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they originate from granulation alterations producing a defect in the closure of the neural tube during the third to sixth week of gestation , at level surface of the ectoderm . at intracranial level
represents 0.21.8% of all tumor lesions ; they can be located intra- or extradurally , .
ieics are very rare ; as the name indicates are found among the two tables of the skull bones , having a slow growth , .
the first ieic was reported by mller in 1838 , cushing in 1922 , also performed a description of an ieic , and the first case of giant ieic in the posterior cranial fossa was reported by rengachary .
two cases are reported , that in a particular way , go to the emergency room because they display intracranial hypertension syndrome , which is very intriguing because of the slow growth of this type of pathology ; however , decompensation occurring in the pathophysiology of space - occupying lesions at intracranial level explain this type of clinical presentation .
male , 42 , with a history of smoking and drug use methamphetamines . presents occipital headache with 3 months evolution that radiates to the frontal region , pulsatile , which increases with valsalva maneuvers with a frequency initially described as sporadic until evolving to daily , in the week prior to admission ; accompanied by vomiting , on the day of admission , with intensified headache intensity . on physical examination ,
the patient exhibits intracranial hypertension symptoms ( papilledema ) without rostrocaudal deterioration , which is accompanied by indifferent lateropulsion ; in imaging studies , extra - axial ( fig .
study protocol is completed , and resection of the lesion is performed by means of a right lateral suboccipital approach . among the findings
an important erosion of the occipital squama was observed due to extra - axial injury , eroding the bone , avascular , not infiltrating with surrounding tissues , of pearly aspect with fatty content inside ( fig .
histopathological studies , reports us soft lesion with granular areas and firm membranous areas with calcifications , reporting an epidermal cyst .
the patient progressed favorably , without neurological deficits with one - year follow - up ( fig .
sine 6 months ago presents painless right frontal region deformation ; since 2 months ago with palpebral edema , which lasted four days , and was treated at another health facility as cellulitis , with antibiotics and anti - inflammatory therapy , resolving favorably ; due to increasing deformity of the right frontal region , consults with private doctor who diagnosed intracranial lesion with respective imaging studies and subsequently refers to our hospital , by the mass effect is admitted ( fig .
4 ) ; at the time of admission the patient develops severe holocraneal cephalea , accompanied by vomiting , papilledema and stupor , therefore is decided to take to the or suite for resection of the lesion , through a centered approach ; during surgery important right frontal bone erosion was observed due to extra - axial injury , not vascularized , not infiltrating with neighboring tissues , with fatty content inside ( fig .
5 ) ; histopathological result is epidermoid cyst ; during the immediate post - surgery period , the patient develops mixed delirium , which is solved in three days ; is discharged without any complications ; craneoplasty is later performed at 3 months after resection surgery of intradiploic epidermoid cyst ( fig .
male , 42 , with a history of smoking and drug use methamphetamines . presents occipital headache with 3 months evolution that radiates to the frontal region , pulsatile , which increases with valsalva maneuvers with a frequency initially described as sporadic until evolving to daily , in the week prior to admission ; accompanied by vomiting , on the day of admission , with intensified headache intensity . on physical examination ,
the patient exhibits intracranial hypertension symptoms ( papilledema ) without rostrocaudal deterioration , which is accompanied by indifferent lateropulsion ; in imaging studies , extra - axial ( fig .
study protocol is completed , and resection of the lesion is performed by means of a right lateral suboccipital approach . among the findings
an important erosion of the occipital squama was observed due to extra - axial injury , eroding the bone , avascular , not infiltrating with surrounding tissues , of pearly aspect with fatty content inside ( fig .
histopathological studies , reports us soft lesion with granular areas and firm membranous areas with calcifications , reporting an epidermal cyst .
the patient progressed favorably , without neurological deficits with one - year follow - up ( fig .
sine 6 months ago presents painless right frontal region deformation ; since 2 months ago with palpebral edema , which lasted four days , and was treated at another health facility as cellulitis , with antibiotics and anti - inflammatory therapy , resolving favorably ; due to increasing deformity of the right frontal region , consults with private doctor who diagnosed intracranial lesion with respective imaging studies and subsequently refers to our hospital , by the mass effect is admitted ( fig .
4 ) ; at the time of admission the patient develops severe holocraneal cephalea , accompanied by vomiting , papilledema and stupor , therefore is decided to take to the or suite for resection of the lesion , through a centered approach ; during surgery important right frontal bone erosion was observed due to extra - axial injury , not vascularized , not infiltrating with neighboring tissues , with fatty content inside ( fig .
5 ) ; histopathological result is epidermoid cyst ; during the immediate post - surgery period , the patient develops mixed delirium , which is solved in three days ; is discharged without any complications ; craneoplasty is later performed at 3 months after resection surgery of intradiploic epidermoid cyst ( fig .
the incidence of intracranial epidermoid cysts is higher in the intradural location , as reported in their work love and kernohan70% and gormley et al.72.7% , .
reported 28 cases of intradural epidermoid cysts , of which the majority are found in the cerebellopontine angle17 ; other locations were in the fourth ventricle , into the cisterna magna , in the fissure of silvio , in the occipital lobe , in the lateral ventricles and diploic space .
the intradiploic extradural epidermoid cysts are located in the cranial vault , destroy the internal table and thin the external table .
the most common presentation of ieic is the prominence at the level of the soft tissues and then presenting less frequently local pain and cephalea ; rarely the size of the lesion can cause focal neurological signs , .
they can be located in the paranasal sinus , in the temporal and sphenoid bone , however it occurs most frequently at the level of the occipital , frontal and parietal bones .
the differential diagnosis of an intradiploic cranial lesion includes the aneurysmal bone cyst , dermoid cyst , cavernous hemangioma , langerhans cells histiocytosis , fibrous dysplasia and eosinophilic granuloma , .
histologically , it is covered by fibrous tissue capsule , in which are included keratin , cholesterol , proteins and detritus crystals .
malignant transformation of epidermoid cysts is very rare ; the possibility of squamous cell carcinoma originated from a ieic is described with poor prognosis even after radio or chemotherapy , .
resection of these lesions are done to protect the patient from mass effect on intracranial mass , abscess formation and to avoid potential complications such as bleeding and malignant transformation of ieic , , .
these benign lesions although they are of low incidence , are seen very rarely in intradiploic locations and above all , of significant size , may produce significant mass effect in patients , which was initially tolerated because of its slow growth , however , they may become decompensate and cause intracranial hypertension syndrome .
the name of the ethics committee is collegial session neurosurgery , with number 213892 for case 1 and214450 for case 2 , of the neurology and neurosurgery national institute .
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 .
| highlightsepidermoid cysts are benign tumors -ieic - that originate in the spine or intracraneally.intradural compose 75% of epidermoid cysts and 25% are extradural in the diploic spaceieic.these benign lesions although they are of low incidence , are seen very rarely in intradiploic locations and above all , of significant size , may produce significant mass effect in patients , which was initially tolerated because of its slow growth , however , they may become decompensate and cause intracranial hypertension syndrome . |
genital herpes is the most common sexually transmitted disease ( std ) in hiv - seropositive individuals . the severity of herpes infection increases in the presence of immunosuppression .
necrotic ulceration may appear at unusual sites and more extensive and chronic lesions may occur .
chronic ulcers caused by herpes of more than 1 month duration are an aids - defining illness .
a 27-year - old female patient presented with non - healing painful ulcer in the left hand of 6 months duration .
an ulcer measuring 10 6 cm was present over the ulnar aspect of the dorsa of left hand which extended to the first three fingers and the palmar aspect of left hand [ figure 1 ] .
liver function , renal function , and chest x - ray were within normal limits .
mantoux , smear for afb , koh for fungus , and tzanck smear were negative .
a biopsy of the ulcer showed neutrophilic vasculitis , necrosis , and ill - defined granulomas .
oral steroids were added to the dose after 5 days as the lesions increased in size .
after 15 days , her ulcer did not show any improvement but pain was aggravated .
cyclosporine 100 mg was advised and after 5 days she reported with vesicles and ulcers over perianal region . on genital examination , multiple vesicles and ulcers with polycyclic border were seen in the perianal region and vesicles were present near the ulcer in the left hand .
tzanck smear from perianal vesicle showed multinucleated giant cells whereas from the vesicles on the hand showed only neutrophils .
pus culture showed staphylococcus and e. coli , and linezolid 600 mg twice daily was started according to sensitivity pattern .
a workup for std revealed hiv 1 elisa to be positive , and result was confirmed by western blot .
she had dry cough for 3 days and chest x - ray showed patchy haziness on both sides .
after 5 days of famciclovir , her perianal lesions resolved and her hand ulcer showed some improvement with subsidence of pain . the ulcer over the hand healed in a span of 25 days [ figure 2 ] .
atypical presentations of herpes such as hemorrhagic deep painful ulcers , vegetating plaque with ulceration , hyperkeratotic verrucous plaques , erythema multiforme , like eruption or generalized erythematous and papulovesicular rash , are found in hiv - affected individuals . in these cases tzanck
herpetic infection of the hands usually presents as whitlow , erythema multiforme , or cellulitis .
the probable reasons for the development of herpetic ulcer over the hand in our patient have been the transfer of herpes virus from the perianal region to the left hand and subsequent involvement of perianal region .
famciclovir in hiv - infected persons with genital hsv infection results in significant reduction in symptoms associated with hsv infection and the symptomatic and asymptomatic shedding of hsv .
response to acyclovir is excellent except in few patients with very low cd4 count ( < 50 ) . in such cases ,
recurrence rate is very high and treatment with higher doses for longer period is required .
pyoderma gangrenosum presents as rapidly progressive , painful , suppurative cutaneous ulcers with edematous , blue , undermined , and necrotic borders .
ulcers due to arterial or venous disease , vasculitis , cancer , infection , and trauma can be misdiagnosed as pyoderma gangrenosum .
a biopsy of an ulcerative variant of pyoderma gangrenosum demonstrates central necrosis and ulceration of epidermis , superficial dermal edema , and dermo - epidermal neutrophilic infiltrate with abscess .
pyoderma gangrenosum is a diagnosis of exclusion , and the misdiagnosis of pyoderma gangrenosum and its treatment can result in complications in patients who have other causes of severe cutaneous ulceration .
our patient was initially diagnosed as pyoderma gangrenosum and treated with immunosuppressive which might have prompted the increase in ulcer size and appearance of perianal vesicles and gave the clue to the diagnosis of herpes .
chronic non - healing large ulcer mimicking pyoderma gangrenosum needs to have a thorough workup prior to initiation of immunosuppressants .
although screening for hiv was carried out , the intial omission of this investigation led to misdiagnosis of pyoderma gangrenosum in our case .
close long - term follow - up and workup are recommended for all patients with suspected pyoderma gangrenosum . | retroviral infection is known to cause varied manifestation of any disease .
we report a 27-year - old female who presented with non - healing painful ulcer in the left hand of 6 months duration .
the patient was diagnosed as pyoderma gangrenosum and treated with immunosuppressant drugs .
one month later , she developed perianal vesicles and tzanck smear from the vesicles showed multinucleated giant cells .
a workup for sexually transmitted disease revealed hiv 1 elisa to be positive .
diagnosis of herpetic ulcer was made and treated with famciclovir and hand ulcer gradually healed in 25 days .
we report this case to highlight the occurrence of herpetic ulcer in an unusual site in a hiv patient and may lead to misdiagnosis of pyoderma gangrenosum |
levetiracetam ( lev ) is an antiepileptic drug that is approved for use as adjunct treatment for partial epilepsy in patients aged 4 years and older , as well as for juvenile myoclonic epilepsy in patients aged 12 years and older .
irritability and agitation are known behavioral side effects of lev , and it is reported in almost 13% of patients across all age groups .
levetiracetam use can be associated with insomnia , agitation , anxiety , emotional lability , and hyperactivity .
some of these adverse events , however , are reported in patients with preexisting behavioral issues .
furthermore , these adverse effects can lead to lev discontinuation in a small percentage of patients .
previous case series from children and adolescents have indicated that pyridoxine ( vitamin b6 ) can alleviate behavioral side effects related to the use of lev .
, we sought to explore the potential benefits of pyridoxine supplementation for the treatment of behavioral side effects induced by lev in our patients attending the epilepsy clinic .
we retrospectively reviewed our electronic medical records to identify all patients attending our epilepsy clinic and being treated with both lev and pyridoxine .
fifty - one adult patients were identified using pyridoxine supplementation for the management of either irritability or agitation following the introduction of lev .
age , gender , epilepsy classification , duration of seizures , dosages of lev and pyridoxine , and the effect of the addition of pyridoxine on behavioral symptoms were carefully analyzed .
the epilepsy was classified as idiopathic in 15 patients ( 29.5% ) and as partial in 36 ( 70.5% ) .
levetiracetam monotherapy was used for 26 patients ; the others were taking an average of two additional antiepileptic drugs .
pyridoxine was started at 50 mg / day , and if no or partial benefit was noted , the dose was increased to 100 mg / day .
however , the average dose to control the behavioral symptoms in our study was 54.5 mg / day ( sd = 18.2 ) .
most patients had reported behavioral problems within the first weeks after the addition of lev .
none of our patients , however , had preexisting behavioral issues prior to the use of lev .
none of the patients who did not receive pyridoxine supplementation reported any significant improvement in behavioral symptoms throughout the course of treatment with lev . on average , the effects of pyridoxine supplementation were reported during the first 2 weeks . on a separate note , none of the patients who received pyridoxine supplementation reported any significant improvement in seizure frequency throughout the course of treatment with lev .
to our knowledge , this is the first study that suggests a possible benefit of pyridoxine supplementation in the treatment of lev - induced behavioral side effects in the adult population .
previous reports in the pediatric population have demonstrated the potential beneficial effect of pyridoxine to control the lev - induced behavioral symptoms .
miller was able to control the behavioral disturbances caused by lev completely , in 5 of 6 children aged between 2 and 10 years , by supplementing pyridoxine at an average dose of 7 mg / kg / day . in another study to examine the use of pyridoxine , major et al .
twenty - two patients started pyridoxine after being on lev , due to behavioral side effects , and significant behavioral improvement was observed in nine ( 41% ) , no effect in eight ( 36% ) , deterioration in four ( 18% ) , and an uncertain effect in one .
the effects of pyridoxine supplementation , similar to our study , were observed during the first week of its introduction .
our study used a retrospective , chart review method and lacked a placebo - controlled approach .
our results demonstrated that 66% of patients who started pyridoxine after being on lev had a significant behavioral improvement , and in the remaining one - third , pyridoxine was not helpful in preventing lev - related behavioral side effects . interestingly , and similar to previous reports in pediatric populations , the improvement occurred during the first few weeks after pyridoxine supplementation , probably supporting its potential effect .
pyridoxine is a water - soluble cofactor in more than 100 enzyme - catalyzed reactions in the body , including many involved in the synthesis or catabolism of neurotransmitters .
there is no known pharmacokinetic or pharmacodynamic interaction between lev and pyridoxine that could explain its potential clinical benefit . on that note
, it can be said that the underlying mechanism(s ) that would explain its efficacy in controlling the behavioral adverse effects of lev is largely unknown , and its recommended use to control these symptoms is empirical at best .
