to help this patient survive by mscs therapy are likely to be the following .
first , his pulmonary fibrosis was so advanced that it could not respond to the stem cell therapy .
its effectiveness may depend mainly on paracrine activity rather than regeneration by stem cell engraftment .
thus , a long - term effect would not be expected due to his advanced stage of fibrosis .
second , his bpf - related repeat infections may have caused the lack of improvement in his lung compliance .
since the pathogens that were responsible for the infection were multidrug - resistant to current antibiotics , a proper antimicrobial treatment could not be performed in advance of the stem cell therapy . a third possible reason concerns the dose and the number of stem cells administered .
we intratracheally infused 110/kg stem cells on one occasion but no study regarding the proper number of cells for intrapulmonary administration in humans has thus far been reported .
accordingly , our dose was based on that used intravenously in a trial in acute graft - versus - host - disease after allogeneic hematologic stem cell transplantation ( 5 ) .
we decided to infuse using a local route as we expected more intrapulmonary action and less systemic adverse effects than for an intravenous injection .
however , our dose or the one - time - only administration may have been insufficient to produce a significant effect .
this report has a number of notable limitations that hinder the interpretation of the definite effect of mscs in post - ards pulmonary fibrosis .
first , we did not check the level of cytokines or other soluble factors that have been shown to be associated with the functions of mscs in preclinical studies .
in addition , our interpretation in this case study relied on clinical parameters and radiological images only .
thus , it is difficult to precisely link the patient 's short - term clinical improvement to the definite action of the mscs . finally ,
even though the clinical studies of msc therapy for acute lung injury are currently underway , there is no definite evidence so far .
thus , our decision on the msc therapy may have some ethical problems although we had no other way as a salvage therapy for his fibrotic stage of ards .
our current findings suggest the possibility of using msc therapy in an ards patient and it is the first clinical case of ucb - mscs therapy ever reported . for a clear verification of msc therapy in ali ,
|
umbilical cord blood ( ucb)-derived mesenchymal stem cells ( mscs ) have been introduced as a possible therapy in acute lung injury and acute respiratory distress syndrome ( ards ) .
this case history is reported of a 59-yr - old man who was treated with mscs in the course of ards and subsequent pulmonary fibrosis .
he received a long period of mechanical ventilation and weaning proved difficult . on hospital day 114 , he underwent the intratracheal administration of ucb - derived mscs at a dose of 1 106/kg .
after cell infusion , an immediate improvement was shown in his mental status , his lung compliance ( from 22.7 ml / cmh2o to 27.9 ml / cmh2o ) , pao2/fio2 ratio ( from 191 mmhg to 334 mmhg ) and his chest radiography over the course of three days .
even though he finally died of repeated pulmonary infection , our current findings suggest the possibility of using mscs therapy in an ards patient .
it is the first clinical case of ucb - derived mscs therapy ever reported .
|
INTRODUCTION
CASE DESCRIPTION
DISCUSSION
|
in 1982 chang and der , two postdoctoral fellows working in geoffrey cooper 's laboratory , discovered kristen rat sarcoma virus and murine sarcoma virus ; retroviral oncogenes related to rodent sarcoma virus genes . the human kras gene is a homolog of these two oncogenes
. a normal form of human c - ras has been called kras or kras2 ( kristen rat sarcoma viral oncogene homolog or alternatively kristen murine sarcoma virus2 homolog ) . in 1983
, der described an abnormal form of the p21 protein expressed by colon and lung carcinoma cell lines and showed that the gene encoding this protein is able to transform nih3t3 cells .
this finding was later confirmed by parada and weinberg , who described the transformation of nih3t3 cells by an activated kras oncogene .
aberrant p21 proteins were encoded by the altered kras gene and their expression in carcinoma tissue was causally linked to an abnormal state of activation . since then , it has been accepted that kras is one of front - line sensors that initiate the activation of an array of signalling molecules allowing the transmission of transducting signals from the cell surface to the nucleus , thus affecting cell differentiation , growth , chemotaxis , and apoptosis . a signal transduction cascade initiated by the activated form of kras
kras elicits changes in the cytoskeleton and consequently affects cell shape , adhesion and migration [ 4 , 5 ] . in the following paragraphs , kras protein , gene , oncogenesis , and cancer therapy is reviewed
kras belongs to a group of small gtp - binding proteins , known as the ras superfamily or ras - like gtpases .
the entire ras superfamily is characterised by the presence of a catalytic g domain , but includes members with distinct evolutionary specializations with respect to different cellular process .
the ras subfamily ( ras , rho , rab , arf , rac , and ran ) includes the most frequently studied proteins , such as harvey - ras ( h - ras ) , neuroblastoma - ras ( n - ras ) , and two variants of kristen - ras ( k - ras)one , known as kras4a , which is weakly expressed in human cells and the dominant form , known as kras4b , which is much more highly expressed .
the kras gene product , kras protein , contains 188 amino acid residues with a molecular mass of 21.6 kd and participates in intracellular signal transduction .
as mentioned above , the kras protein remains inactive until it binds to gtp , as depicted in figure 2 .
once the gtp is bound to the kras protein , kras undergoes conformational changes that involve two regions of the protein , thus activating it .
these two important regions are known as switch 1 ( aminoacids 3038 ) and switch 2 ( aminoacids 5967 ) , which form an effector loop , controlling the specificity of the binding of this gtpase to its effector molecules .
this conformational change in the kras protein affects its interactions with multiple downstream transducers gtpase - activating proteins ( gaps)which amplify the gtpase activity of the ras protein 100,000-fold .
the change also affects interactions with guanine - exchanging / releasing factors ( gefs / grfs ) promoting the release of gtp .
the kras protein also has intrinsic gtpase activity , stimulated by gaps , which acts as a timer associated with direct interactions with the effectors .
mutations found in an oncogenic form of the ras p21 protein impair gtpase activity and make the kras protein unresponsive to gap proteins . mutated forms of p21 rapidly exchange gdp for gtp , which it prefers as a substrate , thus inducing the active state .
such aberrant forms of kras protein deregulate many effectors , thus affecting several important cellular pathways .
many gtp derivatives targeting ras or raf effectors have been developed to repair the defective gtpase activity that influences the aberrant ras signalling .
however , little is known about the specificity and transport of compounds modified by gtps through the plasma membrane .
the first domain includes 85 amino acids at the n - terminus and is identical in the three forms of ras ( kras , nras , and hras ) .
the second domain contains 80 amino acids , with lower sequence identity ( 7080% ) among the three forms of ras protein .
these regions are important for the signalling function of the kras protein and jointly form the g - domain ( amino acids 1165 , figure 3 ) .
the g - domain of the kras protein includes the gtp - binding pocket , where p - loop - phosphate binding loops ( aminoacids 1016 and 5659 ) interact with the b - phosphate and c - phosphate of gtp .
the region between amino acids 32 and 40 ( the core effector region ) is essential for the interactions between the putative downstream effectors and gaps .
ras protein also contains a hypervariable region ( hvr ) at the c - terminus ( amino acids 165188/189 ; the third domain ) , which guides posttranslational modification and determines plasma membrane anchoring .
this region plays an important role in the regulation of the biological activity of ras protein .
switch regions i and ii play important roles in the binding of regulators and effectors .
the phosphate binding pocket - p loop permits temporary binding of gtp to the ras protein .
this is also the region of gtpase activity , which negatively regulates the ras protein via a ras - gtp hydrolysis reaction and binding of guanosine diphosphate .
the kras protein acts like a plasma membrane - localized molecular switch , regulating multiple signal transduction pathways .
it is synthesized in the cytosol , where it is farnesylated by farnesyl transferase at the cysteine residue of the carboxy - terminal motif caax ( where c represents cysteine , a is an aliphatic amino acid , and x is any amino acid ) .
the aax amino acid motif is cleaved by proteases , whereas the c - terminal carboxyl residue of the kras protein is methylated .