similarly , pyridoxine , based on empirical grounds , has been used to treat other medical conditions such as premenstrual depression . on the other hand ,
pyridoxine has been tried based on our current understanding for treatment of other underlying metabolic disorders .
for example , pyridoxine - dependent epilepsy , a rare autosomal recessive error of metabolism characterized by neonatal seizures , responds to pyridoxine .
intravenous administration of 50100 mg of pyridoxine promptly controls the seizures , but lifelong supplementation is required ( 5300 mg / kg / day ) .
the average dose to control the behavioral symptoms in our study was less than 100 mg / day .
toxicity of this supplementation tends to occur at a dose higher than 1000 mg / day .
we realize the limitation of our study , being retrospective and from a single center .
however , this preliminary study suggests that pyridoxine might be used safely to control lev- induced behavioral side effects in adults . whether vitamin b6 has the same efficacy in controlling behavioral symptoms in patients with epilepsy in general or it shows the same efficacy in those patients who are treated with other aeds remains to be carefully examined . a prospective , placebo - controlled study is needed to confirm our finding and to address these unanswered questions .
| behavioral symptoms are known side effects of levetiracetam . previous case series in children and adolescents
have demonstrated the potential effect of pyridoxine in ameliorating these symptoms .
we retrospectively reviewed the charts of 51 patients treated with pyridoxine to control agitation and irritability following the introduction of levetiracetam .
these symptoms were relieved in 34 patients ( 66.6% ) .
seventeen patients did not appear to benefit from this supplementation .
this preliminary study suggests that pyridoxine might be an effective option across all ages for patients suffering from levetiracetam - induced behavioral side effects . |
clinical protocol - the protocol for this trial is available as
supplementary
data . the clinical study protocol and informed
consent were reviewed and approved by the ethical committee at the university of
campinas ( caae 3652.0.000.146 - 08 ) .
this committee is accredited by the brazilian
national council on ethics in research of the ministry of health .
the clinical study was
conducted in accordance with the helsinky declaration revised in 2000 , good clinical
practice ( ich 1996 ) , guidance on the investigation of bioavailability and bioequivalence
( cpmp / ewp / qwp/1401 1998 ) and brazilian legislation ( res 196/96/ms / cns 1996 , res
251/97/ms / cns 1997 , res 1170/06/ms / cns 2006 ) . written informed consent was obtained for
every subject prior to enrolment .
all of the subjects were informed about the nature of
the trial , the possible risks and that they were free to withdraw their consent of
participation at any time .
the clinical part of the study was conducted at the
synchrophar clinical unit at irmos penteado hospital , campinas , state of so paulo ,
brazil , which is accredited by the brazilian health surveillance agency ( anvisa ) .
the trial was designed as a single centre , randomised , single dose , open label , fasting ,
two - phase crossover bioequivalence study with an washout period of seven days ( > 7
half - life ) .
the trial was registered at the brazilian national information system of
pharmaceutical equivalence and bioequivalence studies ( sineb ) ( registry cef 03/08 ) and
in the clinicaltrials.gov database ( registry nct02043314 ) .
the authors confirm that all
of the ongoing and related trials for this intervention are registered .
the inclusion criteria were predefined as follows : healthy men and women , aged between
18 - 50 years and body - mass - index ( bmi ) between 18.5 - 29.9 kg / m .
the exclusion
criteria included known hypersensitivity to inh , pregnancy , use of other drugs other
than contraceptives , health problems , alcohol or drug abuse and any other condition that
could alter absorption , distribution , metabolism and excretion of inh .
study procedures - the study participants were recruited and enrolled
from the study volunteers pool at synchrophar .
all of the volunteers met the inclusion
and the exclusion criteria defined in the study protocol .
healthy volunteers were
considered to be eligible for the study based on medical history , demographic data ,
medication history , physical examination and clinical laboratory tests [ haematology ,
biochemistry , urinalysis , hiv and hepatitis b and c , parasitological faeces test , -hcg
( female subjects before each administration of study drugs and post study ) and a 12-lead
electrocardiogram ] .
the volunteers were recruited and included in the study on the 21 october 2008 , the
second phase was conducted after the washout period of seven days and the follow - up
adverse event ( ae ) evaluation was conducted 10 days after the last blood collection .
each subject was assigned to one of
two study treatment sequences ( rt or tr ) .
after 10 h of fasting overnight , each subject received either the test
formulation ( inh 300 mg manufactured by farmanguinhos - batch 06102382 ) or three tablets
of the reference formulation ( inh 100 mg manufactured by aeronautics chemical and
pharmaceutical laboratory - batch 07090034 ) .
subjects continued to fast until after 4 h after the
administration and controlled meals were served afterwards .
fluid intake was allowed
ad libitum until 6 h before dosing and 2 h after dosing .
serial blood samples for the pharmacokinetic assessments were collected over a period
that was greater than three half - lives at the following time points : 0 ( pre - dose 30 min
before dosing ) , 0:20 , 0:40 , 1 , 1:20 , 1:40 , 2 , 2:30 , 3 , 3:30 , 4 , 5 , 6 , 8 , 12 and 24 h
after oral administration of each treatment .
blood samples ( 7.5 ml each ) were drawn into
heparinised tubes for further processing .
immediately after blood collection , the tube
was inverted by hand to ensure thorough mixing with the anticoagulant and centrifuged
for 10 min at 3,000 rpm ( + 4c ) .
after centrifugation , the plasma samples were
immediately transferred to separate appropriately labelled plastic cryotubes and stored
at -25c until analysis .
aes were assessed either by periodic physical examination or by clinical laboratory
tests at baseline and at the end of the study .
registration of aes included duration ,
severity , intensity and causality relationship with the drug as well as the outcome .
. 2 : mass spectrum and fragmentation route of fluconazole ( flc ) - internal
standard .
fig .
drug analysis - the analytical part of the study was performed from 11
december 2008 - 14 january 2009 .
chemicals and reagents - the inh reference standard and fluconazole
( flc ) [ internal standard ( is ) ] were identified and supplied by the united states
pharmacopoeia ( usa ) .
methanol , acetonitrile and isopropanol were of chromatographically
pure grade and the formic acid was of analytical grade .
ultra purified water ( type i ) was separated and used
for preparing the solutions and the mobile phase study .
instrumentation and operating conditions - liquid chromatography was
performed using a varian system ( usa ) equipped with two varian 212-lc pumps .
chromatographic separation was conducted using an
inertsil sil 100a ( 5 m , 50 x 2.1 mm i.d . ) column at an ambient temperature .
the mobile
phase consisted of a mixture of the following two solutions : ( i ) water containing formic
acid 0,046% and ( ii ) isopropanol - acetonitrile .
the composition of the mobile phase was
20a/80b v / v and was set at a flow rate of 0.3 ml / min .
the mass spectrometric detection was performed using a varian 310-ms quadrupole lc - ms / ms
system ( usa ) equipped with an electrospray ion source and operated in the positive
ionisation mode . the ion spray voltage and
the parameters for the other gas source were set , as follows : drying gas
20 psi , nebulizer gas 55 psi and collision induced dissociation 2.00 mtorr .
quantification was performed using multiple reaction monitoring of the fragmentation
transitions m / z 138.50121.30 for inh and m / z 307.30238.00 for flc ( is ) ( figs 2 , 3 ) .
data acquisition and analysis were achieved using the varian ms workstation software
v.6.6 ( usa ) .
preparation of stock and work solutions - the stock solution of inh was
prepared at a concentration of 1.0 mg / ml in ultrapure water .
stock solutions of flc ( is )
were prepared only in methanol / water - 50/50 - v / v at a concentration of 1.0 mg / ml . all
of the solutions were stored at -25c .
the standard working solutions of inh analytes were prepared in 200 , 400 , 1,000 , 2,000 ,
4,000 , 10,000 and 15,000 ng / ml concentrations .
they were obtained by serial dilution of
the stock solution with ultrapure water . quality control ( qc )
working solutions of inh
in 200 , 400 , 4,000 and 10,000 ng / ml concentrations were similarly prepared as the
standard working solution .
the working is solution containing 1,000 ng / ml of flc was
prepared in methanol / water - 50/50 - v / v by diluting the stock solutions . when not in
use , all of the solutions were stored at -25c in an amber glass bottle .
sample preparation - qcs , calibration curve and clinical plasma samples
were extracted using a precipitation extraction technique .
a 0.10 ml aliquot of the
collected plasma sample from healthy volunteers , qcs or calibration curve was pipetted
into a 2 ml polypropylene tube .
the working solution of the is ( 1,000 ng / ml ) and 0.4 ml
of acetonitrile were added and then vortexed for 1 min .
then , the samples were
centrifuged at 14,400 rpm for 5 min at + 4c .
the organic layer was transferred to
another 2 ml polypropylene tube , 0.1 ml ultrapure water and formic acid 0.046% were
added and the mixture was vortexed for 30 s. an aliquot of 0.3 ml was transferred to
vial glasses and 10 l was injected into the lc - ms / ms system .
method validation - the validation process was conducted according to
guidance ( bioanalytical method validation ) provided by the us food drug administration
( fda 2001 ) .
this guidance is in accordance
with resolution 899 of 23 may 2003 by the anvisa ( anvisa 2003b ) .
selectivity was assessed by comparing chromatograms of six different batches of blank
human plasma with the corresponding spiked human plasma . in this test
, no significant
interference was detected in the retention times of the analyte and the is .
intra and
inter - day precision [ the relative standard deviation ( rsd ) ] and accuracy ( the relative
error ) were determined by analysing low , medium and high qc samples ( n = 8) on three
different days .
the extraction recovery was determined by comparing the mean peak areas
of eight extracted samples at low , medium and high qc concentrations with the mean peak
areas of spike after the extraction samples .
stability was assessed by analysing the replicates ( n = 8) of the low and high qc
samples during sample storage and processing procedures .
post - preparation stability was estimated by
analysing qc samples at 24 h at + 4c .
six aliquots of qc samples were stored at -20c
for 63 days and at ambient temperature for 6 h to determine long - term and short - term
stability , respectively .
pharmacokinetics analysis - pharmacokinetic analysis was performed with
the data from subjects who completed both periods of the study using the
winnonlin software v.5.01 ( pharsight co , usa ) .
concentration values below
the quantification limit ( 100 ng / ml ) at the initial absorption or at the terminal
elimination phase were considered to be zero .
the following pharmacokinetic parameters were estimated : maximum concentration of drug
observed in plasma ( cmax ) , area under the plasma concentration vs. time curve
from time zero to the last measurable concentration time t ( auc0-t )
calculated using a linear trapezoidal method , area under the plasma concentration vs.
time curve from time zero to time infinity ( auc0-inf ) calculated as where
cycle threshold is the last concentration of the drug , the time required to reach
maximum concentration of drug in plasma ( tmax ) , elimination rate constant ,
lambda_z and the time it took the plasma concentration to reduce to 50% during the
elimination phase .
the pharmacokinetic parameters were calculated from the plasma concentrations using a
noncompartmental method for inh .
the cmax and the tmax were obtained by
directly reading the concentration results .
statistical analysis - summary statistics were calculated for the
concentration - time data of the test and reference products for all of the
pharmacokinetic parameters , including the arithmetic mean [ mean , sd , coefficient of
variation ( cv ) , minimum ( min ) , maximum ( max ) and median ] .
the bioequivalence acceptance interval was set at 80 - 125% as required by the regulatory
agency ( anvisa 2003b ) .
the 90% confidence interval ( ci ) of the test / reference mean ratio
of inh was computed for the log - transformed pharmacokinetic parameters of
cmax , auc0-t and auc0-inf .
the anova model included
the following factors : sequence , subject with sequence , period and treatment .
tables i , ii show the pharmacokinetic parameters of the test product and the reference ,
respectively .
table imean pharmacokinetic parameters of isoniazid for the test
formulation test
cmax
auc0-t
aucinf
t1/2
tmax
mean4.619.7712.425.5313.375.632.540.75sd2.151.526.177.136.788.751.010.42cv0.470.500.510.400.56minimum1.632.743.554.163.734.031.030.33maximum10.820.8826.327.9427.856.544.262.00auc0-t : time zero to the last measurable concentration
time t ;
aucinf : area under the plasma concentration vs. time curve
from zero to infinity ; cmax : maximum concentration of drug
observed in plasma ; cv : coefficient of variation ; sd : standard deviation ;
t1/2 : time it took the plasma concentration to reduce to 50%
during the elimination phase ; tmax : time required to reach
maximum concentration of drug in plasma .
auc0-t : time zero to the last measurable concentration time t ;
aucinf : area under the plasma concentration vs. time curve
from zero to infinity ; cmax : maximum concentration of drug
observed in plasma ; cv : coefficient of variation ; sd : standard deviation ;
t1/2 : time it took the plasma concentration to reduce to 50%
during the elimination phase ; tmax : time required to reach
maximum concentration of drug in plasma .
table iimean pharmacokinetic parameters of isoniazid for the reference
formulation reference
cmax
auc0-t
aucinf
t1/2
tmax
mean4.366.8712.522.2413.504.982.570.92sd1.475.096.057.476.703.681.010.62cv0.340.480.500.390.68minimum1.482.002.946.623.204.841.050.33maximum7.355.1724.536.5627.024.084.163.50auc0-t : time zero to the last measurable concentration time t ;
aucinf : area under the plasma concentration vs. time curve
from zero to infinity ; cmax : maximum concentration of drug
observed in plasma ; cv : coefficient of variation ; sd : standard deviation ;
t1/2 : time it took the plasma concentration to reduce to 50%
during the elimination phase ; tmax : time required to reach
maximum concentration of drug in plasma .
auc0-t : time zero to the last measurable concentration time t ;
aucinf : area under the plasma concentration vs. time curve
from zero to infinity ; cmax : maximum concentration of drug
observed in plasma ; cv : coefficient of variation ; sd : standard deviation ;
t1/2 : time it took the plasma concentration to reduce to 50%
during the elimination phase ; tmax : time required to reach
maximum concentration of drug in plasma .
drug analysis - inh is a hydrophilic compound that is easily eluted
from a normal reversed phase c18 column .
therefore , to chromatically resolve these
compounds , we used a highly mobile phase and an inertsil sil 100a ( 5 m , 50 x 2.1 mm
i.d . ) column that provided appropriate retention and selectivity for polar
compounds .
the best analysis conditions were achieved using a mixture of solutions , including ( i )
water containing formic acid 0.046% and ( ii ) isopropanol - acetonitrile .
this mixture
provided a good chromatographic peak shape of the analyte and the is as well as their
elution in a short period of analysis when compared with literature ( panchagnula et al . 2003 , wang et al .
however , the analyst chose to perform a
procedure of division in the acquisition of masses at the beginning of the analysis up
to 0.5 min to eliminate potential contaminants that could suppress the signal when in
contact with the ion source ( fig .
4 ) . the
linearity of the method set from the llq of 100 ng / ml to the upper limit of 7,500 ng / ml
( table iii ) was sufficient to define the
pharmacokinetic parameters of the study .
the linearity limits of the bioanalytical
method were defined based on published data of the bioavailability of inh .
flc :
fluconazole ;
table iiiprecision and accuracy data of back calculated concentrations of
calibration samples for isoniazid ( inh ) in plasmaa
analytenominal concentration
( ng / ml)observed
concentration ( ng / ml - mean sd)precision ( rsd )
( % ) accuracy ( % ) inh10097.28 3.323.4197.28 200208.10 15.317.36104.05 500503.12 43.118.57100.62 1,0001.071.81 10.440.97107.18 2,0001.967.41 18.500.9498.37 5,0004.994.55 179.653.6099.89
7,5006.934.19 488.777.0592.46
a : two replicates per batch ; rsd : relative standard
deviation ; sd : standard deviation .
a :
two replicates per batch ; rsd : relative standard
deviation ; sd : standard deviation .
table iii describes the calibration curve of inh
with precision and accuracy according to regulatory agency requirements [ 20% accuracy
for the lower limit of quantification ( llq ) ( 100 ng / ml ) and 15% for the other
concentration values ] ( anvisa 2003b ) .
the llq of 100 ng / ml was established as a function
of signal to noise ratio greater than 10 and quantifiable analytical signal with
acceptable precision and accuracy .
intra - day , inter - day precisions and accuracies were within the pre - defined acceptance
limits ( table iv ) .
the precision and accuracy
were according to the acceptable criteria recommended by the anvisa .
the precision at
each concentration level did not exceed a cv of 15% and its accuracy was within the
range established ( i.e. , 85 - 115% ) for each concentration level tested .
the mean
extraction recoveries of inh ( n = 8) were 80.35% , 88.59% , 94.58% at concentrations of
200 , 2,000 and 5,000 ng / ml , respectively ( table
iv ) .
table ivprecision , accuracy and recovery data for isoniazid ( inh ) quantification by
high - performance liquid chromatography - tandem mass spectrometryvalidation parametersinh quality control
concentration ( ng / ml )
1002002,0005,000precision ( rsd ) ( % ) intra - run ( n = 8)8.1511.687.635.78 inter - run ( n = 24)10.227.205.758.65accuracy ( % ) intra - run ( n = 8)102.06102.1399.0095.20 inter - run ( n = 24)99.15104.49105.99101.81recovery ( % ) ( n = 8)-80.3588.5994.58rsd : relative standard deviation .
the plasma stability tests were reproduced under the conditions of analysis in which the
subject samples were studied .
the results presented in table v were obtained comparing freshly prepared samples with those
undergoing short - term stability tests ( 6 h , room temperature ) , post - preparative
stability ( 48 h , + 7c ) , freeze - thaw stability ( 4 cycles - 96 h , -20c ) and long - term
stability ( 63 days , -20c ) .
table vstability of human plasma samples of isoniazid under various storage
conditions ( n = 8)stabilitymeasured
concentration ( ng / ml)fresh samples ( ng / ml)rsd ( % ) stability samples ( ng / ml)rsd ( % ) assay values ( % ) post - preparative stability
( 48 h , + 7c)200190.368.13200.77
10.04 - 5.47 5,0004.772.496.664.941.09
3.663.53long - term stability ( 63 days , -20c)200199.4211.13191.09
8.57 - 4.18 5,0004.807.863.774.556.39
2.66 - 5.23freeze - thaw stability ( 4 cycles/96 h ,
-20c)200196.604.68204.92
5.534.23 5,0004.670.175.024.766.18
3.143.53short - term stability ( 6 h , room
temperature)200199.204.11198.76
4.17 - 0.22 5,0004.913.084.095.198.38
2.705.81
a : mean of eight replicates ; b : after 48
h ; c : after 1,512 h ; d : after 96 h ;
e : after 6 h ; rsd : relative standard deviation .
a : mean of eight replicates ; b : after 48
h ; c : after 1,512 h ; d : after 96 h ;
e : after 6 h ; rsd : relative standard deviation .
bioequivalence analysis - twenty - eight healthy subjects of both genders
between 21 - 50 years of age ( 33 and sd 8.42 ) , weight 54.3 - 84 kg ( 65.88 and sd 8.29 )
and bmi 19.79 kg / m and 28.04 kg / m ( 24.09 and sd 2.08 ) were
enrolled and dosed in the study
. all of the subjects completed both periods of the
study . the mean plasma concentration - time profile for the test and reference product of inh is
shown in fig .
the mean pharmacokinetic
parameters of inh are provided in tables i , ii for the test and reference product ,
respectively .
fig . 5 : mean plasma concentration - time profile for test and reference product of
isoniazid ( inh ) .
the average bioequivalence was evaluated based on the 90% ci for the intra - individual
mean ratio of log - transformed cmax and auc0-t of the test and
reference formulation .
the 90% ci interval was planned in the protocol and was
recommended by the regulatory agencies ( anvisa 2003a , fda 2006 ) .
the 90% ci for the inh log transformed cmax and
auc0-t was 89.61 , 115.92 and 94.82 - 119.44 , respectively .
hence , all of the
pharmacokinetic parameters were within the accepted bioequivalence range 80 - 125% for
cmax and auc0-t of inh .
the most frequent aes were anaemia or low haemoglobin levels ( 9 cases ) and headache
( 8 cases ) .
the distribution of aes was similar between the reference and test groups , as follows :
12 aes were reported after the administration of the reference formulation and 13 were
reported after the test dosing formulation .
causality was defined as probable in 14 cases , possible in six cases and
unrelated in five cases .
this manuscript addresses a new treatment approach for latent tb that is highly relevant
for clinical practice in brazil . because there have been no radical innovations for tb
treatment for a long time , incremental innovations are important to increase patients
quality of life .
public health policies that reduce pill burden have been recommended to
simplify treatments ( blomberg et al . 2001 )
increase compliance and even prevent the emergence of resistance ( mitchison 1998 ) .
a well - known approach is the use of fixed dose
combinations ( blomberg et al .
because the
new formulation will reduce pill burden compared to the old formulation , it is expected
to improve care by simplifying treatment .
the main limitation of our study is that neither adherence nor multiple doses safety
profile was evaluated .
however , an increased risk of aes is not expected because the
daily and total dose of treatment remains the same .
the bioavailability results confirm
that exposure to the drug using the old formulation and the new formulation is
equivalent . for this bioequivalence study , a sensitive , simple and rapid high - performance liquid
chromatography - tandem mass spectrometry method to determine inh levels in human plasma
was developed and validated .
the described method showed good specificity , precision ,
accuracy and linearity over the range of 100 - 7,500 mg / ml for inh .
the established
llq of 100 ng / ml for inh was adequate to determine concentrations in human plasma during
the pharmacokinetic study .
the stability tests demonstrated that the inh analyte showed no signs of degradation
when present in the biological matrix under plasma conditions of storage and analysis in
short and long term .
the developed method using tandem mass spectrometry demonstrated
the advantage of a short run time and high selectivity for application to high
throughput analysis .
contrary to usual practice , this analytical method is thoroughly
reported in this paper .
researchers will benefit from this simple method , which can be
used to explore the pharmacokinetic aspects of inh in future research studies , clinical
practice and therapeutic drug monitoring . by applying this method , the new formulation of inh 300 mg has been shown to be
bioequivalent to three tablets of the 100 mg reference product . this new product in
brazil may increase adherence to treatment for latent tb . in 2013 , the pnct made the
standard dose formulation recommended worldwide available to brazilians . while the new
formulation is made available in the country , the medical community needs to be aware of
the new treatment strategy adopted by the pnct , namely a single tablet of 300 mg inh
daily for six months .
increasing medical awareness is important to avoid medication
errors when prescribing inh because the wrong use of the new formulation could lead to a
higher frequency of aes .
this bioequivalence study reports important data on the pharmacokinetics of inh to
consider including inh as a class i drug in the biopharmaceutical classification system
( bcs ) .
the bcs may also lower the costs of developing generic products and improve the
population s access to these products .
inh has been regarded as a product that can be
considered as a biowaiver , i.e. , a product can be approved in a regulatory agency based
on in vitro dissolution tests rather than requiring bioequivalence studies in human
subjects ( lindenberg et al .
however ,
regulatory agencies have been refusing this approach because standard permeability tests
have not been defined .
the historic series of bioequivalence studies have been the
actual gold standard to define the permeability of a product . in 2011 ,
the brazilian
regulatory agency published a list of biowaivers that included inh ( anvisa 2011 ) .
this study may provide additional
support for this decision that can lower the costs of future inh formulation
developments .
these results could also support the acceptance of inh as a biowaiver by
regulatory agencies worldwide .
this bioavailability study demonstrates that the new formulation of inh 300 mg and three
100 mg tablets of the reference formulation are interchangeable .
the study led to the
registration of the new product by the brazilian regulatory agency . by increasing
patient adherence to the treatment
, it is expected that the new formulation will improve
patient care for latent tb .
the analytical method reported provides a sensitive , simple and rapid way of measuring
inh in human plasma that will help future research on inh worldwide . | the recommended treatment for latent tuberculosis ( tb ) infection in adults is a daily
dose of isoniazid ( inh ) 300 mg for six months . in brazil ,
inh was formulated as 100
mg tablets .
the treatment duration and the high pill burden compromised patient
adherence to the treatment .
the brazilian national programme for tuberculosis
requested a new 300 mg inh formulation .
the aim of our study was to compare the
bioavailability of the new inh 300 mg formulation and three 100 mg tablets of the
reference formulation .
we conducted a randomised , single dose , open label , two - phase
crossover bioequivalence study in 28 healthy human volunteers .
the 90% confidence
interval for the inh maximum concentration of drug observed in plasma and area under
the plasma concentration vs. time curve from time zero to the last measurable
concentration time t was 89.61 - 115.92 and 94.82 - 119.44 , respectively .
the main
limitation of our study was that neither adherence nor the safety profile of multiple
doses was evaluated .
to determine the level of inh in human plasma , we developed and
validated a sensitive , simple and rapid high - performance liquid chromatography - tandem
mass spectrometry method .
our results showed that the new formulation was
bioequivalent to the 100 mg reference product .
this finding supports the use of a
single 300 mg tablet daily strategy to treat latent tb . this new formulation may
increase patients adherence to the treatment and quality of life . |
colorectal cancer ( crc ) is the third most commonly diagnosed cancer in males and the second in females ( 10% and 9.2% of the total cancer incidence , respectively ) , according to globocan worldwide estimates of cancer incidence for 2012 .
there is wide geographical variation in the incidence across the world , and nearly 55% of cases occur in more developed regions.1 the crc incidence trends in high - income countries have varied over the past 20 years2 ; for instance , rates have recently tended to decline in the united states , whereas high rates have been reported in newly developed countries around the globe , where the risk was once low.3 south korea is one example of a country reporting crc as the cancer with the second highest incidence in both sexes , excluding thyroid cancer overdiagnosed in korea.4 the rates increased continuously from 1999 to 20125 in the korean population and nearly caught up to those in western countries.6 this trend in asian countries may reflect increased risk factors for crc , including obesity , irregular and inadequate dietary habits adapting westernized lifestyle as well as various environmental factors that may potentially affect people physically or psychosocially.2,7 as the world cancer research fund and the american institute for cancer research ( wcrf / aicr ) have reported,8,9 lifestyle factors that very likely confer an increased risk of crc are physical inactivity , obesity , alcohol consumption , and certain dietary habits or patterns . among the diverse crc risk factors ,
a combination of extrinsic and intrinsic factors may cause differences between individuals ; extrinsic factors including a diet containing certain nutrients and food compositions , and intrinsic for having different genetic background , which may alter gene expression or may function in carcinogenesis.10,11 in this review , we will discuss the dietary factors vitamin d and calcium , which are linked to each other , rather than reviewing factors well known to reduce crc risk . in the 2012 wcrf / aicr continuous update project report on food , nutrition , physical activity , and the prevention of cancer : a global perspective ,
the evidence for an effect of vitamin d on decreasing crc risk was classified as limited - suggestive, and calcium was classified as having a probable effect on decreasing crc risk.9 over the course of actively conducting studies to prove that these two dietary factors have real effects , vitamin d has been reported to be a protective agent that induces pro - differentiation , anti - proliferative , and growth - inhibitory effects on carcinogenesis12,13 in association with a decrease in crc risk .
certain previous meta - analyses demonstrated an inverse association between calcium intake and crc risk.14 in addition to dietary factors , genetic changes , known as mutations , will also be discussed , including a review of diet - associated single nucleotide polymorphisms ( snps ) .
the above - described risk factors association with crc and the interaction between dietary and genetic factors will be summarized by reviewing relevant studies .
an article search was conducted using the electronic database pubmed to identify studies published between january 2000 and march 2015 .
the search keywords were combinations of following terms : vitamin d , calcium , crc , and polymorphism .
we selected eligible studies based on several inclusion criteria , as follows : ( 1 ) epidemiological studies including cases and controls as well as cohort studies ; ( 2 ) studies investigating the association between dietary factors / genetic variants and crc including adenoma or polyps ; ( 3 ) studies assessing the gene - diet interaction effect on crc ; and ( 4 ) studies using an or or relative ratio and a 95% ci to estimate crc risk .
we assessed the relevance of the studies using a hierarchical approach based on the title , abstract , and full - text article . a flow chart depicting the literature search and the selection of 21 eligible studies
we excluded experimental studies that aimed to examine the association with colorectal neoplasia risk using non - epidemiological approaches ( n = 5 ) , as well as laboratory studies conducted with animals or cell lines ( n = 273 ) .
the articles comprising a meta - analysis ( n = 3 ) or a systematic review ( n = 8) also were not included in the current study .
we excluded certain studies consisting of randomized clinical trials or survival analyses of risk association between cancer cases and healthy controls ( n = 10 ) .
four articles were additionally included by using the same keywords and checking for eligible references to other articles .
we assessed the study objectives and risk of bias of each selected article by using the national institute for clinical excellence methodology checklist for case - control study.15 the contents of quality assessment included selection of participants , assessment , confounding factors , and statistical analysis .
we summarized previous studies based on dietary factors , vitamin d and calcium and collected relevant findings associated with crc risk .
for each eligible study , we extracted the publication year , the study design , the geographical location , the number of study subjects ( cases and controls ) , any diet of interest and related genetic polymorphism , and gene - diet interactions associated with a modified crc risk .
we identified a total of 21 case - control studies that investigated the association between crc risk and dietary or genetic factors modified by gene - diet interactions .
the majority of the studies were conducted in western countries ( n = 16 ) , such as the united states and european countries , whereas a few studies were performed in non - western countries ( n = 5 , mostly japan ) .
all of the studies were designed as case - control studies , including 5 nested case - control studies .
according to the study subject recruitment , the studies were population based ( n = 12 ) , clinic based ( n = 8) , or family based ( n = 1 ) .
the studies examined dietary and genetic factors in association with crc risk and also observed gene - diet interactions , mainly focusing on vitamin d and calcium , which share a biological pathway .
we grouped the studies by each dietary factor and summarize the findings from the studies in table 11628 and 2.2936 additional study data that are not mentioned in the tables will be discussed separately .
both vitamin d and calcium were determined to be protective agents against crc in many quantitative studies .
several studies showed data with a significant or to represent cancer risk and with a significant p - value for the comparison of dietary intake between cases and controls .
generally , it is known that high levels of vitamin d or calcium intake are associated a decreased risk of developing crc .