cleavage of the axx peptide motif and methylation occur at the cytosolic surface of the endoplasmatic reticulum and are mediated by the ras - converting enzyme rce1 .
c - terminal farnesylation plays an important role in membrane localization . in the splice variant kras4a , the axx motif undergoes additional palmitoylation by palmitoyl transferase , resulting in proper targeting of kras4a to the membrane . however , there is no detectable palmitoylation of the predominant splice variant kras4b , which probably reaches the plasma membrane via a microtubule - dependent mechanism , thus avoiding the golgi apparatus [ 13 , 15 ] .
posttranslational farnesylation and carboxymethylation are believed to be important for the oncogenicity of the ras protein .
treatment with farnesyl transferase inhibitors has been shown to inhibit anchorage - independent growth of both kras - transformed mouse fibroblasts and human tumour cells containing kras and nras mutations .
activation of downstream signalling pathways by kras can also be triggered by signals from subcellular compartments , such as the endoplasmatic reticulum and the golgi apparatus [ 16 , 17 ] .
while wild - type kras usually promotes cell cycle progression , it can also induce growth arrest , apoptosis , and replicative senescence when increased to abnormal levels .
this can be triggered by cellular stress , ultraviolet or ionizing irradiation , heat shock , and some cytokines .
in these circumstances , triggering of growth arrest can represent a defence mechanism against inappropriate activation of ras .
it has been demonstrated that the wild - type kras gene is a tumour suppressor that is frequently lost during tumour progression in many types of cancer .
once the kras gene mutates , it acquires oncogenic properties ( table 1 ) and seems to be causally involved in the development of various human cancers [ 19 , 20 ] .
loss of the wild - type kras allele has been observed in both human and mouse tumours , indicating that absence of the normal allele may facilitate transformation by one copy of the oncogenic kras allele .
oncogenic mutations in the kras gene prevent the hydrolysis of gtp , thus permanently activating the ras molecules .
expression of a mutated kras gene in fibroblasts has been shown to augment metalloproteinase 2 ( mmp2 ) expression in the matrix and enhance the invasion of cancer cells .
overexpression of this mutated form of kras also inhibits glycosylation of the integrin 1-chain , resulting in altered polarisation and increased adhesiveness of colon cancer cells .
in addition , expression of this oncogenic form of kras protein has been shown to be associated with upregulated carcinoembryonic antigen ( cea ) expression and disturbance of epithelial cell polarization .
there are two copies of the kras gene in the human genome , designated kras1 and kras2 .
the mrna encoded by the main kras2 is 5.5 kb long , and differs from transcripts of the transforming kristen murine viral gene by only six codons .
analysis of human placental and embryonic cdna libraries has revealed that 900 bp of the kras1 gene is homologous to the corresponding sequence of the kristen murine sarcoma virus2 homolog , with one intervening sequence , and 300 bp of the kras2 is fully homologous to the viral counterpart . the kras1 gene is a pseudogene derived from kras2 by alternative mrna splicing .
mcbride and colleagues found that the protooncogenes kras1 and kras2 are localized at human chromosomes 6 and 12 , respectively .
later , kras1 and kras2 were mapped by in situ hybridization to chromosome positions 6p11 - 12 and 12p11.1 - 12.1 , respectively .
alternative splicing of exon 4 produces two mrna forms , known as 4a and 4b .
exon 5 can be skipped during alternative splicing , giving rise to isoforms krasa and krasb .
the 6th exon encodes the c - terminal region in krasb and is not translated ( the 3untranslated region , 3utr ) in krasa .
krasb is the predominant splice variant of kras2 , and is referred to , briefly , as kras .
there are indications that allelic losses of chromosome region 12p commonly occur in human cancers , and a frequently deleted region is near the kras gene at position 12p12 - 13 .
further , recent studies on lung adenocarcinoma suggest there is an association between the incidence of allelic losses in the 12p12 - 13 region and kras gene mutation
. diagnostics of kras gene mutations in clinical setting is limited by two factors : first , in the time of testing , kras mutated tumour cells may be in minority , outbalanced by wild type tumour cells and wild type non - tumour cells present in the sample .
second , analytically preferable snap - frozen tumour samples are rarely available for kras mutation testing . instead , formalin fixed paraffin - embedded ( ffpe ) tissue is used . there , integrity of dna may be severely compromised by procedure of formalin fixation ( especially by its long duration and low ph ) .
all the known principles of dna polymorphism detection are applicable to kras mutation detection and demand a dedicated review outside of the scope of this paper .
more than 60 methods described can be divided into sequencing methods [ 3037 ] , methods based on specific interaction with oligonucleotide , methods based on specific interaction with enzyme [ 3840 ] , and conformational methods [ 4147 ] . while many specificity and/or sensitivity enhancement of methods were described as well [ 4853 ] , analytical validation , systematic comparison , and assessment of methods side by side is lacking . to authors
best knowledge , only communaut europene ( ce ) marked kras mutation detection kits are supplied by dxs ( mutations in codons 12 a 13 are tested using principle of arms - pcr and scorpion primers , vienna - lab ( reverse dot blot assay format ) , tib molbiol ( kras lightmix clamped hybridization probes for codon 12 ) , and invigene ( qpcr with sequence suppressor agent stopprimer for the unwanted excess component , applicable for first two nucleotide positions in codons 12 and 13 ) .
kras expression is regulated both during the initiation of transcription by the binding of proteins to its promoter and during transcriptional elongation by micrornas affecting kras mrna stability .
both human and murine kras gene promoters contain a nuclease hypersensitive polypurine - polypyrimidine element ( nhppe ) .
the g - rich strand of nhppe located in the proximal promoter sequence is able to form an intramolecular parallel g - quadruplex , consisting of three g - tetrads and three loops , which recognizes and binds nuclear proteins that are involved in transcriptional repression of kras expression .
accordingly , it has been reported that sequestration of nuclear proteins that bind to nhpp by an oligonucleotide mimicking the kras g - quadruplex resulted in 40% inhibition of kras transcription , compared to controls .
the transcription of kras is regulated , in part , by an interaction between the promoter region and the 65 kda esxr1 protein and , in part , by micrornas ( mirnas ) .
esxr1 is a human protein with an n - terminal homeodomain in the nucleus and a c - terminal proline - rich repeat region i in the cytoplasm .
the n - terminal fragment of esxr1 binds to the taatgttatta consensus sequence in exon-1 of the kras gene , thus inhibiting its mrna expression .
mirnas contain a 21 - 22 nucleotide long noncoding sequence that is able to regulate gene expression . in 2005
it was estimated that there are more than 500 mirnas , which collectively regulate approximately 30% of all human genes , including the ras gene family .
regulation of gene expression by mirnas probably occurs as a result of imperfect hybridization of the mirna to the complementary sequences located in the 3untranslated region ( 3utr ) of target messenger rna ( mrna ) species .
this interaction between mirna and mrna both decreases mrna stability and represses protein synthesis by preventing access to ribosomes .
interestingly , many altered mirnas have been identified in human cancers [ 6163 ] , including some of the most thoroughly analyzed mirnas
members of one group , the oncomirs , are upregulated in cancer and can act like oncogenes .
the second group , the anti - oncomirs , probably act as tumour suppressors by targeting oncogenes , repressing the cell cycle and division of cancer cells .
for example , mirna - let7 is an oncogene - antioncomir pair that negatively regulates ras protein levels and decreases cell proliferation rates [ 68 , 69 ] .
kras , nras , and hras harbour multiple let-7 mirna complementary sites ( lcss ) in their 3utrs .
zhang et al . found that reducing the activity of let-7 in hela cells resulted in a 70% increase in ras protein levels , while takamizawa et al .
found that let-7 expression was 80% lower in 60% of lung cancer adenocarcinoma and squamous cell carcinoma lines than in normal lung tissue .
moreover , a correlation between low levels of let-7 mirna and significantly higher ras protein expression has been found in lung squamous cell carcinomas .
these results suggest that let-7 is able to downregulate the expression of ras in human carcinomas .
these molecular findings provide a strong rationale for developing novel therapeutic treatments aimed at decreasing kras protein expression in cancer cells . in many cases kras protein expression
is dramatically increased due to mutations in the kras gene sequence , thus making cells refractory to current therapies , such as those involving use of epidermal growth factor receptor inhibitors .
such oncogenic forms of the kras gene are prevalent in pancreatic carcinomas ( > 80% ) , colon carcinomas ( 4050% ) , and lung carcinomas ( 3050% ) , but are also present in biliary tract malignancies , endometrial cancer , cervical cancer , bladder cancer , liver cancer , myeloid leukemia [ 73 , 74 ] and breast cancer .
mutations in the kras gene have important effects on the process of carcinogenesis , which depend on the cells and tissues involved .
the mutations found most frequently in the kras gene of cancer cells are located at positions 12 and 13 in exon 1 , and less frequently in codons 61 , 63 , 117 , 119 , and 146 [ 77 , 78 ] .
allelic mutations result in amino acid changes , namely gly to asp , ala , arg , ser , val , or cys in codon 12 and gly to asp in codon 13 .
somatic missense mutations at positions 12 , 13 , 61 , and 63 enable perturbation of the intrinsic gtpase activity of the kras protein , resulting in reductions in gtp hydrolysis capacity .
mutations in codons 12 ( figure 4 ) or 13 are known to lead to conformational changes in the kras protein .
mutation in codon 12 of the kras gene causes the encoded kras protein to freeze in its active state for a much longer duration than its nonmutated counterpart .
mutations resulting in the substitution of amino acids 116 , 117 , 119 , and 146 reduce the nucleotide affinity of the kras protein , thereby affecting the rate of gdp / gtp exchange .
the oncogenic forms of the ras protein have a profound effect on the downstream effector pathways , resulting in much higher proliferation rates of cancer cells expressing such forms .
the transforming ability of the kras oncogene may result from overexpression of the mutant kras allele or from deletion of the wild - type allele .
overexpression of kras can also be induced by the loss of p16ink4 ( cdkn2a ) , p19ink4 ( cdkn2d ) , or p53 .
( 2001 ) have shown that the wild - type kras allele can suppress the oncogenic function of the mutated allele .
in addition , the radiosensitivity of tumour cells is altered by oncogenic ras expression , probably as a result of the effect of the kras mutation on several intercommunicating pathways .
the prevalence of mutations in the kras gene at the time of diagnosis is highest in pancreatic cancers ( > 80% of cases ) , notably pancreatic adenocarcinomas predominantly harbour kras forms with a guanine to thymine transversion in codon 12 .
wei and colleagues examined samples collected from 30 patients with pancreatic cancer and found that 24 of them showed mutations at codon 12 and only one at codon 13 . however , concurrent kras mutations frequently occur in patients with pancreatic cancer . a positive association has been found in patients with pancreatic cancer between tobacco exposure and mutations in the kras gene .
similar associations have also been reported for coffee drinking , and milk , butter , and alcohol consumption [ 88 , 89 ] .
however , no direct evidence of a causal relationship between these dietary components and mutations in the kras gene has been presented .
the second highest incidence ( about 50% of cases ) of mutations in the kras gene is found in colon cancers [ 90 , 91 ] .
the first stage is characterized by the development of a small , benign tubular type of adenoma or polyp with sporadically detectable kras mutation(s ) .
the second stage is more aggressive and is usually associated with patches of definitive carcinoma cells , which may grow into invasive cancers characterising the third stage .
mutations of the kras gene have been identified in tissues from both adenoma and carcinoma cases , but at much lower frequencies in colon adenoma tissues than in carcinoma tissues [ 93 , 94 ] .
the incidence of mutation in the kras gene has been found to be low and to occur mainly in the small adenomas of patients with familial adenomatous polyposis , who have a predisposition to colon cancer in the kras gene associated with colon cancer appear most often in codons 12 ( 28% ) and 13 ( 8% ) of exon 1 and less frequently in codon 61 . in colorectal cancer ,
the main substitution ( gly to asp ) has been found to occur in codon 12 .
mutation from ggt ( gly ) to gtt ( val ) in codon 12 has been observed more frequently in primary metastatic carcinoma , suggesting that this mutation may confer a more aggressive phenotype in colorectal carcinoma .
a mutation in codon 13 , resulting in the substitution of gly with asp , observed in colon cancer has been shown to be associated with reduced survival rates .
this kind of kras gene mutation has also been shown to occur more frequently in unstable , than in stable , colon tumours [ 97 , 98 ] .
losses of kras wild type alleles in both mouse and human lung adenocarcinomas and squamous carcinomas have been found in many studies , notably in 67% to 100% of chemically induced murine lung adenocarcinoma cases harbouring a mutant kras . in humans ,
kras mutations appear in 1030% of lung carcinoma cases , demonstrating strong associations with a history of smoking and poor prognosis [ 100 , 101 ] . among both current and former smokers ,
further , although some researchers have found sporadic kras mutations in non - smokers with early onset of cancer , smoking history is an important factor and is correlated with increased occurrence of mutations in the kras gene in lung cancer cases .
mutations in the kras gene in codons 12 and 13 were detected in 21% of nsclc ( non - small cell lung cancer ) tumour samples examined in the tribute iii trial .
nsclc patients have a tendency to accumulate activating mutations in either the egfr or kras genes .
however , a clinical study has shown that mutations of these two genes are , in general , mutually exclusive .
although higher kras mutational frequency is primarily found in cancers of the pancreas , colon and lung , possible links between kras hyperactivity and human breast cancer have been explored recently .
hollestelle at al . found mutations in 12.5% of cases but the sanger cosmic database version 28 ( http://www.sanger.ac.uk/genetics/cgp/cosmic/ ) records only a 5% incidence .
the lower frequency of kras mutations in breast cancer cell lines suggests that the gene mutation may be less important in breast cancer carcinogenesis than in other forms of cancer , although mutations at a hotspot in the kras gene have been found in a small subset of breast cancers .
many clinical trials have shown that a poorer response to chemotherapy , a shorter time - to - progression , and worse overall survival are consistently associated with specific mutations in oncogenes .
kras is one of the most frequently mutated oncogenes in many cancers , and it is also one of the most important predictors of resistance to targeted therapy using egfr1 tyrosine kinase inhibitors ( egfr - tkis ) .
two of the most important egfr - specific tkis are gefitinib ( iressa , zd1839 ) and erlotinib ( tarceva ) .
the first indications of the predictive strength of the association between the kras gene and therapeutic responses to the egfr - tki gefitinib were originally observed in nsclc patients with tumours bearing the wild - type form of the kras gene and constitutively activated egfr1 gene , due to activating mutations in exons 18 to 21 or high copy number / amplification of the egfr1 gene .
clinically , better responses to tyrosine kinase inhibitor treatment were observed in patients with adenocarcinomas and well - differentiated tumours , female patients , non - smokers , and people of asiatic origin [ 107109 ] .
clinical research data show that gefitinib monotherapy is well tolerated and active against a wide range of tumour types , including colon , head , neck , breast , prostate , and lung cancers , especially nsclcs .
egfr - tkis are usually used as the second line therapy in patients after failure of chemotherapy .
however , gefitinib did not pass the registration procedure in the european union because insufficient clinical benefit was demonstrated , probably because european clinical trials did not include sufficient good responders
clinical data also suggest that the drug represents a new therapeutic option for nsclc patients with brain metastases .
after the successful tribute and talent clinical trials , erlotinib ( tarceva ) was also approved by the us fda in 2002 as a second or third line treatment for nsclc after failure of standard chemotherapy . however , molecular analysis revealed that patients who have activating mutations in the kras gene ( exon 1 : codons 12 , 13 , or 61 ) with or without increases in egfr copy numbers did not derive benefit from this therapy and had about a 96% chance of disease progression .