atoum and tchoporyan16 found that 75% of crc patients were vitamin deficient , with a significant decrease in serum 25-hydroxy - vitamin d ( p < 0.05 ) .
the same study also reported that vitamin d deficiency was associated with an approximately 19-fold higher risk among jordanian patients compared with controls ( p 0.05 ) .
similar to the dietary calcium - related studies listed in table 2 , another study by gong et al.17 found that patients with sporadic colorectal adenomas had significantly lower calcium intake compared with the control group , with a p - value of 0.02 .
takeshige et al.18 found that calcium intake was associated with a decreased risk of colon cancer ( p = 0.06 ) and rectal cancer ( p = 0.10 ) ( the corresponding data from gong et al.17 and takeshige et al.18 are not shown in the tables ) .
the effect of genetic factors was determined for several representative gene polymorphisms related to vitamin d , calcium , or both , as follows : vitamin d receptor ( vdr ) ,
vitamin d 24-hydroxylase ( cyp24a1 ) , vitamin d 25-hydroxylase ( cyp2r1 ) , calcium sensing receptor ( casr ) , transient receptor potential cation channel ( trpm7 ) , and cyclin d1 protein ( ccnd1 ) polymorphisms.37,38 the vdr gene polymorphisms were diversely studied with regard to crc development . among the reviewed studies , many examined tru9i , bsmi , apai , taqi , foki , and group - specific component / vitamin d - binding protein ( gc ) .
several , such as tru9i and bsmi , were found to be reliably associated with a decreased risk , whereas for others , such as taqi and gc , there was no significant a relationship between the gene polymorphism and crc risk .
based on the allele frequency in genotypes , the frequency of certain alleles distinctly increased cancer risk .
the difference between the vdr foki ff and ff genotypes was shown in a study by wong et al.29 boyapati et al.19 reported that the calcium - colorectal adenoma association varied with the vdr bsmi genotype , indicating that individuals with at least one b allele were at much lower risk , with or = 0.25 ( 0.080.80 ) , p trend = 0.02 ( these data are not shown in the tables )
. however , there have been different results regarding the allelic expression of the same gene ( vdr apai ) in different populations .
takeshige et al.18 determined that vdr apai aa / aa was associated with a decreased risk of rectal cancer reporting or = 0.75 ( 0.560.99 ) in a japanese population , whereas kupfer et al.20 demonstrated that the vdr apai t allele was associated with an increased crc risk reporting or = 1.15 ( 1.001.33 ) among the population of african americans .
two studies listed in table 2 , or those by dong et al.30 and peters et al.31 ( 2004 ) , reported significant associations in analyses by cancer anatomic site and diplotype , in which both studies examined the casr gene polymorphism by stratified analyses due to statistical insignificance . for proximal colon , casr ivs3 + 1048tt was associated with an increased risk with or = 1.35 ( 1.011.18 ) and casr ivs6 + 16cc with a decreased reporting or = 0.43 ( 0.190.97).30 by the diplotype analysis conducted with casr , a decreased risk was observed with or = 0.56 ( 0.360.88).31 in combination with dietary and genetic factors , genetic polymorphism can influence the dietary absorption level , which can affect the risk of cancer .
atoum and tchoporyan16 found that the absorption of vitamin d among crc patients can be affected by the t allele of vdr taqi . compared with the healthy control group ,
the case group had a lower level of vitamin d in a comparison based on the frequency of the t allele .
vitamin d absorption levels in tt- and tt - genotyped cases were significantly lower than those in controls ( table 1 ) .
in contrast , the dietary intake level can modify the effect of genetic polymorphism on the risk of cancer development .
for instance , takeshige et al.18 indicated that vdr foki did not have a significant association with cancer risk but seemed to be associated with a decreased risk of colon cancer , especially among individuals with high intake of vitamin d. in the same study , it was also found that vdr apai aa / aa showed a decreased rectal cancer risk , with or = 0.75 ( 0.560.99 ) , p = 0.04 . among the cases with the same genotype and a high calcium intake , a higher degree of reduction
was detected , with or = 0.53 ( 0.360.79 ) , p = 0.05 ( these data are not shown in the tables ) . in a cohort of singaporean chinese ,
wong et al.29 observed the effect of vdr foki only in the lower dietary calcium category , and this effect depended on the f allele in a gene dose - dependent manner ( these data are shown in table 2 ) .
this review summarizes the previous epidemiological studies that have investigated the effects of dietary factors and genetic polymorphisms on crc risk . to understand the variety of potential factors that may influence crc risk ,
the gene - diet interactions among diverse study populations have been examined according to stratification by dietary intake or allele frequency in gene expression .
dietary factors are commonly recognized as modifiable factors that can have a profound influence on cancer and tumor behavior.8 therefore , the relationship between dietary factors and crc risk has long attracted a great deal of attention .
the effects of vitamin d and calcium on crc risk and its incidence have been extensively studied.9 mounting evidence supports an inverse association of vitamin d with colorectal adenoma or cancer incidence,3942 and a long - term inadequate vitamin d status might lead to a progressive increase in crc incidence in humans.10 high intake of calcium has also consistently been reported to lower the risk of crc.14,4345 as protective nutrients against crc , vitamin d and calcium are closely related to each other due to the role of vitamin d in maintaining calcium levels .
calcium homeostasis is critical for the inhibition of cancerous tumor development , and the active metabolites of vitamin d , 1,25(oh)2d3 play a mediating role in intestinal calcium absorption to prevent homeostatic disruption.19,30 in addition to preventing carcinogenesis in the colon and rectum , vitamin d and calcium are diversely linked to biological responses , including dna synthesis and inhibition of double - strand breaks caused by endogenous or exogenous factors.10,19 the association between vitamin d and calcium is also well known to account for the physiologic functions and regulation of genes responsible for cell proliferation , differentiation , angiogenesis , and apoptosis.4648 nutrient metabolism - associated gene variants have been studied to investigate their implication in crc.4952 various genes , such as casr , cyp2r1 , cyp27a1 , cyp27b1 , cyp24a1 , vdr , and gc , have been identified to be activated by different forms of vitamin d metabolites.21,53 certain genes , such as vdr and casr , can mediate the interaction between dietary factors and can possibly modify physiologic pathways .
vitamin d exerts its effects on calcium metabolism through binding the vdr gene , which regulates the transcription of genes involved in calcium absorption46 and which is involved in the wnt/ catenin signaling pathway , a central pathway in crc development.11,32,53 additionally , the promoter region of the casr gene contains a vitamin d response element that may also modify the calcium uptake level.50,54 determination of crc risk is further complicated by interaction with genetic factors in conjunction with environmental stimuli , such as dietary intake .
previous studies have demonstrated that gene - diet interactions may influence cancer risk . dietary intake and the polymorphic genotype of different genes
interact with each other , modulating crc risk.12,16,18,19,29,55,56 the interaction between diet and genes may contribute to modulating the risk of crc or adenoma .
guerreiro et al.32 reported that high calcium intake was more markedly associated with lowering crc risk in those carrying the polymorphic allele ( dv / vv ) of the apc gene ( adenomatous polyposis coli ; tumor suppressor gene ) than in those without that allele in the portuguese population .
kupfer et al.20 reported a significant result in african americans , suggesting that there was a significant vdr gene - vitamin d interaction in lowering crc risk . in both studies
, dietary intake seemed to modulate the associations that were originally not present in the gene - crc and diet - crc comparisons .
the frequency of certain alleles that differentiate genetic expression can alter the absorption level of nutrients in subjects .
atoum and tchoporyan16 conducted a study in jordanians and reported an inverse association with crc risk in a gene dose - dependent manner .
nutrient absorption may be inhibited based on the frequency of a certain type of allele in both crc patients and controls . in this study ,
the expression of more t alleles resulted in a lower level of vitamin d absorption , which was linked to a higher crc risk .
the findings suggest that the type of allele has its own effects ; therefore , the allele frequency in a gene may modulate the ultimate results , with additive effects of dietary intake .
boyapati et al.19 reported that those with at least one b allele , representing the restriction site , appeared to have a reduced risk of colorectal adenoma under the condition of high vitamin d intake .
although many previous epidemiological studies have investigated the association with susceptibility to crc , the results show wide ethnic variation by location or population.49,52,53,5759 most of the studies discussed above were conducted in different populations with diverse ethnic and environmental backgrounds .
analyses of the effects of the same polymorphisms on crc association yielded unreliable results , occasionally providing inverse results between different study populations .
this may imply that in investigations of crc , all people do not share the same background . to achieve a better understanding of crc in specified populations , environmental differences and genetic variations that exist within groups of individuals must be considered .
this current study represented the lack of studies related diet - gene interaction in asian countries , compared to western countries .
this suggests the necessity of further researches on asian populations that may different from western countries and their culture . in conclusion
, this review demonstrates that dietary consumption of vitamin d and calcium may decrease the risk of crc or adenoma .
the findings suggest that gene - diet interactions may possibly alter the associations among dietary intake , genetic polymorphisms , and crc risk
. however , the observations regarding effect modification are still controversial , which may be partly due to different exposures for each individual or population .
further studies conducted with different target populations are actively needed to investigate the implications of gene - diet interactions for crc .
identification of modifiable risk factors in various locations may help to effectively and broadly reduce the disease burden from an epidemiological or public health point of view .
dietary factors are commonly recognized as modifiable factors that can have a profound influence on cancer and tumor behavior.8 therefore , the relationship between dietary factors and crc risk has long attracted a great deal of attention .
the effects of vitamin d and calcium on crc risk and its incidence have been extensively studied.9 mounting evidence supports an inverse association of vitamin d with colorectal adenoma or cancer incidence,3942 and a long - term inadequate vitamin d status might lead to a progressive increase in crc incidence in humans.10 high intake of calcium has also consistently been reported to lower the risk of crc.14,4345 as protective nutrients against crc , vitamin d and calcium are closely related to each other due to the role of vitamin d in maintaining calcium levels .
calcium homeostasis is critical for the inhibition of cancerous tumor development , and the active metabolites of vitamin d , 1,25(oh)2d3 play a mediating role in intestinal calcium absorption to prevent homeostatic disruption.19,30 in addition to preventing carcinogenesis in the colon and rectum , vitamin d and calcium are diversely linked to biological responses , including dna synthesis and inhibition of double - strand breaks caused by endogenous or exogenous factors.10,19 the association between vitamin d and calcium is also well known to account for the physiologic functions and regulation of genes responsible for cell proliferation , differentiation , angiogenesis , and apoptosis.4648 nutrient metabolism - associated gene variants have been studied to investigate their implication in crc.4952 various genes , such as casr , cyp2r1 , cyp27a1 , cyp27b1 , cyp24a1 , vdr , and gc , have been identified to be activated by different forms of vitamin d metabolites.21,53 certain genes , such as vdr and casr , can mediate the interaction between dietary factors and can possibly modify physiologic pathways .
vitamin d exerts its effects on calcium metabolism through binding the vdr gene , which regulates the transcription of genes involved in calcium absorption46 and which is involved in the wnt/ catenin signaling pathway , a central pathway in crc development.11,32,53 additionally , the promoter region of the casr gene contains a vitamin d response element that may also modify the calcium uptake level.50,54
determination of crc risk is further complicated by interaction with genetic factors in conjunction with environmental stimuli , such as dietary intake .
previous studies have demonstrated that gene - diet interactions may influence cancer risk . dietary intake and the polymorphic genotype of different genes
interact with each other , modulating crc risk.12,16,18,19,29,55,56 the interaction between diet and genes may contribute to modulating the risk of crc or adenoma .
guerreiro et al.32 reported that high calcium intake was more markedly associated with lowering crc risk in those carrying the polymorphic allele ( dv / vv ) of the apc gene ( adenomatous polyposis coli ; tumor suppressor gene ) than in those without that allele in the portuguese population .
kupfer et al.20 reported a significant result in african americans , suggesting that there was a significant vdr gene - vitamin d interaction in lowering crc risk . in both studies
, dietary intake seemed to modulate the associations that were originally not present in the gene - crc and diet - crc comparisons .
the frequency of certain alleles that differentiate genetic expression can alter the absorption level of nutrients in subjects .
atoum and tchoporyan16 conducted a study in jordanians and reported an inverse association with crc risk in a gene dose - dependent manner .
nutrient absorption may be inhibited based on the frequency of a certain type of allele in both crc patients and controls . in this study ,
the expression of more t alleles resulted in a lower level of vitamin d absorption , which was linked to a higher crc risk .
the findings suggest that the type of allele has its own effects ; therefore , the allele frequency in a gene may modulate the ultimate results , with additive effects of dietary intake .
boyapati et al.19 reported that those with at least one b allele , representing the restriction site , appeared to have a reduced risk of colorectal adenoma under the condition of high vitamin d intake .
although many previous epidemiological studies have investigated the association with susceptibility to crc , the results show wide ethnic variation by location or population.49,52,53,5759 most of the studies discussed above were conducted in different populations with diverse ethnic and environmental backgrounds .
analyses of the effects of the same polymorphisms on crc association yielded unreliable results , occasionally providing inverse results between different study populations .
this may imply that in investigations of crc , all people do not share the same background . to achieve a better understanding of crc in specified populations , environmental differences and genetic variations that exist within groups of individuals must be considered .
this current study represented the lack of studies related diet - gene interaction in asian countries , compared to western countries .
this suggests the necessity of further researches on asian populations that may different from western countries and their culture . in conclusion
, this review demonstrates that dietary consumption of vitamin d and calcium may decrease the risk of crc or adenoma .
the findings suggest that gene - diet interactions may possibly alter the associations among dietary intake , genetic polymorphisms , and crc risk . however , the observations regarding effect modification are still controversial , which may be partly due to different exposures for each individual or population .
further studies conducted with different target populations are actively needed to investigate the implications of gene - diet interactions for crc .
identification of modifiable risk factors in various locations may help to effectively and broadly reduce the disease burden from an epidemiological or public health point of view . | the incidence trends of colorectal cancer have varied over time , and there is wide geographical variation across the world . regarding colorectal cancer , diverse modifiable environmental or intrinsic risk factors have been investigated .
this review summarizes the effects of both dietary intake of vitamin d and calcium in particular and diet - associated genetic factors on colorectal cancer risk .
we searched the electronic database pubmed for articles published between january 2000 and march 2015 .
we reviewed case - control studies that included dietary factors , genetic polymorphisms , and gene - diet interactions in association with colorectal cancer risk .
overall , 21 studies were selected as eligible studies .
these studies demonstrated that dietary consumption of vitamin d and calcium may decrease the risk of colorectal cancer or adenoma .
colorectal carcinogenesis was discussed in conjunction with dietary factors and mediating genetic factors .
the epidemiological findings suggested that the gene - diet interactions may possibly alter the associations between dietary intake , genetic polymorphisms , and the risk of colorectal cancer .
however , the reported effects of the same potential factors on colorectal cancer risk were inconsistent , depending on the study population and geographical location .
this finding may imply the necessity of considering the environmental differences and genetic variations existing between individuals or specified populations .
therefore , further studies are required to investigate modifiable risk factors in diverse locations to derive useful implications for colorectal neoplasia . |
monolayers of transition
metal dichalcogenides are interesting
materials for optoelectronic devices due to their direct electronic
band gaps in the visible spectral range . here , we grow single layers
of mos2 on au(111 ) and find that nanometer - sized patches
exhibit an electronic structure similar to their freestanding analogue .