similarly , eberhard et al . first observed the relationship between kras mutations and the outcome of erlotinib therapy in a randomized phase iii clinical trial in which the drug was used , in combination with first line gold standard chemotherapy ( carboplatin and paclitaxel ) , to treat advanced nsclc patients .
patients with the kras mutation exhibited a shorter time to progression ( three months ) and a shorter overall survival ( four months ) when treated with a combination of erlotinib and first line chemotherapy , such as treatment with cisplatin , compared to the group with wild - type kras , for whom the time - to - progression was 12 months .
most nsclc patients in the erlotinib treatment study had expressed wild - type kras , and their kras status had greater prognostic than predictive value as a biomarker .
however , in colorectal cancer , mutations in the kras gene are important predictive ( as well as prognostic ) biomarkers , since the effectiveness of treatment with cetuximab and panitumumab is impaired in tumours with the activating mutation .
information regarding the status of the kras gene allows the selection of appropriate therapies for patients who do not display activating mutations and the selection of alternative therapies for patients with mutations . although results pertaining to the role of kras in the prognosis of clinical outcome or prediction of therapeutic responses to egfr1 tyrosine kinase inhibitors are interesting , they need to be validated in larger and prospective trials , using standardized and sensitive mutation detection techniques .
if the associations are confirmed , knowledge of the mutation status of kras in nsclc tumours could help physicians decide which patients should receive gefitinib and/or erlotinib .
interestingly , kras gene mutations also seem to provide strong predictive indication of therapeutic responses to other classes of tyrosine kinase inhibitors , as recently demonstrated for the imatinib mesylate ( glivec ) .
imatinib is the standard drug for patients with chronic myeloid leukaemia ( cml ) and patients with gastrointestinal stromal tumours ( gists ) , expressing the bcr - abl fusion protein and tyrosine kinase receptor c - kit , respectively .
further , drug resistance to imatinib is usually attributed to mutation of the imatinib - binding sites of these proteins , although amplification of the bcr - abl fusion gene or overexpression of multidrug resistance proteins may be involved in some cases . however , a recent study by agarwal et al .
colorectal cancer is another frequent neoplasia that is associated with activation of the egfr1 pathway , so it is not surprising that novel and successful therapeutic strategies for this condition involve egfr1 protein kinase inhibition .
in contrast to nsclc , two monoclonal antibodies against egfr1 , rather than small molecular inhibitors of egfr1 , are generally used for treating colorectal cancer : cetuximab ( erbitux ) and panitumumab ( vectibix ) . in accordance with the effect of small molecular egfr1 inhibitors in nsclc , kras alterations play a critical role in the response of colorectal cancer patients to such therapeutic monoclonal antibodies .
indeed , kras mutation status is the most important predictor of resistance to cetuximab or panitumumab ; both the median progression - free survival of cetuximab - treated patients and overall survival was recently found to be superior in a kras wild - type group than in a kras mutant group ( 31 versus 10 weeks , and 16 versus 7 months , respectively ) . on september 27 , 2006 , the us fda approved the completely humanized monoclonal anti - egfr1 igg2 antibody panitumumab ( vectibix ) for clinical use in the third line treatment of patients with metastatic colorectal carcinoma who had progressed after standard chemotherapy .
kras panitumumab therapy was tested in a randomized study involving 463 patients , and the results showed that the wild - type kras gene is essential for its therapeutic activity .
progression - free survival of patients with wild - type versus mutant kras gene tumours was 12 versus seven weeks , while response rates obtained in another study were 17% versus 0% .
these findings strongly indicate that kras gene status should be routinely tested as a critically important diagnostic biomarker to determine which patients will derive therapeutic benefit from egfr1 inhibition .
indeed , analysis of the kras gene status in colorectal cancer cases has become conditio sinequa non for deciding whether or not to apply cetuximab or panitumumab therapy in routine clinical practice and fda has updated vectibix and erbitux labels in 2009 to include this information .
surprisingly , the effects of kras gene mutations on tumour sensitivity to cytotoxic chemotherapies and radiation have only been explored in a few studies . however , expression of a 12 val mutated form of kras has been shown to increase the resistance of cancer cells to radiation therapy .
similarly , the presence of oncogenic kras has been found to significantly increase the sensitivity of cells to a novel class of anticancer agents , cucurbitacins , in a p53- or p21-dependent manner .
in contrast , an ovarian cancer cell line tov-21 g bearing a mutant allele of kras is reportedly significantly more sensitive to cisplatin and radiation , but not to paclitaxel or campthotecin , than the corresponding kras wild type line .
however , results of clinical studies by rosell and colleagues ( 1995 ) showed that patients with a mutation in the kras gene had poorer clinical responses to paclitaxel monotherapy than wild type controls , suggesting that kras gene status is a predictive marker of paclitaxel resistance . in a phase iii retrospective study on nsclc patients ( tribute ) ,
randomly treated with carboplatin and paclitaxel with erlotinib or placebo , patients with kras mutant tumours showed poorer clinical outcomes when treated with erlotinib plus chemotherapy compared to chemotherapy alone . an updated clinical trial ( crystal ) involving 540 metastatic colorectal cancer patients demonstrated that cetuximab in combination with folfiri ( folic acid , fluorouracil , and irinotecan ) in first line therapy is highly effective against kras wild type , but not mutant , tumours .
however , further analysis of the data showed that neither the response nor the progression - free survival of patients treated with chemotherapy alone were significantly affected by kras gene status , although the overall survival of patients with kras mutant tumours was significantly shorter than that of patients with kras wild type tumours .
recently we have also shown that egfr may represent a predictive molecular marker for poor response to preoperative chemoradiotherapy in locally advanced gastric carcinoma .
responses to chemoradiotherapy were found in 60% of egfr - negative patients , but only 13% of egfr - positive patients ( p = .044 ) , and pathologic complete responses were observed in 29% of patients with egfr - negative staining , but none ( of eight ) egfr - positive patients ( p = .16 ) .
the above findings regarding the role of the kras gene in tumour responses to cytotoxic therapies appear to conflict somewhat .
the predictive and prognostic significance of oncogenic kras seems to have been mixed in many studies , and the contributions of variations in the gene to clinical outcome appear to differ according to tumour types and therapeutic interventions . clearly , further studies are urgently needed to confirm and clarify the findings in large prospective biomarker - oriented clinical trials .
other clinical trials have also demonstrated that activating mutations in the kras gene can contribute to tumour progression by affecting the expression of vascular endothelial growth factor ( vegf ) , which plays a critical role in tumour angiogenesis .
inhibition of kras expression by selected kras antisense oligonucleotides has been shown to be associated with significantly reduced secretion of vegf - a165 into the medium of colorectal cancer cell cultures .
in addition , in a cohort of patients with pancreatic tumours , 25/33 ( 76% ) with kras mutations showed higher vegf expression , and their median survival was shorter , than those with tumours expressing the wild - type allele .
similar findings have also been reported from a study of nsclcs , in which higher vegf expression was observed in 50% of tumours bearing a kras gene mutation .
although these studies suggest that kras gene status could play an important role in responses to anti - vegf targeted antiangiogenic therapy , a recent study by hurwitz and saini showed that groups of patients bearing either kras mutant or wild - type tumours derive therapeutic benefit from first line application of the anti - vegf monoclonal antibody bevacizumab ( avastin ) .
furthermore , although both groups of patients ( i.e. , those with wild - type kras and mutated kras genes ) benefited from adding bevacizumab to chemotherapy , both progression - free survival and survival was better for wild - type kras patients , both with chemotherapy alone and with chemotherapy plus bevacizumab .
bevacizumab did not increase the percentage of patients with mutated kras who responded to treatment .
an optimal therapeutic drug should be able to specifically target the mutated kras gene or its product , have minimal systemic toxicity and be orally active .
unfortunately , drugs like this remain to be developed and less efficient strategies need to be used in clinical trials .
however , in addition to the cancer therapies mentioned above , several therapeutic agents and strategies can directly suppress the activating mutant form of the kras gene , and thus improve the efficiency of chemotherapy and biological therapy .
one possible approach for inhibiting kras expression is to use antisense oligonucleotides or viral constructs delivering antisense sequences in order to inactivate the mutant oncogene rna message .
in addition , synthesis of mutated kras protein has been repressed by applying a small interfering adenovirus - mediated rna ( sirna ) , and the specifically designed sirna was shown to have prolonged anti - proliferative effects against various tumour cancer cell lines expressing mutated kras proteins .
another , similar strategy to target mutant kras mrna is based on designing an mrna ribozyme that specifically interacts with a mutated form of the kras mrna and encodes catalytic rna molecules that bind to the mrna substrate by base - pair complementation , leading to translation arrest and/or degradation of the specific mrna .
the kras - specific ribozyme strategy has also been shown to suppress successfully the proliferation of kras - mutated tumour cells .
recently an interesting novel strategy employing farnesyltransferase inhibitors ( ftis ) was shown to inhibit the biochemical transactivation initiated by the mutated kras gene .
farnesyl transferase is an enzyme that primarily regulates zinc metabolism by the addition of a farnesyl group to the cysteine residue of a protein .
at least 30 proteins ( including kras ) require posttranslational farnesylation to reach their membrane positions and function properly in cell signalling .
farnesyl inhibitors represent a novel class of biologically active anticancer drugs that inhibit cell growth .
after the discovery that ras proteins have to be farnesylated to become functionally active , several farnesyl inhibitors were developed .
however , phase ii and phase iii clinical trials conducted to date have found that kras ftis might not be sufficient to inhibit the mutated - overactive forms of kras protein .
the reason for this is probably incomplete inhibition of farnesylation , because garnesylation of kras protein by geranyltransferase i leads to suppression of the effects of farnesyltransferase inhibitors [ 131 , 132 ] .
it should be noted that although kras inhibitory strategies have shown promise in preclinical trials and have been partially successful in clinical trials , there are insufficient data on their efficacy in combination with anti - egfr1 strategies to recommend their routine use as yet .
the evidence from various studies summarized in this review demonstrates that the kras protein is an important signal transducer involved in the regulation of various cellular responses during cell proliferation , differentiation , and survival .
a pivotal function of kras protein in the regulation of the mapk and pi3k / akt pathways is its effect on the proliferation rate of both normal and cancer cells . activating mutations of the kras protein , which frequently occur in cancer cells ,
overall , this review summarizes novel approaches allowing the management of cancers with or without kras mutations , and highlights the importance of early identification of somatic mutations in the kras gene in cancer biopsies .
|
the kras gene ( ki - ras2 kirsten rat sarcoma viral oncogene homolog ) is an oncogene that encodes a small gtpase transductor protein called kras .
kras is involved in the regulation of cell division as a result of its ability to relay external signals to the cell nucleus . activating mutations in the kras gene
impair the ability of the kras protein to switch between active and inactive states , leading to cell transformation and increased resistance to chemotherapy and biological therapies targeting epidermal growth factor receptors .
this review highlights some of the features of the kras gene and the kras protein and summarizes current knowledge of the mechanism of kras gene regulation .
it also underlines the importance of activating mutations in the kras gene in relation to carcinogenesis and their importance as diagnostic biomarkers , providing clues regarding human cancer patients ' prognosis and indicating potential therapeutic approaches .
|
1. Introduction
2. KRAS Protein
3. The
4. KRAS in Oncogenesis
5. KRAS and Cancer Therapy
6. Conclusions
|
hepatitis c virus ( hcv ) is a major public health problem all over the world ; it is currently estimated that about 85% of those infected with hepatitis c virus will become a chronic carrier and may develop severe end stage liver diseases including cirrhosis and hepatocellular carcinoma . currently , the most important risk factor for infecting by hepatitis c is intravenous drug use ( idu ) which is the most associated with the sharing of drug injection equipments especially needles , syringes and other paraphernalia .
infection with hcv among prison inmates is usually higher than that among the general population mainly because of past history of intravenous drug use and possibly high risk addiction - related behaviors in prison . during the last few years
, much attention has been given to the prevalence of blood born diseases including hepatitis c among prisoners .
according to limited research studies in iran , prevalence of hcv infection among prisoners with a history of drug injecting varies between 31.5% to 47% in different parts of the country.[68 ] however , a study in a local prison in fars province revealed 78% prevalence rates of hcv infection among incarcerated drug users . to address the often hidden phenomenon of hcv infection in prisons ,
considering the limited data on the epidemiology of hcv infection and related risk behaviors in our region , in this study we have focused on this infection and transmission risk factors among prisoners with history of idu in isfahan that could potentially be incorporated into current and future harm reduction initiatives us in iran .
in a cross - sectional study , according to self - report and confirm by head of prison 's healthcare , the prison inmates who had intravenous drug history or current idus entered into study at march 2009 .
since the most of our enrollees had sclerosis in peripheral vessels , blood sampling was mostly taken from the femoral vein by the health personnel of the prison setting .
blood samples were then sent to the laboratory of infectious diseases research center during 3 hours in cold box to be tested by eilsa ( diapro - italy ) for the presence of hcv antibody . incarcerated with idu histories
were also asked about their demographic characteristics and hcv - related risk behaviors with an interviewer assisted questionnaire .
face and content validity of the questionnaire were evaluated by specialists and its reliability was confirmed by chronbach alpha = 0.74 .
the research protocol was approved by the ethical committee of isfahan university of medical sciences in iran .
all available means were used to guarantee privacy during interviews and confidentiality . instead of a block for the patient 's name , each questionnaire and test tube had an anonymous identification code , which was used for reporting laboratory results , too .
statistical analysis was performed using spss for windows ( version 16.0 , 2007 , spss inc , chicago , il , usa ) .
univariate analysis was used to assess association between being hcv positive and related risk factors .
the variables that were significant in the univariate analysis ( p - value < 0.05 ) were included in the multiple logistic regression to estimate adjusted odds ratio ( aor ) and 95% confidence intervals ( ci ) .
a total of 943 incarcerated idus ( 938 male and 5 female ) participated in the study . the median age of participants was 32.6 years ( range : 18 - 67 ) .
the majority of participants ( 98.6% ) were iranian and 523 ( 55.5% ) had ever been married . among
the married persons 451 ( 92% ) had been married once , 35 ( 7% ) had been married twice and 5 ( 1% ) had been married more . 143 ( 15.1% ) cases mentioned a history of traveling to another countries .
the socio - demographic characteristics of participants overall , the prevalence of hcv - ab seropositivity was 41.6% [ table 1 ] .
the median frequency of iv injections was 30 times per month ( range 1500 ) , the median duration of addiction was 12 years ( range 0.5 - 57 ) , the median frequency of incarceration was 3 times ( range 1 - 37 ) , and the median of total duration of incarceration was 3.66 years ( range 0.08 - 35 ) .
frequency of hcv transmission related factors within study population 71.76% of the participants had illegal sex ( contact with other than spouse ) . among whom , 43% of men had history of having sex with another man ( msm ) , 64.2% had an intercourse with commercial sex workers and 30.9% of them had idu sexual partner . according to self reports , only 31% of them had ever used a condom during sex . within all
studied samples with idu history , 13.3% had a single sexual partner and the others had 2 or more , in their lifetime .
table 3 shows the odds ratio for some risk characteristics of idus using the logistic regression model .
the results of this study indicated that overall prevalence of hcv ab seropositivity among idu inmates in isfahan province is 41.6% . in similar studies in iran , similar results were observed . in zakizad
study , the seroprevalence of hepatitis c infection in sari addict prisoners has been reported as 30.8% . in another study by khani et al .
the prevalence of hcv was 47.7% among drug addict prisoners in zanjan . in a study which was done in 3 provinces in iran ( isfahan , chahamahal bakhtiary and lorestan ) in 2003 , 34.7% of male prisoners who had been imprisoned for various drug - related offenses including purchase or sale and consumption , were hcv ab positive . in mashhad ,
the seroprevalence of hcv in incarcerated idus had been reported as 59.4% . in mohammad alizadeh
study , the hcv antibody positivity among drug abusers in the central prison of hamedan was 30% . in a study on drug abusers admitted to prison in guilan province , of 460 inmates , 45.4% were hcv antibody positive which in intravenous drug abusers the prevalence reached to 88.9% .