we ascribe the electronic decoupling from the au substrate to the
incorporation of vacancy islands underneath the intact mos2 layer .
excitation of the patches by electrons from the tip of a
scanning tunneling microscope leads to luminescence of the mos2 junction and reflects the one - electron band structure of
the quasi - freestanding layer . | |
nearly two decades ago , brand - miller and colagiuri published the carnivore connection hypothesis , proposing that dietary carbohydrate , both quantity and quality , played a critical role in the natural history of type 2 diabetes .
we proposed that low glucose intake associated with a low - carbohydrate , high - protein carnivorous diet during the ice ages which dominated the last two million years of human evolution led to insulin resistance becoming a survival and reproductive advantage .
when food energy was abundant , but dietary carbohydrate scarce , those with greater inherent insulin resistance were able to redirect glucose from maternal use to fetal metabolism , increasing birth weight and survival of offspring .
in certain groups of people , other factors such as geographic isolation or starvation may have contributed further to positive selection for insulin resistance genes .
we also hypothesized that the selective pressure for insulin resistance was relaxed with the advent of agriculture and increased amounts of carbohydrate in the diet .
domestication of cereals first began around 12,000 years ago in the middle east and spread throughout europe before it was developed elsewhere .
populations that have only recently adopted agriculture are therefore likely to have a higher prevalence of genes for insulin resistance compared with those exposed for longer timeframes . in susceptible populations , lifestyle changes , including higher carbohydrate intake , weight gain , and sedentary habits , worsen insulin resistance and expose the genetic predisposition to type 2 diabetes .
westernization is associated with a high - carbohydrate , high - glycemic index ( gi ) diet , which produces sustained postprandial hyperinsulinemia , high insulin secretory capacity , and potentially -cell dysfunction .
the highest prevalence of type 2 diabetes is seen in recent hunter - gatherer populations who have rapidly westernized , including pima indians , nauruans , and australian aboriginals [ 24 ] .
europeans and their descendants appear to be the only group with a relatively low predisposition to type 2 diabetes , even in the midst of the current epidemic of obesity . since the publication of the original hypothesis in 1994 , there have been notable changes in the prevalence of type 2 diabetes and new findings relating to glucose metabolism in pregnancy and in response to different types of diet .
the revised carnivore connection hypothesis is shown in figure 1 and hinges on five ( four old and one new ) lines of evidence . during the last two million years of evolution
, humans were increasingly carnivorous , that is , consumed a low - carbohydrate , high - protein diet . a low - carbohydrate
, high - protein diet requires profound insulin resistance to maintain glucose homeostasis , particularly during reproduction .
genetic differences in insulin resistance and predisposition to type 2 diabetes can be explained by differences in exposure to carbohydrate during the past 12,000 years .
changes in the quality of carbohydrate can explain the higher prevalence of type 2 diabetes in susceptible populations .
habitual consumption of a high - glycemic - load diet worsens insulin resistance and contributes to the obesity and type 2 diabetes in all populations .
over 2.5 million years ago , our prehuman ancestors in africa lived in a warm , moist environment in which carbohydrate derived from fruit and berries was an important source of energy .
however , with the beginning of the pleistocene 2.5 million years ago , came at least 11 distinct glaciations , the last of which ended 12,000 years ago , with the start of the holocene .
with the first severe ice age , global temperatures fell dramatically and resulted in moist african forest becoming dry , open woodland and savannah .
the first stone tools in the fossil record coincide with the existence of homo habilis 2 million years ago , suggesting that they may have supplemented a vegetarian diet with scavenged or hunted meat .
h. erectus who lived 1.5 million years ago is known to have actively hunted and was the first species to systematically make tools and use fire . in africa and eurasia ,
hunted animals displaced gathered plant foods as the principal source of food , leading to a diet low in carbohydrate and high in protein for most of the year .
increased meat intake from wild terrestrial and marine animals would have also provided greater amounts of omega-3 fatty acids such as docosahexaenoic acid essential for brain development , facilitating the larger brain size of h. sapiens [ 9 , 10 ] .
hunting and fishing continued to dominate the way of life through subsequent ice ages in a variety of geographical environments . even during the warmer interglacials , parts of the world remained cold and humans maintained a hunting / fishing existence , for example , the historical eskimo diet of arctic canada , alaska , greenland , and russia that was almost devoid of readily absorbable carbohydrate .
wild plant foods such as berries and root vegetables would have supplied some carbohydrate especially at lower latitudes and during interglacials .
however , much of the carbohydrate was unavailable and elicited a low glycemic response in part due to a large amount of fiber .
the amount of carbohydrate may have ranged from as little as 10 g up to 125 g a day , much lower than the typical 250 to 400 g per day consumed in modern diets .
the diets of contemporary hunter - gatherers probably contained around 2035% energy from protein and 2040% of energy from carbohydrate .
reconstruction of paleolithic diets in east africa incorporating different foraging strategies generates similar values : 2530% energy from protein and 40% energy from carbohydrate .
the advent of agriculture 12,000 years ago led to increased cereal consumption and high intakes of available starch for the first time in human evolution .
agriculture began in the middle east , and , after spreading rapidly to europe , it extended gradually throughout asia and the americas .
however , some groups such as the meso - americans and pima indians adopted agriculture only recently , that is , within the last 5,000 years , while australian aboriginal people never developed agriculture .
the early settlers of the nauru and other pacific atolls consumed a diet high in fish and coconuts supplemented at various times by root crops .
certain metabolic adaptations were necessary to accommodate low carbohydrate intake because the brain and reproductive tissues had evolved a specific requirement for glucose as a source of fuel [ 18 , 19 ] .
the phenotypic expression of this adaptation is insulin resistance in the liver and peripheral tissues .
long - term consumption of a low - carbohydrate , high - protein diet increases insulin resistance , with a rise in hepatic glucose production ( mediated through an increased carbon flux through the gluconeogenic pathway ) and a decrease in peripheral glucose utilization resulting from hypoinsulinemia . in acute experiments using euglycemic hyperinsulinemic clamps ,
amino acids stimulate postprandial insulin and glucagon secretion without a commensurate change in plasma glucose . in isoenergetic diets ,
insulin sensitivity as assessed by clamp was 33% lower after 6 wk of a high - protein diet than after 6 wk of a high - carbohydrate diet .
high - protein intake was associated with a tendency to increased protein expression in adipose tissue of the translation initiation factor serine - kinase-6 - 1 , which is known to mediate amino - acid - induced insulin resistance . in a low - carbohydrate environment ,
mild insulin resistance may have increased survival , but reproduction probably required greater degrees of intrinsic insulin resistance .
glucose is the main fuel for fetal growth [ 2426 ] , and low glucose availability would compromise fetal survival .
even though increased gluconeogenesis is one of the first adaptations to pregnancy , maternal glucose levels decline in early pregnancy [ 27 , 28 ] . greater insulin resistance in the maternal tissues would increase the likelihood of higher maternal glucose concentration . in healthy pregnant women , fetal growth
is directly correlated with maternal glucose concentration , and larger infants ( with greater likelihood of survival ) are born to women with higher glucose concentrations .
insulin - sensitive animals may not be able to meet the increased demand for preformed glucose during pregnancy .
obtaining adequate glucose for fetal growth from gluconeogenesis can be difficult , especially in younger women , but may be possible if dietary protein is sufficiently high [ 25 , 26 ] .
offspring survival could also be affected due to the increased demand for glucose during lactation . in rats ,
utilization of glucose and other lipogenic precursors by the mammary gland is facilitated by muscular insulin resistance [ 30 , 31 ] .
hence , a reproductive and survival advantage appears to exist in individuals with a greater degree of insulin resistance on a low - carbohydrate diet .
this inherited insulin resistance may involve different genes / enzymes than dietary - induced insulin resistance .
insulin - sensitive subjects seem to find a low - carbohydrate , high - protein diet difficult to tolerate , with side effects such as nausea and headache [ 32 , 33 ] . on the other hand , insulin resistant subjects [ 34 , 35 ] and genetically obese animals appear to tolerate a high - protein diet very well .
studies have shown high - protein diets produce the most desirable metabolic profile in individuals with type 2 diabetes [ 3739 ] .
we propose that the selection pressure for insulin resistance was relaxed first in europeans when dietary carbohydrate increased 12,000 years ago with the advent of agriculture . in accordance with this long - term exposure
, europeans have experienced a lower prevalence of diabetes , even when overweight and obese ( see section 6 ) , compared to other population groups .
high carbohydrate intake over the past millennia may have also increased positive selection for genes associated with higher -cell mass . in swedish and finnish populations , variants in 11 genes ( tcf7l2 , pparg , fto , kcnj11 , notch2 , wfs1 , cdkal1 , igf2bp2 , slc30a8 , jazf1 , and hhex )
are significantly associated with the risk of type 2 diabetes independently of clinical risk factors .
populations that adopted agriculture more recently such as the pima indians are likely to have a higher prevalence of genes transcribing insulin resistance or impaired -cell function than those exposed for thousands of years . the carnivore connection hypothesis has been tested recently in sample populations from the asian steppes .
ten candidate genes for insulin resistance , anthropometry , and physiological measures , including homa insulin resistance , were compared in traditional herders ( pastoralists = high - protein diet ) and farmers ( agriculturalists = high - carbohydrate diet ) .
while none of the genes tested showed causal mutations with higher frequency in herders , tests of neutrality showed some genes ( slc30a8 , lepr , and kcnq1 ) could have been involved in past adaptations to diet .
consistent with the carnivore connection hypothesis , the prevalence of insulin resistance was significantly greater in herders compared to farmers , despite no major differences in current diet .
environmental pressures such as geographic isolation and/or starvation may have led to further increases in the prevalence of insulin resistance gene(s ) in certain population groups .
geographic isolation has led to genetic bottlenecks and reduced genetic diversity in the nauruans and pima indians .
both populations have also been exposed to food shortages and starvation in the recent past . as occurs with low carbohydrate intake , starvation results in the need for increased gluconeogenesis and peripheral insulin resistance .
this may have selected for those with a profound degree of insulin resistance which was inherited by future offspring .
women with polycystic ovarian syndrome are known to be exceptionally insulin resistant and may represent a group that was highly fertile in a low - carbohydrate environment . insulin resistance has previously been proposed to be the mechanism for coping with variable food intake during evolution [ 46 , 47 ] .
( postprandial hyperinsulinemia ) as a mechanism to increase fat stores during food abundance and available during food scarcity .
an alternative hypothesis by reaven suggests that muscle insulin resistance was the key to survival during food scarcity because it conserved glucose by minimizing gluconeogenesis and preserving lean body mass .
both these hypotheses are based on the assumption that there were challenging periods of food scarcity prior to the advent of agriculture . however , this is not supported by the scientific literature . while hunter gatherers would have been exposed to seasonal and geographical changes in food supply , severe food shortages or starvation were rare and more likely to occur after the transition to agriculture ( preindustrialization ) .
specific mechanisms for coping with low carbohydrate intake , rather than total dietary energy , probably afforded the greatest reproductive and survival advantages .
genome - wide scans and other research have been directed towards discovering the gene(s ) associated with insulin resistance and type 2 diabetes .
however , the complexity of human metabolism means that there are likely to be multiple gene systems involved , including those that influence insulin sensitivity and others that influence -cell function .
pc-1 ( plasma cell membrane glycoprotein-1 ) , which interferes with insulin receptor tyrosine kinase activity , thereby inhibiting subsequent cellular signalling , has been associated with insulin resistance .
reduced expression of the susceptibility gene capn10 ( calpain-10 ) has been linked to decreased glucose uptake in skeletal muscle .
recently , the acad10 ( acyl coenzyme a dehydrogenase 10 ) gene has been associated with fatty - acid - induced insulin resistance as it may catalyze mitochondrial fatty - acid oxidation .
dietary carbohydrate quality changed markedly with the industrial revolution in the 17th century . prior to this , cereals were typically eaten whole or coarsely ground or flaked , with large particles of intact fiber and starchy endosperm that was less easily gelatinized during cooking . as a result , cereal carbohydrates were slowly digested and absorbed , eliciting small postprandial glucose and insulin responses [ 57 , 58 ] .
irrespective of gi , the high intake of insoluble cereal fiber likely reduced the burden on the -cell by enhancing insulin sensitivity , perhaps via acceleration of gip ( glucose - dependent insulinotropic polypeptide ) [ 59 , 60 ] .
thus , the -cells probably coped well on the postagriculture high - carbohydrate low - gi diet .
most traditional hunter - gatherer carbohydrate foods have been shown to be low in gi [ 61 , 62 ] .
while traditional hunter - gather diets did not provide cereal fiber , they would have contained soluble and insoluble fiber derived from fruits and vegetables , which acts to slow glucose absorption and enhance glycemic control .
the industrial revolution marked the era of the high - gi , high - glycemic load diet .
new high - speed steel roller mills allowed cereal grains to be more finely ground and the fiber separated and removed .
the degree of gelatinization during cooking increased and thus the rate of carbohydrate digestion and absorption , producing greater postprandial increases in glycemia and insulinemia [ 58 , 65 ] .
high - gi varieties of potatoes were introduced to western diets at this time , contributing to a higher dietary glycemic load .
the modern high - carbohydrate , high - gi diet therefore elicits more insulin secretion , that is , a higher insulin demand .
oxidative stress within the -cell could therefore contribute to increased apoptosis and gradual reduction in -cell mass .
eventually , in susceptible individuals with higher degrees of insulin resistance and reduced -cell mass , impaired glucose tolerance and type 2 diabetes develop [ 40 , 68 ] .
those with inborn , inherent insulin resistance face the greatest challenges when exposed to westernization [ 69 , 70 ] .
hales and barker suggest that -cell dysfunction manifests in individuals exposed in utero and in early life to suboptimal nutrition . but both high- and low - birth - weight infants are predisposed to greater insulin resistance at birth .
high - carbohydrate - weaning diets are associated with greater insulin resistance and risk of type 2 diabetes in animal models . in gestational diabetes ,
exposure in utero to hyperglycemia stimulates fetal insulin secretion and fetal growth , resulting in high birth weight and increased appetite .
the u - shaped association between birth weight and risk of type 2 diabetes has been documented in the pima indians .
consequently , both the quantity and quality of the carbohydrate in modern diets are relevant to the prevention of type 2 diabetes .
alternate dietary strategies that limit postprandial hyperglycemia , including high - protein diets , mediterranean - style diets , and low - glycemic load diets , are associated with greater weight loss and metabolic advantages in overweight individuals . in meta - analyses ,
low - gi and low - glycemic - load diets have been associated with reduced risk of developing type 2 diabetes [ 68 , 78 ] .
low - gi diets may assist weight control by improving satiety and increasing the utilization of stored fuels . in both animals
[ 80 , 81 ] and humans [ 8284 ] , low - gi diets have improved weight management .
in recent decades , overweight and obesity have risen to epidemic proportions in both developed and developing countries .