the prominent aspect of our study in comparison with other similar studies in iran , is the high number of participants whom all of them had history of intravenous addiction . as a matter of fact , this is one of the first large prevalence studies of blood- born infections among incarcerated idus in iran . in this study ,
the history of shared drug injection inside prison was one of the main incarceration - related risk factors .
this association , has also been reported in other studies in the world.[1315 ] it seems that , the lack of access new needle / syringe in prisons and consequently needle sharing practice is the main reason of the hepatitis c epidemic in prisoners .
our data , highlight for comprehensive and integrated interventions for incarcerated idus to prevent hcv transmission among idu population and community .
effectiveness of needle programs in reducing needle sharing among idus has been shown in many countries . in our country ,
an outreach program for blood born infections prevention was supported by the united nation and the ministry of health of iran in 2003 .
multiple incarcerations was the another independent risk factor in our subjects which show that the high rates of arrests and incarcerations lead to drug - related offences or other confounding factors inside prison , such as violence .
our findings suggest that preventive interventions are compelling now for idus to ensure their safe passage throughout incarceration . in our study ,
other important modes of transmission were frequency and length of drug injection . according to these results ,
harm reduction strategies need to be expanded to prevent new hcv infections , particularly among young injectors.[1920 ] in our study , history of marriage was a protective factor for hcv - infection in the subjects after controlling for all other variables in the logistic - regression model .
sexual transmission could play a role in sporadic or community - acquired hcv infections . however , there is contradictory finding regarding the association between marriage and hcv infection in the world .
vandelli and colleagues indicated that the risk of sexual transmission of hcv within heterosexual monogamous couples is extremely low or even null .
it is shown that , those who have multiple sexual partners , including female sex workers , men having sex with men , and attendees of sexually - transmitted diseases ( stds ) clinics are the main risk groups for hepatitis c. in the acute hepatitis surveillance study in usa , 18% of newly infected individuals reported sexual contact with an hcv - infected person or multiple sexual partners as their only risk factor for hcv acquisition .
for the individual with chronic hcv infection , the estimated risk of sexual transmission of virus was 0% to 0.6% per year for those in monogamous relationships , and 1% per year for those with multiple sexual partners .
so , it will be useful to encourage young people to get married to prevent sexually transmitted diseases in our society .
in conclusion , according to progressive reported prevalence rates of hcv in the world and the importance of the current and potential burden of hcv - related complications , it is important to primary prevention of hcv infection that will be addressed through implementation of safe injection practices .
the mortality associated with hepatitis c is expected to double in the next 10 - 20 years .
it is conservatively estimated that the direct costs related to hepatitis c will be $ 10.3 billion during the years 2010 - 2020 .
the indirect costs of hepatitis c ( eg , the loss of productivity during that era ) are estimated to total another $ 54.3 billion due to premature death and $ 20.6 billion due to disability .
in addition , the total cost of therapy for hepatitis c is estimated to be $ 10,000-$12,000 .
so , primary prevention will be more cost - effective than secondary prevention of morbidity and mortality from hcv infection through provision of interferon - based therapy .
it is good opportunities in prison to have large number of idus over longer period to do specific preventive activities such as needle / syringe exchange and counsel imprisoned idus .
|
objectives : hepatitis c virus ( hcv ) infection is a major public health problem worldwide with serious complications . according to the importance of intravenous drug use ( idu ) as the main risk factor for hcv infection and transmission and prison as the main source of risky behaviors , this study conducted to define hcv infection and related risk factors in prison inmates with history of idu in isfahan province ,
iran.methods:this is a cross sectional study which the prison inmates with idu history in voluntary basis were enrolled .
a validated questionnaire was asked and blood sample was obtained from each subject for the presence of hcv antibody .
odds ratio and logistic regression were used for data analysis and p - value < 0.05 considered significant.results:i943 inmates with history of idu participated in the study .
the overall prevalence of hcv antibody was 41.6% .
the main independent risk factors were number of injection in the month [ or : 1.006 ( 1.002- 1.011 ) ] , length of drug addiction [ or : 1.05 ( 1.004 - 1.098 ) ] , multiple incarceration [ or : 1.15 ( 1.05 - 1.23 ) ] and use of needle / syringe share inside prison [ or : 4.19 ( 2.22 - 7.9 ) ] . in our study ,
marriage was a protective factor for hcv infection [ or : 0.34 ( 0.18 - 0.64 ) ] as well.conclusions:according to relatively high prevalence of hcv infection and associated risk factors which observed in this study it is important to primary prevention in prisons through syringe / needle exchange and counsel with imprisoned idus .
|
INTRODUCTION
METHODS
RESULTS
DISCUSSION
CONCLUSION
|
mucocele of the appendix ( collection of mucus within the appendiceal lumen ) is a rare lesion , found in only 0.2% to 0.3% of 43,000 appendectomies reviewed .
currently , the assessment of pelvic masses relies heavily on usg as the primary diagnostic tool .
in such cases , clinical findings and other investigative modalities are warranted to aid the diagnostic process . in spite of extensive preoperative investigations ,
the diagnosis may still remain elusive and may only be made at the time of surgery . some regard this lesion as benign , a result of obstruction of the proximal lumen by fibrosis ; others believe it to be a neoplasm of the appendix .
is the method of choice in the management of simple mucocele and for cystadenoma with an intact base .
several studies ( mostly case reports ) on laparoscopic resection of mucocele have been reported .
a 60-year - old female presented with pain in lower part of abdomen and palpable tender lump in the right ileac fossa .
ultrasound of the abdomen reports a cystic mass of size 12 15 cm with thin internal septations in the right adnexa .
the pneumoperitoneum was created with veress needle using carbon dioxide and the pressure was kept at 11 mmhg .
a 0 telescope was introduced through the umbilical port for the complete examination of the abdomen .
diagnostic laparoscopy revealed approximately 14 15 cm large bluish mucocele of the appendix with omental adhesions .
two 5-mm ports were placed in the supra pubic area below the pubic hair line as the working port .
the mucocele of the appendix was isolated after separating the mesoappendix from it with the help of bipolar cautery . following this ,
mucocele of the appendix [ figure 1 ] was retrieved out in a plastic bag through the umbilical port .
hemostasis was obtained and a suction drain left in situ which was removed when non - productive .
cut section showed appendix was filled with mucin - like material [ figure 2 ] .
she was started orally after 4 hours of operation and solid food on the next day .
appendicular lump from the distal portion of appendix after removal the appendicular lump filled with mucinus material
mucocele of the appendix is a descriptive term for an appendix distended by mucus , secondary to mucinous cystadenoma ( 63% ) , mucosal hyperplasia ( 25% ) , mucinous cystadenocarcinoma ( 11% ) , and retention cyst
. clinical presentation may include right lower quadrant pain , change in bowel habits , per rectal bleeding , or a palpable mass .
approximately 23 - 50% of patients are asymptomatic , with the lesions being discovered incidentally during surgery , radiological evaluations , or endoscopic procedures .
the preoperative clinical diagnosis of appendiceal mucoceles can therefore be difficult because of this lack of clinical symptomotology .
the initial detection of the lesion may be facilitated by radiological , sonographic , or endoscopic means . on barium enema ,
the lesion may be seen as a sharply outlined sub - mucosal or extrinsic mass indenting the cecum and laterally displacing it .
purely cystic lesions with anechoic fluid , hypoechoic masses with fine internal echoes as well as complex hyperechoic masses can be seen depending on the contents .
ct of the abdomen usually shows a cystic well - encapslated mass sometimes with mural calcification , in the expected location of the appendix .
it may be causing extrinsic pressure on the cecal wall without any surrounding inflammatory reaction .
colonoscopic findings include the volcano sign , the appendiceal orifice seen in the center of a firm mound covered by normal mucosa or a yellowish , lipoma - like submucosal mass . in our case , usg was unable to provide a preoperative diagnosis . in our case , the decision for excision of the appendiceal mucocele was made as a result of diagnostic laparoscopy and a need to rule out malignancy .
therefore mucocele of the appendix can mimic an adnexal mass and prove to be a diagnostic challenge . in a woman presenting with right iliac fossa mass and with clinical features not indicative of gynecological pathology , an appendiceal origin should be considered in the differential diagnosis .
surgery is the treatment of choice and should be done early as tumor can not be ruled out as the causative factor for the mucocele .
pre - operative diagnosis is important to avoid unintended rupture and the development of pseudomyxoma peritonei during surgery .
however , laparoscopic dissection , grasping of the appendix specimen , pneumoperitoneum , or transport of the specimen through the abdominal wall might contribute to peritoneal dissemination of a tumor , if present .
these setbacks can be avoided by taking precautions like using bowel holding graspers ( non - traumatic ) to handle the mucocele and using a non - permeable bag to deliver the specimen out of the port .
mucocele of the appendix can mimic an adnexal mass and prove to be a diagnostic challenge .
laparoscopic resection of mucocele of the appendix is feasible in spite of the danger of malignancy , provided necessary precautions are taken .
|
mucocele of the appendix is an aseptic dilatation secondary to obstruction .
the preoperative clinical diagnosis of appendiceal mucoceles can therefore be difficult because of this lack of clinical symptomotology .
surgical excision is the treatment of choice in benign mucocele .
we report a case presenting to the surgeons where initial clinical findings and investigations suggested a cyst in the right adnexa . diagnostic laparoscopy revealed mucocele of the appendix and laparoscopic appendicectomy
was done .
|
INTRODUCTION
CASE REPORT
DISCUSSION
CONCLUSION
|
village indigenous birds are constantly exposed to immunosuppressive conditions such as aflatoxicosis and infectious bursal disease virus .
in addition , management and ecological factors such as confinement , climatic and seasonal fluctuations , poor feeding , and worm infestations have been associated with stress and reduced immune response .
stressful factors have been reported to cause functional and morphological changes in chickens . in tanzania , it was observed that newcastle disease ( nd ) was a greater problem in villages with ducks .
earlier reports indicated that newcastle disease virus ( ndv ) persisted for a long time in a flock of ducks in a village situation in indonesia .
however , the factors leading to shedding of the virus by the carrier ducks are not well documented .
it was hypothesized that immunosuppression of immunised carrier ducks does not influence persistence of ndv in these birds . in this experiment ,
thus , the aim of the present study was to determine the effect of immunosuppression on the viral persistence and immune status of ducks .
it was designed to simulate field situation where ducks that have varying levels of ndv antibodies undergo immunosuppression in the presence of high ndv challenge .
one - day - old indigenous ducklings were hatched from the duck flock maintained at the university of nairobi premises .
all the birds were reared in isolation and transferred to experimental units at one year of age .
they were wing tagged , tested , and confirmed to be free of ndv and respective antibodies .
water and food were provided ad libitum . a kenyan virulent newcastle disease virus isolate ( vndv )
was obtained from the repository maintained at the university of nairobi and characterized by standard methods .
the inactivated vaccine was prepared by mixing 40% formalin and allantoic fluid with a titer of 2 of vndv in a ratio of 1 : 40 , that is , formalin to virus .
the reparation was kept at room temperature ( 24c to 26c ) for 24 hours before use .
all the ducks were vaccinated via an initial dose of 1 ml of the vaccine intramuscularly on the thighs and a booster of 0.5 ml of the same vaccine 16 days later .
the live virulent kenyan newcastle diseases virus isolate , previously characterized by standard methods , was later used to challenge vaccinated and naive ducks .
dexamethasone ( dexamethasone sodium phosphate and sodium methyl hydroxybenzoate , coophavet , france ) was used to stress ducks in this study .
the respective groups of ducks were injected intramuscularly with the dexamethasone , following the protocol of corrier et al .
modified as follows : the dosage was given at the rate of 2 mg per kilogram of body weight per day for 4 days continuously , then the ducks were rested for 2 days and the injections resumed at the same dosage for 2 more days .
sixty - four ducks were vaccinated with 1 ml of inactivated nd vaccine intramuscularly and 14 days later , they were bled from the brachial vein and sera prepared .
they were later boosted with a single dose of 0.5 ml of the nd inactivated vaccine and bled 7 days later .
seven days after the booster dose , the ducks were divided into two groups , each with 32 birds , namely , low antibody level group ( 1 : 32 ) and medium antibody level group ( 1 : 64 ) .
each group of 32 ducks was further subdivided into 4 minigroups , as follows : ( i ) immunosuppressed and challenged ( 1a , 2a ) , ( ii ) immunosuppressed only ( 1b , 2b ) , ( iii ) challenged only ( 1c , 2c ) , and ( iv ) not challenged nor immunosuppressed ( 1d , 2d ) . another group ( group 3 ) of 30 nonimmunized ducks were subdivided into 4 groups .
groups 3a and 3c had 12 ducks each while 3b and 3d had 3 birds each .
immunosuppression was done before respective groups were inoculated intranasally with 0.2 ml of undiluted amnioallantoic fluids of vndv having a titer of 1 : 1024 .
five birds from each of the challenge groups and all the 3 ducks from each control group were sampled throughout the experimental period ( 28 days ) .
the samples were taken on days 0 , 1 , 4 , 8 , 14 , and 28-after inoculation ( p.i . ) .
blood for serum was sampled each time from the five ducks in each challenge group , and the three ducks from each of the controls .
further , two ducks from each of the ndv challenged groups were killed serially and brain , kidney , lung , cecal tonsils , liver , and spleen collected separately from each bird .
the tissues were processed for nd viral recovery using chicken embryo fibroblasts , while serum samples were tested for ndv - specific antibodies by hemagglutination inhibition ( hi ) test .
virus recovery from the tissues was carried out in primary chicken embryo fibroblasts as described by oie .
analysis of variance was performed using sas software ( sas institute inc . , cary , nc , usa , 2002 - 2003 ) to determine the treatments ' main effects and the interaction between time ( days ) and treatment , on various responses .
immunosuppressed virus - challenged ducks ( group 1a ) had low mean antibody levels ( 5.0 ) up to day 4 after - inoculation ( p.i . ) compared with day 0 ( 4.5 ) .
thereafter , there was marked increase ( from 4.5 to 7.0 ) in antibody titers up to 14 days p.i .
after day 14 p.i . , there was a slight decrease ( 6.9 ) in antibody levels up to 28 days p.i . although the levels were still higher than any period between day 0 and 8 p.i .
the nonimmunosuppressed virus - challenged group ( 1c ) had a moderate increase ( 5.0 to 6.0 ) in antibody levels from day 1 up to day 14 p.i .