rapid increases in body weight among children and younger age groups indicate that environment and lifestyle , not just genetic predisposition , play critical roles in the etiology of chronic disease . declining physical activity and energy expenditure worsen insulin resistance and the likelihood of incremental weight gain . while the causes of the obesity epidemic are complex and multifactorial , worldwide dietary trends over the last three decades , including lower protein intake as a proportion of energy , higher consumption of refined cereals , and the substitution of carbohydrate for saturated fat , have led to substantial increases in average dietary glycemic load . in a predominantly overweight and sedentary population , refined carbohydrates may cause greater metabolic damage than saturated fat . while weight gain will occur whenever energy intake surpasses energy expenditure , diet composition has important influences on appetite , hunger / satiety , and therefore weight control .
the protein leverage hypothesis proposes that animals will overeat carbohydrate and fat in an effort to achieve a specific protein target .
overconsumption of energy on low - protein diets has been reported for insects , fish , birds , rodents , nonhuman primates , and humans , providing another example of metabolic pathways conserved by evolution . in large ,
well - designed randomized controlled trials , higher - protein diets have been associated with greater weight loss and prevention of weight regain .
diet composition also influences fuel partitioning , that is , the use of fatty acids versus glucose as the substrate for energy production . diets that increase postprandial hyperglycemia and hyperinsulinemia have been shown to promote carbohydrate oxidation at the expense of fat oxidation , an effect that may be conducive to increased adiposity [ 94 , 95 ] .
low - gi diets may therefore enhance weight control by several mechanisms , including higher - satiety , higher - insulin sensitivity , and lower postprandial hyperinsulinemia .
this hypothesis is supported by intervention studies showing that ad libitum consumption of low - gi foods promotes greater weight loss and prevention of weight regain than macronutrient matched diets based on high gi foods [ 92 , 96 , 97 ] .
long - term studies in animal models also show that high gi starch diets promote weight gain , visceral adiposity , and higher levels of lipogenic enzymes compared with isoenergetic , macronutrient - controlled low - gi starch diets [ 96 , 97 ] .
finally , in healthy pregnant women , a high - gi diet is associated with higher birth weight and infant ponderal index than a nutrient - balanced low - gi diet . in this way
, epigenetic changes brought about by differences in diet composition during pregnancy could promote acceleration of obesity from one generation to the next .
individuals that are capable of high postload insulin secretion may be the group most likely to accumulate fat on a high - glycemic - load diet and find weight loss difficult .
thus , although europeans may be less genetically susceptible to type 2 diabetes than other populations , the increasing prevalence of insulin resistance associated with obesity increases the likelihood of -cell dysfunction and type 2 diabetes . any healthy diet that reduces postprandial glycemia and insulinemia , including high - protein , lower - carbohydrate diets , mediterranean - style diets , and low - glycemic - load diets ,
may be preferable to the conventional high - carbohydrate , low - fat diet . in a large european study ,
a modest increase in protein intake and a modest reduction in gi was associated with maintenance of weight loss and greater participant satisfaction .
future research on fuel metabolism during pregnancy , gestational diabetes , and polycystic ovarian syndrome will hopefully shed light on the origins of intrinsic insulin resistance in humans .
the new field of metabolomics in which multiple plasma metabolites can be monitored in the postprandial state offers exciting opportunities .
anthropological studies in traditional hunter gatherers versus farmers and further animal studies , particularly in the pregnant state in carnivorous versus omnivorous mammals , will also provide data in support ( or not ) of the carnivore connection .
the carnivore connection argues that a scarcity of carbohydrate , rather than food energy , over the course of human evolution is intimately linked to the population risk of type 2 diabetes .
we hypothesize that low glucose intake associated with a low - carbohydrate , high - protein diet during the ice ages led to positive selection of intrinsic insulin resistance as a survival and reproductive advantage .
alternate hypotheses , such as the thrifty genotype hypothesis , postulate that total dietary energy selected for insulin resistance , but there is currently no evidence that regular periods of food scarcity occurred prior to the advent of agriculture .
only the carnivore connection hypothesis explains the relatively low susceptibility to type 2 diabetes in europeans versus other population groups .
consistent with the hypothesis , a recent trial found the prevalence of insulin resistance significantly greater in pastoralists compared to agriculturalists on the asian steppes .
further research is required to determine whether genes associated with insulin resistance and -cell function vary between recent hunter gatherers and long - standing farming populations . in the interim , protein intake and carbohydrate quality and quantity are relevant to the prevention and management of obesity and type 2 diabetes . | the carnivore connection hypothesizes that , during human evolution , a scarcity of dietary carbohydrate in diets with low plant : animal subsistence ratios led to insulin resistance providing a survival and reproductive advantage with selection of genes for insulin resistance .
the selection pressure was relaxed at the beginning of the agricultural revolution when large quantities of cereals first entered human diets .
the carnivore connection explains the high prevalence of intrinsic insulin resistance and type 2 diabetes in populations that transition rapidly from traditional diets with a low - glycemic load , to high - carbohydrate , high - glycemic index diets that characterize modern diets .
selection pressure has been relaxed longest in european populations , explaining a lower prevalence of insulin resistance and type 2 diabetes , despite recent exposure to famine and food scarcity .
increasing obesity and habitual consumption of high - glycemic - load diets worsens insulin resistance and increases the risk of type 2 diabetes in all populations . |
today , burnout syndrome is defined as a psychological syndrome of emotional exhaustion , depersonalization , and reduced personal accomplishment that can occur among people who work with the general public in some capacity .
a key aspect of burnout syndrome is an increased feeling of emotional exhaustion because as emotional resources are depleted , caregivers feel that they are no longer able to give support at a psychological level .
another aspect of burnout is the development of depersonalization / disengagement , which occurs when the helping professional develops a negative demeanor toward his or her client and/or work - related responsibilities .
the final aspect of burnout , reduced personal accomplishment , refers to the tendency of the caregivers to evaluate themselves negatively , such that they are no longer effective in working with clients and in fulfilling their job responsibilities .
maslach et al have described burnout as a syndrome of emotional exhaustion , depersonalization , and reduced personal accomplishment that can occur among individuals who work with people in some capacity .
to date , a search in specialized scientific databases such as psychlit and pubmed reveals thousands of scientific publications with burnout in the title .
based on this wealth of available research , burnout is highly prevalent in the literature of medical and nursing professions , and seems to affect other health care professionals including dentists , physical therapists , and occupational therapists . although burnout has been studied comprehensively in a number of helping professions , information on burnout within the chiropractic profession does not appear to exist .
the importance of exploring burnout in the chiropractic profession is grounded in the theoretical and empirical evidence that is available in similar health care professions .
currently , 2 theory - based models are consistently used in the literature to guide burnout studies .
the conservation of resources ( cor ) model and the job demands - resource ( jd - r ) model ( fig 1 ) .
both theories offer an explanation for the relationship between job resources , job demands , and burnout .
job resources refer to valued things such as employment , job security , job enhancement opportunities , autonomy , participation in decision making , and supervisor support .
job demands include physical and psychological demands ( ie , workload , work pace , and time pressures ) and role conflict .
both the cor model and jd - r model agree that when job demands are high and job resources are limited , there is an increased potential for burnout .
similar helping professionals within the health care system , such as dentists , physical therapists , occupational therapists , and chiropractors , may be exposed to a unique source of stressors ( increased demands and reduced resources ) because of the autonomous and isolated nature of their practices ; their strong reliance on technical skills ; their dependence on third - party reimbursements ; their constant contact with people and the connected emotional involvement with clients ; their relatively high incidence of work - related injury ; and their struggles with market competition , regulations , and managed care .
in addition , many of these practitioners may also assume the role of private business owner , which usually involves a personal financial risk and a significant dependence on third - party reimbursements .
these similar occupational characteristics appear to significantly influence the prevalence of burnout in the physical therapy , occupational therapy , and dentistry professions . as such
, it may be appropriate to assume that if these occupational characteristics contribute to burnout in the physical therapy , occupational therapy , and dentistry professions , they may also contribute to burnout in the chiropractic profession .
if this presumption holds true , then it is appropriate to suggest that the chiropractic profession may be negatively and unknowingly impacted by the consequences of burnout .
in addition to the above - mentioned shared characteristics that predispose various health professions to burnout , chiropractic physicians may also be exposed to a host of other profession - specific stressors that may further increase their risk for burnout .
therefore , the purpose of this literature review is to discuss unique profession - specific stressors and the potential impact of burnout on the chiropractic profession .
the foundation of the proposed conceptual framework was based upon a 2-part investigation . the first part involved reviewing burnout prevalence information in similar health professions . as such
, a literature search in pubmed using the terms burnout , job stress , physical therapy , occupational therapy , dentistry , and manual therapy was conducted .
the search revealed an abundance of systematic reviews , observational and exploratory studies , and experimental investigations from 1974 to 2011 .
the second part of the search involved literature exploring occupational stressors of the chiropractic profession as applied to the jd - r and cor models .
a thematic search of pubmed and psychlit , as well as the cochrane library , professional governing bodies of the chiropractic profession , trade magazines , and research conferences and symposium proceedings , was used .
the chiropractic profession has overcome a variety of struggles since its inception in the mid-1890s . from that time , numerous chiropractors were prosecuted and jailed under the pretense of being a threat to society and for practicing medicine without a license .
other hardships included attempts by the american medical association to contain and eliminate the chiropractic profession , including a wide - ranging prejudice by many within and outside of mainstream health care . since the early 1900s , the profession has made substantial advances , such as obtaining conventional health care recognition , gaining authenticity and access to insurance and managed care plans , receiving research funding by the national institutes of health , and including chiropractic physicians in the department of defense and the veterans administration health care system . yet despite these advances , the chiropractic profession in the united states continues to struggle with unique profession - specific stressors that may place its members at increased risk for burnout .
examples of these unique profession - specific stressors involve philosophical and political differences among leadership groups within the profession , professional - identity inconsistencies , poor public perception and negative media coverage , unique physical demands that result in a risk of suffering from a work - related injury , market competition from other manual therapists ( acupuncturists and massage therapists ) , student loan default rate , threats to autonomy , and various ethical and morality issues .
the essence of this exploration is to review these unique profession - specific stressors combined with the typical stressors ( close interaction with clients , lack of supervisor support , strong reliance on technical skills , profitability ) that similar helping professions are exposed to and apply them to the jd - r and cor models as a means of explaining the potential for burnout in chiropractic profession . as such , consistent with the jd - r model ,
the main job demands of practicing chiropractors include physical workload , mental and emotional demands , and role stress ( role ambiguity and role conflict ) ; and the main job resources involved in the practice of chiropractic include job autonomy and social support climate ( lack of supervisor support ) .
a source of stress within the chiropractic profession , as well as the physical therapy , occupational therapy , and dentistry professions , is a relatively high incidence of work - related injuries . for many professions ,
injury history , area of practice specialization , or work environment has been identified as potential risk of work - related injuries .
the chiropractic , physical therapy , and dentistry professions may be at increased risks for work - related injury because of their exposure to repetitive movements , hand force , static loading , and awkward postures in their work .
lorme and naqvi found that when performing treatments , doctors of chiropractic were subject to dynamic forces that increase spinal loading and could increase risk of injury .
studies investigating the musculoskeletal demands of chiropractic therapies have suggested a high prevalence of low back pain ranging from 57% to 87% .
mior and diakow report that 41% of the chiropractors interviewed felt that the postures they assumed while treating patients was the most influential agent in the development of back pain , and that 82% thought that their back pain was further aggravated by continued practice .
homack 's survey of practicing chiropractors in new york state suggests that patient handling and delivery of side - posture manipulative procedures are the activities that most frequently result in work - related injury to the practicing chiropractors .
bisiachhi and huber report differences in the prevalence of musculoskeletal injuries between male and female chiropractors in the united states . among female chiropractors ,
the low back was the most common site of injury reported , whereas male chiropractors reported the neck as the most common site of injury .
the study by holm and rose found a moderate prevalence ( 41% ) of workplace musculoskeletal injuries among doctors of chiropractic , particularly of the wrist , hand , fingers , and shoulder .
furthermore , holm and rose note that injuries occurred most commonly in the first to fifth years of practice and that side - posture manipulation seemed to cause the greatest percentage of these injuries .
the few studies that have investigated injuries associated with chiropractic practice appear to agree that using manual therapy , particularly side - posture manipulation , increases the amount of biomechanical and postural demands on the practitioner applying the therapy .
side - posture manipulation is a trademark modality in the chiropractic profession and is used for the treatment of low back pain . as a result of injury
, some chiropractors may change their treatment techniques or their frequency or duration of work , or may choose to leave the profession .
this raises the following question : what is the impact of chiropractic - specific work - related injuries on burnout of chiropractic practitioners ? despite the lack of literature on this issue , it may be reasonable to hypothesize that if work - related injuries are as substantial as the literature suggests , then sustaining a chiropractic - specific work - related injury may partially contribute to not only a physical injury but a sense of increased exhaustion , increased depersonalization , and reduced personal accomplishment . within the job demand category of the jd - r model , concepts such as role ambiguity and role conflict
role ambiguity typically refers to an individual 's lack of clarity about his / her expected behavior from a job ; whereas role conflict refers to a type of social conflict caused by an individual being forced to take on separate and incompatible roles ( ie , business owner and physician ) .
the construct of role stress and its influence on burnout within the chiropractic profession may involve the profession 's unique public perception and identity stressors . within the united states , the chiropractic profession has struggled with establishing a clear identity that is both accepted and relevant to the public .
this struggle may , in part , be due to the presence of 2 opposing philosophical and political forces within the profession .
two predominate governing bodies in the united states , the american chiropractic associate and the international chiropractic association , represent the profession at large , often resulting in contradictory scenarios with regard to lobbying , public relations , leadership , and advancement of the profession .
although the situation is more complex , the 2 opposing views are sometimes simplistically referred to as straights vs mixers .
although each side sees chiropractic potentially addressing a wide range of diseases and conditions , the
chiropractic subluxations that interfere with the flow of vital energy , whereas the mixer view supports a broader scope of treatment ( eg , a mixer will mix in other therapies in addition to manipulation , such as physiotherapeutics , diet , exercise ) and has a more musculoskeletal and evidenced - based practice focus . these philosophical differences
clash during lobbying efforts among leadership groups within the profession and , as a result , may further exacerbate the professional - identity inconsistencies and public perception uncertainties that shadow the profession .
these political conflicts resulting in inherent poor public perception and identity discrepancies stressors may have prevented the chiropractic profession from establishing its cultural authority over any specific domain of health care and , therefore , may be the source of significant stress among its practitioners .
delinquencies in creating its cultural authority may be the result of the varying public perceptions of the chiropractic profession and the chiropractic profession 's internal struggle with establishing a universal identity . within the profession
, an individual chiropractor may specialize in a number of available techniques and methods in his or her practice .
the practice of chiropractic is multifaceted and , for many , involves a vast array of healing arts and modalities such as manipulation , massage , strength training , various physical therapy modalities , traction , and nutritional consultation . these varying views and practice styles
may result in confusion for the public in that patients , medical physicians , the managed care industry , and individual chiropractic practitioners themselves have different perspectives on defining the profession .
thus , identity problems combined with poor public comprehension may threaten the future vitality of the profession .
the end result of these incongruencies makes it plausible to hypothesize that the identity and public perception discrepancies that shadow the chiropractic profession may partially contribute to a sense of increased emotional exhaustion , increased depersonalization , and reduced personal accomplishment .