, after which there was a decrease to day 0 level titers by day 28 p.i .
the immunosuppressed group ( 1b ) had marked decrease in antibody titers from day 1 to 4 and gradual decrease ( 5.0 to 3.8 ) up to day 28 p.i .
the immunosuppressed virus - challenged group for the medium antibody level ducks ( 2a ) had a gradual decline ( 6.0 to 5.7 ) in antibody titers up to day 4 followed by an increase in antibody titers ( 6.9 ) up to day 14 p.i .
this was followed by a marked decrease ( 6.1 ) and by day 28 p.i .
the nonimmunosuppressed virus - challenged group ( 2c ) showed a slight decrease ( from 6.0 to 5.9 ) in the antibody titer followed by a gradual decrease and then an increase up to the end of the experiment . from day 1 up to day 4 after - inoculation , the immunosuppressed , immunised noninfected ( 2b ) group showed a more rapid decrease ( 6.0 , 5.2 , 4.8 , 3.7 , 2.3 , and finally 2.0 ) in antibody levels as compared ( 6.0 , 4.7 , 4.5 , 3.8 , 2.7 , and finally 2.2 ) to the nonimmunosuppressed controls ( 2d ) .
in general , all the nonchallenged , but immunized control ducks showed decrease in antibody titers with time ( figure 1(b ) ) .
the immunosuppressed virus - challenged group ( 3a ) had a gradual antibody response ( from 0.0 to 6.5 ) up to the end of the experimental period .
the nonimmunosuppressed virus - challenged group ( 3c ) showed a massive increase ( 0.0 to 6.6 ) in antibody levels similar to immunosuppressed virus - challenged group 3a .
the group 3c also had a marked decrease ( from 6.6 to 4.6 ) in antibody titres after day 14 p.i . and by 28 days p.i . , the titers were quite low .
negative control ducks ( 3b and d ) , sampled at the same time , were negative for ndv antibodies ( figure 1(c ) ) . for days 4 , 8 , 14 , and 28 p.i .
antibody titres of the following groups were found to be significantly different ( p < 0.05 ) . when considering the antibody levels elicited in the different duck - groups , with respect to their initial antibody levels , both sets ( low - antibody group and medium - antibody group ) , and both the immunosuppressed and non - immunosuppressed ducks showed higher responses in ducks that were challenged with virulent virus than in those that were not challenged .
all the control naive ducks ( groups 3b and 3d ) did not sero - convert .
immunosuppressed medium - antibody - level , challenged with ndv ducks ( group 2a ) versus immunosuppressed medium - antibody - level , nonchallenged ( group 2b ) , was lowest in the latter group .
nonimmunosuppressed low antibody level , challenged with ndv ducks ( group 2c ) versus 2d , the latter group had lower levels of antibodies .
in addition , antibody titres of group 1a versus 1d were significantly different ( p < 0.05 ) on day 14 , being lower in the latter .
all the control nave ( groups 3b and 3d ) birds did not seroconvert . on day 1 after inoculation ,
ndv titers were recorded in liver tissues of group 1a ( low - antibody - level group , immunosuppressed and challenged with vndv ) ducks only . on day 4
, high titres of the ndv were recorded in the kidneys ( figure 2(a ) ) . on day 8 p.i .
, ndv was isolated in the liver , kidneys , caecal tonsils , and lungs of all treatment groups ( figure 2(b ) ) .
the highest ndv titers were recorded in the liver and kidney tissues of immunosuppressed medium ( 2a ) and nonimmune ( 3a ) challenged ducks and nonimmunosuppressed , low - antibody - level challenged ducks ( 1c ) .
no ndv was isolated by day 14 p.i . in the brain and spleen from any of the groups .
high ndv titres were recorded from the liver tissues of ducks in all treatment groups .
however , titres were recorded in the cecal tonsils only in group 2a on day 14 p.i . and in groups 1a and 1c at day 28 p.i .
in addition , the immunosuppressed ducks of groups 1a ( low - antibody - level group , immunosuppressed and challenged with vndv ) and 2a ( medium - antibody - level group , immunosuppressed and challenged with vndv ) yielded the highest ndv titres as compared to other treatment groups .
newcastle disease virus was recovered from the brain and lung on day 28 p.i from immunosuppressed ducks only ( figure 2(d ) ) .
there have been comparatively few sequential virological studies on the pathogenesis of nd in ducks and the reported studies involved only fully susceptible chickens [ 8 , 9 ] .
reports in other studies have documented frequent isolation of virulent ndv from captive caged birds , healthy wild birds , and village chickens [ 1012 ] . in some cases , the ducks expressed clinical nd as a result of confinement that was understood to have induced stress .
our findings indicate that ndv carrier status in nonnatural hosts such as ducks would possibly , under stress , recrudescence virulent virus from sequestered sites in the kidney , liver , and caecal tonsils , leading to virus release in faecal and respiratory exudates .
the excreted virus would set up an infectious index case in chickens , thus maintaining ndv endemicity .
immunosuppression induced by injection of dexamethasone in the three treatments influenced the pattern of antibody response and the ndv recovery rate .
the immunosuppressed ducks that had low and medium antibody level showed a decrease in antibody titers up to day 4 after challenge with ndv .
the nonimmunosuppressed virus - challenged ducks of low - to - medium antibody level had an increase in antibody titres up to day 14 p.i .
the nonimmunized ducks manifested increased antibody titres after day 4 p.i . and had a massive increase in antibody levels as compared to immunosuppressed challenged group . in the present study ,
the number of immunosuppressed ducks that yielded the nd virus was higher compared to the nonimmunosuppressed .
the prechallenge antibody titers may therefore play a significant role in shedding off the virus as well as clinical manifestation of the disease .
. noted that a few hours of treatment with low concentrations of synthetic glucocorticoid ( analogue dexamethasone ) are sufficient to inhibit the synthesis of interferon , a virus inhibitor .
this may , in part , explain the observation that treatment with glucocorticoids increased virus yield and lethality in mice infected with coxsackie virus .
our present study using ducks concurs with those of asdell and hanson who showed that prior treatment of chickens with dexamethasone lead to massive nd virus multiplication .
there was significant difference in geometric mean antibody titers between the immunosuppressed ducks of group 1a and nonimmunosuppressed counterparts ( group 1d ) and also between immunosuppressed ducks of group 3a and nonimmunosuppressed group 3d .
this means that whereas dexamethasone seems to have an effect on immune system of ndv - infected ducks , the prechallenge titres also play a major role in the immune response of immunosuppressed birds in that immunosuppression of ducks with high viral titers allows virus multiplication making ducks better carriers .
the effect of immunosuppression with dexamethasone in ducks appears to be the same as that induced by aflatoxin in chicks with ndv infections .
chickens fed on aflatoxin produced lower antibody levels when compared to the non - aflatoxin - treated ones .
the nonchallenged preimmunized ducks had a progressive decrease in antibody levels suggesting that if they were to be exposed to the virus , they could come down with the nd or if the antibody titers were within the protective levels ( 2 to 2 ) , they might not develop clinical disease but instead may remain as virus carriers .
the fact that the ducks in these experiments had high levels of antibodies may not necessarily prevent subclinical infection and excretion of virulent virus as supported by other studies elsewhere . based on these results ,
it is possible to assume that immunosuppressed ducks carrying ndv are more likely to shed virus under stress than nonimmunosuppressed ducks .
furthermore , the ducks showed progressively declining antibodies titres to very low levels making them more susceptible to infection by challenge nd virus , thus leading to clinical disease and virus excretion .
the excreted virus would contaminate the birds ' environment and be transferred to susceptible chickens and other birds .
thus , immunosuppression seems to have epidemiologically significant effects on ndv carrier ducks having varying antibody titer levels .
|
this study was carried out to verify the possibility that ducks are sources of newcastle disease ( nd ) virus infection for chickens in mixed flocks .
immunosuppressed ( is ) and non immunosuppressed ( nis ) birds , at three different antibody levels ( medium , low and absent ) were used ; the titres having been induced through vaccination , and immunosuppression done using dexamethazone .
each of the 3 respective groups was further divided into 2 groups of about 12 ducks each : one challenged with velogenic nd virus ; the other not challenged . selected ducks from all groups
had their antibody titres monitored serially using hemagglutination inhibition test , while two birds from each of the challenged groups were killed and respective tissues processed for nd viral recovery , using chicken embryo fibroblasts . in general , antibody titres of is and nis challenged ducks were significantly higher than their unchallenged counterparts ( p < 0.05 ) .
non - challenged pre - immunised ducks had a progressive decrease in antibody levels ; non - immunised ducks did not seroconvert .
newcastle disease virus was isolated from livers and kidneys of the challenged ducks throughout the experimental period ; indicating a possibility of viral excretion , especially when the birds are stressed .
it , therefore , provides another possible model of viral circulation within mixed flocks .
|
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
|
Subsets and Splits
No community queries yet
The top public SQL queries from the community will appear here once available.