increased susceptibility for burnout in health care professionals may be the result of spending their careers focusing on the needs of others .
in addition to the mental and emotional demands involved in direct patient care , doctors of chiropractic also battle issues that threaten their ability to maintain a satisfactory income ( eg , managed care , medicare regulation , scope of practice restrictions , national health care reform ) . these issues may serve as a source of mental stress and thus may have an impact on the prevalence of burnout . in an attempt to understand profitability and viability insecurities , zhang and colleauges sought to investigate chiropractic students ' knowledge and attitudes of education and financial investments .
these authors , along with coulter et al , suggest that the present - day chiropractic student invests a similar amount of time , effort , and finances to pursue a career in chiropractic as does the present - day medical student .
these investment issues raise an important line of questioning , especially in terms of return on investment and career expectations .
edwards et al suggest that differences between what medical doctors might have reasonably expected for their career and how their career actually turned out may have implications on burnout .
furthermore , edwards et al suggest that students and practitioners , in general , who invest time , effort , and finances into their education would expect to be rewarded with a profitable career
. market competition may serve as a source of mental and emotional stress within the chiropractic profession .
whereas the number of practitioners is growing and competition from other healing professions is increasing , insurance reimbursements to chiropractors seem to be decreasing .
data from the institute for alternative futures suggest that a slowing economy , along with the recent increase in health insurance premiums , has a dampening effect on the rate of growth in demand for chiropractic services .
in addition , cherkin et al suggest that patients seeking manual treatment of musculoskeletal conditions , in a form that is similar to chiropractic spinal manipulation therapy , may choose to seek services from massage therapists who outnumber chiropractors and are growing in both numbers and market share .
competition from acupuncturists , osteopaths , and medical physicians , some of whom are also demonstrating an increased interest in manual therapy , further adds pressure .
in addition , there may be a supply and demand issue regarding the number of chiropractors within the profession .
data from the us department of commerce indicate that the total reported net income for chiropractic offices and clinics rose from $ 6.56 billion in 1992 to $ 7.68 billion in 1998 , which is about 2.8% per year . because the number of practicing chiropractors has been increasing
, these figures may show that chiropractic income may be decreasing steadily , thus adding to burnout factors . as we look to the future to strengthen the profession and develop our research agenda ,
these burnout factors include those that are associated with physical workload , role stress , and mental and emotional demands .
investigations into these complex factors should include a focus on intervention as well as prevention . using the cor model and
this article provides a discussion of the topic of burnout and the chiropractic profession and was not intended to be a systematic review of the literature ; therefore , key articles related to this topic may have not been included .
terms describing psychological stress and burnout are sometimes used inconsistently in the literature , which is a potential additional limitation to this study .
the evidence available for the chiropractic - specific stressors has not been held to a high level of academic rigor .
the data within the chiropractic literature have generally suffered from sampling biases and are vulnerable to biases stemming from subjectivity and imprecision .
in addition , some experts may question the approach of comparing chiropractic to dentistry , physical therapy , and occupational therapy .
arguments over the autonomous nature of physical therapy also reduce the strength of the presented potential occupational correlations .
the chiropractic profession has overcome a variety of struggles since its inception in the mid-1890s . from that time , numerous chiropractors were prosecuted and jailed under the pretense of being a threat to society and for practicing medicine without a license .
other hardships included attempts by the american medical association to contain and eliminate the chiropractic profession , including a wide - ranging prejudice by many within and outside of mainstream health care . since the early 1900s , the profession has made substantial advances , such as obtaining conventional health care recognition , gaining authenticity and access to insurance and managed care plans , receiving research funding by the national institutes of health , and including chiropractic physicians in the department of defense and the veterans administration health care system . yet despite these advances , the chiropractic profession in the united states continues to struggle with unique profession - specific stressors that may place its members at increased risk for burnout .
examples of these unique profession - specific stressors involve philosophical and political differences among leadership groups within the profession , professional - identity inconsistencies , poor public perception and negative media coverage , unique physical demands that result in a risk of suffering from a work - related injury , market competition from other manual therapists ( acupuncturists and massage therapists ) , student loan default rate , threats to autonomy , and various ethical and morality issues .
the essence of this exploration is to review these unique profession - specific stressors combined with the typical stressors ( close interaction with clients , lack of supervisor support , strong reliance on technical skills , profitability ) that similar helping professions are exposed to and apply them to the jd - r and cor models as a means of explaining the potential for burnout in chiropractic profession . as such , consistent with the jd - r model ,
the main job demands of practicing chiropractors include physical workload , mental and emotional demands , and role stress ( role ambiguity and role conflict ) ; and the main job resources involved in the practice of chiropractic include job autonomy and social support climate ( lack of supervisor support ) .
a source of stress within the chiropractic profession , as well as the physical therapy , occupational therapy , and dentistry professions , is a relatively high incidence of work - related injuries . for many professions ,
injury history , area of practice specialization , or work environment has been identified as potential risk of work - related injuries .
the chiropractic , physical therapy , and dentistry professions may be at increased risks for work - related injury because of their exposure to repetitive movements , hand force , static loading , and awkward postures in their work .
lorme and naqvi found that when performing treatments , doctors of chiropractic were subject to dynamic forces that increase spinal loading and could increase risk of injury .
studies investigating the musculoskeletal demands of chiropractic therapies have suggested a high prevalence of low back pain ranging from 57% to 87% .
mior and diakow report that 41% of the chiropractors interviewed felt that the postures they assumed while treating patients was the most influential agent in the development of back pain , and that 82% thought that their back pain was further aggravated by continued practice .
homack 's survey of practicing chiropractors in new york state suggests that patient handling and delivery of side - posture manipulative procedures are the activities that most frequently result in work - related injury to the practicing chiropractors .
bisiachhi and huber report differences in the prevalence of musculoskeletal injuries between male and female chiropractors in the united states . among female chiropractors ,
the low back was the most common site of injury reported , whereas male chiropractors reported the neck as the most common site of injury .
the study by holm and rose found a moderate prevalence ( 41% ) of workplace musculoskeletal injuries among doctors of chiropractic , particularly of the wrist , hand , fingers , and shoulder .
furthermore , holm and rose note that injuries occurred most commonly in the first to fifth years of practice and that side - posture manipulation seemed to cause the greatest percentage of these injuries .
the few studies that have investigated injuries associated with chiropractic practice appear to agree that using manual therapy , particularly side - posture manipulation , increases the amount of biomechanical and postural demands on the practitioner applying the therapy .
side - posture manipulation is a trademark modality in the chiropractic profession and is used for the treatment of low back pain . as a result of injury
, some chiropractors may change their treatment techniques or their frequency or duration of work , or may choose to leave the profession .
this raises the following question : what is the impact of chiropractic - specific work - related injuries on burnout of chiropractic practitioners ? despite the lack of literature on this issue , it may be reasonable to hypothesize that if work - related injuries are as substantial as the literature suggests , then sustaining a chiropractic - specific work - related injury may partially contribute to not only a physical injury but a sense of increased exhaustion , increased depersonalization , and reduced personal accomplishment . within the job demand category of the jd - r model , concepts such as role ambiguity and role conflict
role ambiguity typically refers to an individual 's lack of clarity about his / her expected behavior from a job ; whereas role conflict refers to a type of social conflict caused by an individual being forced to take on separate and incompatible roles ( ie , business owner and physician ) .
the construct of role stress and its influence on burnout within the chiropractic profession may involve the profession 's unique public perception and identity stressors . within the united states , the chiropractic profession has struggled with establishing a clear identity that is both accepted and relevant to the public .
this struggle may , in part , be due to the presence of 2 opposing philosophical and political forces within the profession .
two predominate governing bodies in the united states , the american chiropractic associate and the international chiropractic association , represent the profession at large , often resulting in contradictory scenarios with regard to lobbying , public relations , leadership , and advancement of the profession .
although the situation is more complex , the 2 opposing views are sometimes simplistically referred to as straights vs mixers .
although each side sees chiropractic potentially addressing a wide range of diseases and conditions , the
chiropractic subluxations that interfere with the flow of vital energy , whereas the mixer view supports a broader scope of treatment ( eg , a mixer will mix in other therapies in addition to manipulation , such as physiotherapeutics , diet , exercise ) and has a more musculoskeletal and evidenced - based practice focus . these philosophical differences
clash during lobbying efforts among leadership groups within the profession and , as a result , may further exacerbate the professional - identity inconsistencies and public perception uncertainties that shadow the profession .
these political conflicts resulting in inherent poor public perception and identity discrepancies stressors may have prevented the chiropractic profession from establishing its cultural authority over any specific domain of health care and , therefore , may be the source of significant stress among its practitioners .
delinquencies in creating its cultural authority may be the result of the varying public perceptions of the chiropractic profession and the chiropractic profession 's internal struggle with establishing a universal identity . within the profession
, an individual chiropractor may specialize in a number of available techniques and methods in his or her practice .
the practice of chiropractic is multifaceted and , for many , involves a vast array of healing arts and modalities such as manipulation , massage , strength training , various physical therapy modalities , traction , and nutritional consultation . these varying views and practice styles
may result in confusion for the public in that patients , medical physicians , the managed care industry , and individual chiropractic practitioners themselves have different perspectives on defining the profession .
thus , identity problems combined with poor public comprehension may threaten the future vitality of the profession .
the end result of these incongruencies makes it plausible to hypothesize that the identity and public perception discrepancies that shadow the chiropractic profession may partially contribute to a sense of increased emotional exhaustion , increased depersonalization , and reduced personal accomplishment .
increased susceptibility for burnout in health care professionals may be the result of spending their careers focusing on the needs of others .
in addition to the mental and emotional demands involved in direct patient care , doctors of chiropractic also battle issues that threaten their ability to maintain a satisfactory income ( eg , managed care , medicare regulation , scope of practice restrictions , national health care reform ) . these issues may serve as a source of mental stress and thus may have an impact on the prevalence of burnout . in an attempt to understand profitability and viability insecurities , zhang and colleauges sought to investigate chiropractic students ' knowledge and attitudes of education and financial investments .
these authors , along with coulter et al , suggest that the present - day chiropractic student invests a similar amount of time , effort , and finances to pursue a career in chiropractic as does the present - day medical student .
these investment issues raise an important line of questioning , especially in terms of return on investment and career expectations .
edwards et al suggest that differences between what medical doctors might have reasonably expected for their career and how their career actually turned out may have implications on burnout .
furthermore , edwards et al suggest that students and practitioners , in general , who invest time , effort , and finances into their education would expect to be rewarded with a profitable career
. market competition may serve as a source of mental and emotional stress within the chiropractic profession .
whereas the number of practitioners is growing and competition from other healing professions is increasing , insurance reimbursements to chiropractors seem to be decreasing .
data from the institute for alternative futures suggest that a slowing economy , along with the recent increase in health insurance premiums , has a dampening effect on the rate of growth in demand for chiropractic services .
in addition , cherkin et al suggest that patients seeking manual treatment of musculoskeletal conditions , in a form that is similar to chiropractic spinal manipulation therapy , may choose to seek services from massage therapists who outnumber chiropractors and are growing in both numbers and market share .
competition from acupuncturists , osteopaths , and medical physicians , some of whom are also demonstrating an increased interest in manual therapy , further adds pressure .
in addition , there may be a supply and demand issue regarding the number of chiropractors within the profession .
data from the us department of commerce indicate that the total reported net income for chiropractic offices and clinics rose from $ 6.56 billion in 1992 to $ 7.68 billion in 1998 , which is about 2.8% per year . because the number of practicing chiropractors has been increasing
, these figures may show that chiropractic income may be decreasing steadily , thus adding to burnout factors . as we look to the future to strengthen the profession and develop our research agenda ,
these burnout factors include those that are associated with physical workload , role stress , and mental and emotional demands .
investigations into these complex factors should include a focus on intervention as well as prevention . using the cor model and
the chiropractic profession has overcome a variety of struggles since its inception in the mid-1890s . from that time , numerous chiropractors were prosecuted and jailed under the pretense of being a threat to society and for practicing medicine without a license .
other hardships included attempts by the american medical association to contain and eliminate the chiropractic profession , including a wide - ranging prejudice by many within and outside of mainstream health care . since the early 1900s , the profession has made substantial advances , such as obtaining conventional health care recognition , gaining authenticity and access to insurance and managed care plans , receiving research funding by the national institutes of health , and including chiropractic physicians in the department of defense and the veterans administration health care system . yet despite these advances , the chiropractic profession in the united states continues to struggle with unique profession - specific stressors that may place its members at increased risk for burnout .
examples of these unique profession - specific stressors involve philosophical and political differences among leadership groups within the profession , professional - identity inconsistencies , poor public perception and negative media coverage , unique physical demands that result in a risk of suffering from a work - related injury , market competition from other manual therapists ( acupuncturists and massage therapists ) , student loan default rate , threats to autonomy , and various ethical and morality issues .
the essence of this exploration is to review these unique profession - specific stressors combined with the typical stressors ( close interaction with clients , lack of supervisor support , strong reliance on technical skills , profitability ) that similar helping professions are exposed to and apply them to the jd - r and cor models as a means of explaining the potential for burnout in chiropractic profession . as such , consistent with the jd - r model ,
the main job demands of practicing chiropractors include physical workload , mental and emotional demands , and role stress ( role ambiguity and role conflict ) ; and the main job resources involved in the practice of chiropractic include job autonomy and social support climate ( lack of supervisor support ) .
a source of stress within the chiropractic profession , as well as the physical therapy , occupational therapy , and dentistry professions , is a relatively high incidence of work - related injuries . for many professions ,
injury history , area of practice specialization , or work environment has been identified as potential risk of work - related injuries .
the chiropractic , physical therapy , and dentistry professions may be at increased risks for work - related injury because of their exposure to repetitive movements , hand force , static loading , and awkward postures in their work .
lorme and naqvi found that when performing treatments , doctors of chiropractic were subject to dynamic forces that increase spinal loading and could increase risk of injury .
studies investigating the musculoskeletal demands of chiropractic therapies have suggested a high prevalence of low back pain ranging from 57% to 87% .
mior and diakow report that 41% of the chiropractors interviewed felt that the postures they assumed while treating patients was the most influential agent in the development of back pain , and that 82% thought that their back pain was further aggravated by continued practice .
homack 's survey of practicing chiropractors in new york state suggests that patient handling and delivery of side - posture manipulative procedures are the activities that most frequently result in work - related injury to the practicing chiropractors .
bisiachhi and huber report differences in the prevalence of musculoskeletal injuries between male and female chiropractors in the united states . among female chiropractors ,
the low back was the most common site of injury reported , whereas male chiropractors reported the neck as the most common site of injury .
the study by holm and rose found a moderate prevalence ( 41% ) of workplace musculoskeletal injuries among doctors of chiropractic , particularly of the wrist , hand , fingers , and shoulder .
furthermore , holm and rose note that injuries occurred most commonly in the first to fifth years of practice and that side - posture manipulation seemed to cause the greatest percentage of these injuries .
the few studies that have investigated injuries associated with chiropractic practice appear to agree that using manual therapy , particularly side - posture manipulation , increases the amount of biomechanical and postural demands on the practitioner applying the therapy .
side - posture manipulation is a trademark modality in the chiropractic profession and is used for the treatment of low back pain . as a result of injury
, some chiropractors may change their treatment techniques or their frequency or duration of work , or may choose to leave the profession .
this raises the following question : what is the impact of chiropractic - specific work - related injuries on burnout of chiropractic practitioners ? despite the lack of literature on this issue
, it may be reasonable to hypothesize that if work - related injuries are as substantial as the literature suggests , then sustaining a chiropractic - specific work - related injury may partially contribute to not only a physical injury but a sense of increased exhaustion , increased depersonalization , and reduced personal accomplishment .
within the job demand category of the jd - r model , concepts such as role ambiguity and role conflict are emphasized .
role ambiguity typically refers to an individual 's lack of clarity about his / her expected behavior from a job ; whereas role conflict refers to a type of social conflict caused by an individual being forced to take on separate and incompatible roles ( ie , business owner and physician ) .
the construct of role stress and its influence on burnout within the chiropractic profession may involve the profession 's unique public perception and identity stressors . within the united states ,
the chiropractic profession has struggled with establishing a clear identity that is both accepted and relevant to the public .
this struggle may , in part , be due to the presence of 2 opposing philosophical and political forces within the profession .
two predominate governing bodies in the united states , the american chiropractic associate and the international chiropractic association , represent the profession at large , often resulting in contradictory scenarios with regard to lobbying , public relations , leadership , and advancement of the profession .
although the situation is more complex , the 2 opposing views are sometimes simplistically referred to as straights vs mixers .
although each side sees chiropractic potentially addressing a wide range of diseases and conditions , the
chiropractic subluxations that interfere with the flow of vital energy , whereas the mixer view supports a broader scope of treatment ( eg , a mixer will mix in other therapies in addition to manipulation , such as physiotherapeutics , diet , exercise ) and has a more musculoskeletal and evidenced - based practice focus .
these philosophical differences clash during lobbying efforts among leadership groups within the profession and , as a result , may further exacerbate the professional - identity inconsistencies and public perception uncertainties that shadow the profession .
these political conflicts resulting in inherent poor public perception and identity discrepancies stressors may have prevented the chiropractic profession from establishing its cultural authority over any specific domain of health care and , therefore , may be the source of significant stress among its practitioners .
delinquencies in creating its cultural authority may be the result of the varying public perceptions of the chiropractic profession and the chiropractic profession 's internal struggle with establishing a universal identity . within the profession
, an individual chiropractor may specialize in a number of available techniques and methods in his or her practice .
the practice of chiropractic is multifaceted and , for many , involves a vast array of healing arts and modalities such as manipulation , massage , strength training , various physical therapy modalities , traction , and nutritional consultation . these varying views and practice styles
may result in confusion for the public in that patients , medical physicians , the managed care industry , and individual chiropractic practitioners themselves have different perspectives on defining the profession .
thus , identity problems combined with poor public comprehension may threaten the future vitality of the profession .
the end result of these incongruencies makes it plausible to hypothesize that the identity and public perception discrepancies that shadow the chiropractic profession may partially contribute to a sense of increased emotional exhaustion , increased depersonalization , and reduced personal accomplishment .
increased susceptibility for burnout in health care professionals may be the result of spending their careers focusing on the needs of others .
in addition to the mental and emotional demands involved in direct patient care , doctors of chiropractic also battle issues that threaten their ability to maintain a satisfactory income ( eg , managed care , medicare regulation , scope of practice restrictions , national health care reform ) . these issues may serve as a source of mental stress and thus may have an impact on the prevalence of burnout . in an attempt to understand profitability and viability insecurities , zhang and colleauges sought to investigate chiropractic students ' knowledge and attitudes of education and financial investments .
these authors , along with coulter et al , suggest that the present - day chiropractic student invests a similar amount of time , effort , and finances to pursue a career in chiropractic as does the present - day medical student .
these investment issues raise an important line of questioning , especially in terms of return on investment and career expectations .
edwards et al suggest that differences between what medical doctors might have reasonably expected for their career and how their career actually turned out may have implications on burnout .
furthermore , edwards et al suggest that students and practitioners , in general , who invest time , effort , and finances into their education would expect to be rewarded with a profitable career
. market competition may serve as a source of mental and emotional stress within the chiropractic profession .
whereas the number of practitioners is growing and competition from other healing professions is increasing , insurance reimbursements to chiropractors seem to be decreasing
. data from the institute for alternative futures suggest that a slowing economy , along with the recent increase in health insurance premiums , has a dampening effect on the rate of growth in demand for chiropractic services .
in addition , cherkin et al suggest that patients seeking manual treatment of musculoskeletal conditions , in a form that is similar to chiropractic spinal manipulation therapy , may choose to seek services from massage therapists who outnumber chiropractors and are growing in both numbers and market share .
competition from acupuncturists , osteopaths , and medical physicians , some of whom are also demonstrating an increased interest in manual therapy , further adds pressure .
in addition , there may be a supply and demand issue regarding the number of chiropractors within the profession .
data from the us department of commerce indicate that the total reported net income for chiropractic offices and clinics rose from $ 6.56 billion in 1992 to $ 7.68 billion in 1998 , which is about 2.8% per year . because the number of practicing chiropractors has been increasing
, these figures may show that chiropractic income may be decreasing steadily , thus adding to burnout factors . as we look to the future to strengthen the profession and develop our research agenda ,
these burnout factors include those that are associated with physical workload , role stress , and mental and emotional demands .
investigations into these complex factors should include a focus on intervention as well as prevention . using the cor model and
this article provides a discussion of the topic of burnout and the chiropractic profession and was not intended to be a systematic review of the literature ; therefore , key articles related to this topic may have not been included .
terms describing psychological stress and burnout are sometimes used inconsistently in the literature , which is a potential additional limitation to this study . the evidence available for the chiropractic - specific stressors
the data within the chiropractic literature have generally suffered from sampling biases and are vulnerable to biases stemming from subjectivity and imprecision . in addition , some experts may question the approach of comparing chiropractic to dentistry , physical therapy , and occupational therapy .
arguments over the autonomous nature of physical therapy also reduce the strength of the presented potential occupational correlations .
doctors of chiropractic may be exposed to a unique source of stressors because of the autonomous and isolated nature of their practices , their close contact with patients , and their strong reliance on technical skills .
many of these practitioners may assume the role of private business owner , which usually involves a personal financial risk and a significant dependence on third - party reimbursements .
doctors of chiropractic have seen major changes in the health care environments in which they practice in many countries . in summary , as the chiropractic profession faces many of the same challenges as other health care providers with decreased reimbursement , changing patient payer types , changing patient and payer expectations , and diminished profit margins , it also faces a unique set of profession - specific stressors such as increasing market competition challenges ( both internally and externally ) , identity and poor public perceptions inconsistencies , a relatively high prevalence of work - related injuries , and a high student loan default rate .
common challenges within the health care arena , combined with the noted chiropractic - specific stressors and the chronic work - related stressors of caring for individuals , may pose a hazard to the chiropractic profession at large .
as chiropractors face increasing conflicts , both internally and externally , the need for assessing and measuring burnout and attrition within the chiropractic profession becomes increasingly apparent .
| objectivethe objective of this narrative review is to discuss the potential for burnout in chiropractic practitioners .
this discussion is grounded in the job demands - resource model , the conservation of resources model , the unique profession - specific stressors experienced by chiropractors , and information from similar health care professions.methodsa search using both the indexed ( pubmed and psychlit ) and nonindexed psychosocial literature was used .
other resources included the cochrane library , articles from governing bodies of the chiropractic profession , trade magazines , and research conferences and symposium proceedings . articles
were analyzed following the grounded theory principles : open coding and memos for conceptual labeling , axial coding and memos for category building , and selective coding for model building.resultspotential stressors unique to doctors of chiropractic include factors associated with physical workload , role stress , and mental and emotional demands.conclusionsthere are unique chiropractic - specific occupational characteristics that possibly contribute to burnout in the chiropractic professionals .
these findings emphasize the need for assessing and measuring burnout and attrition within the chiropractic profession . |
mutagenesis and protein preparation an expression vector containing full - length rat wild - type p97 ( ptrc his b p97 ) with an n - terminal his6 tag was constructed , and point mutations to the walker a motif ( k251a and k524a ) and the srh motif ( r359a and r635a ) were introduced by the quikchange method ( stratagene ) . all constructs were expressed at 37 c in the escherichia coli rosetta 2 ( de3 ) strain ( novagen ) .
clarified lysates were loaded onto a hitrap chelating column ( ge healthcare ) charged with ni and eluted by imidazole gradient .
the purity and oligomeric state of preparations was confirmed by sds and non - denaturing page .
samples , where required , were digested with apyrase enzyme ( potato apyrase , grade vii , sigma - aldrich ) as described previously ( 19 ) .
apyrase was physically separated from p97 by a dialysis membrane during the digest to prevent contamination of p97 .
all binding experiments were carried out in the following buffer : 50 mm tris ( ph 8) , 20 mm mgcl2 , 2 mm edta , and all concentrations of p97 are of monomers .
quantification of bound nucleotide by heat denaturation bound nucleotide was released by incubating 75 l of 100 m
the precipitated protein was removed by centrifugation , the supernatant was decanted , and the volume was measured .
the concentration of adenine nucleotide was calculated from absorbance at 260 nm ( = 15 400 m cm ) , and the amount of nucleotide released was expressed as the ratio of nucleotide to p97 monomer . error bars in the legends for figs .
the stoichiometry of atps bound by p97 was estimated by mixing 1 ml of 10 m p97 with a moderate excess of atps ( sigma - aldrich ) and spin - concentrating to 10% of the original volume .
protein concentration and volume were recalculated , and the total amount of nucleotide in the sample was determined .
the concentration of unbound atps ( assumed to be equal to the starting concentration of atps ) was subtracted from the total nucleotide concentration , giving the amount of nucleotide bound to p97 .
molar excesses greater than 4-fold were not used as the total nucleotide concentration measurements were dominated by unbound nucleotide .
fluorescence spectroscopy fluorescence measurements were made at 25 c with a fluoromax-3 spectrofluorimeter ( jobin yvon horiba ) , and data were fitted using grafit ( erithacus software ) .
fluorescence anisotropy experiments used 50 nm bodipy - labeled adp ( b - adp ) ( invitrogen ) in a glass cuvette ( ex : 595 nm , slits 3 nm ; em : 620 nm , slits 10 nm ) .
each adp / b - adp competition experiment used a constant concentration of adp ( 1 , 5 , 10 , or 20 m ) and titrated p97 , mixing fresh p97 , adp , and b - adp for each point to correct for prebound adp .
the apparent kd ( app.k d ) at each of the four adp concentrations ( [ itot ] ) was plotted , and linear regression calculated the kd of b - adp ( k d ) and adp ( k d ) for p97 by the following equation ( adapted from ref .
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\begin{equation*}\;app{\cdot}k_{d}^{{\mathrm{l}}}=k_{d}^{{\mathrm{l}}}\left(1+\frac{[i_{tot}]}{k_{d}^{{\mathrm{i}}}}\right)\end{equation*}\end{document } tryptophan fluorescence intensity measurements were made by adding atps to 500 l of 1 m p97 in a quartz cuvette ( ex : 295 nm , slits 2 nm ; em : 345 nm , slits 5 nm ) .
fluorescence intensity changes as atps increased were fitted to a single site or cooperative binding equation ( 23 ) ( mean and standard deviation of multiple titrations are listed in supplemental table 2 ) .
isothermal titration calorimetry ( itc ) measurements were made with a microcal vp - itc microcalorimeter ( microcal ) equilibrated at 25 c , typically titrating nucleotide against 50300 m p97 .
data were analyzed using origin ( originlab ) and fitted to models describing one and two types of binding site and competition between two ligands ( 24 , 25 ) .
( competition models were fitted using the concentration of prebound adp determined by heat denaturation experiments . )
as data did not show features suggesting more complex binding modes and fitted well to two - site and competition binding models giving results consistent with prior knowledge , higher dimensional models were not fitted .
fitting results of at least three repetitions of each experiment were averaged , and the mean and standard deviations are listed in supplemental table 1 ) .
mutagenesis of walker a and srh motifs we sought to explore nucleotide binding to recombinant rat p97 by studying the d1 and d2 domains separately . to selectively reduce nucleotide binding affinity of individual aaa domains , we mutated the walker a motif ( gxxxgkt ) that interacts with bound nucleotide ( 17 ) ( fig .
1 ) . lysine to alanine mutation of the walker a motif was made in d1 ( k251a ) and d2 ( k524a ) both individually and together ( k251a , k524a ) . to also probe potential communication around the hexamer ring ,
an arginine finger in the srh motif was mutated to alanine in both d1 and d2 ( r359a , r635a ) . in d1 at least ,
crystal structures ( e.g. pdb code 1e32 ) show that this arginine residue protrudes into the active site of the neighboring monomer ( fig . 1 ,
1 ) . introduction of these mutations to recombinant p97 did not alter the oligomeric structure ( see supplemental fig .
2a ) ; however , these mutations did affect atpase activity ( see supplemental fig .
similar to findings of other groups , p97 carrying the walker a mutation to the d1 domain alone ( k251a ) showed a moderate decrease in atpase activity , whereas p97 carrying mutations in the d2 domain ( k524a , k251a , k524a , r359a , r635a ) showed little atpase activity when compared with wild type ( 13 , 26 , 27 ) .
this is consistent with the previously suggested hypothesis that the d2 domain is the major site of atpase activity ( 27 , 28 ) .
the figure shows the crystal structure ( pdb code 1e32 ) of the active site of p97 d1 domain ( 17 ) showing adp bound at the interface of two monomers ( blue and gray ) .
conserved residues crucial for nucleotide binding ( walker a lys-251 ) and hydrolysis ( walker b glu-305 ) are shown , as are two arginine residues ( arg-359 and arg-362 ) contributed by the srh motif of the neighboring monomer .
the figure shows the crystal structure ( pdb code 1e32 ) of the active site of p97 d1 domain ( 17 ) showing adp bound at the interface of two monomers ( blue and gray ) .
conserved residues crucial for nucleotide binding ( walker a lys-251 ) and hydrolysis ( walker b glu-305 ) are shown , as are two arginine residues ( arg-359 and arg-362 ) contributed by the srh motif of the neighboring monomer .
a , heat denaturation measurements of the amount of adp prebound to wild type and walker a mutants show that adp is prebound to d1 .
b , fluorescence anisotropy measurements of b - adp binding to wild type and walker a mutants confirm that b - adp binds selectively to d1 .
c , linear regression of the apparent kd of b - adp at different concentrations of adp allows calculation of the actual kd of b - adp and adp .
d , itc shows that adp binds to two types of sites with distinct affinities ( fitted to a two - site model ) .
e , adp binding to p97 pretreated with apyrase ( to remove prebound adp ) was measured by itc and fitted to a two - site model ( as in panel d ) .
the calculated kd ( , ) and h ( , ) of d1 and d2 do not vary significantly from untreated p97 ( shown in purple ) .
a , heat denaturation measurements of the amount of adp prebound to wild type and walker a mutants show that adp is prebound to d1 .
b , fluorescence anisotropy measurements of b - adp binding to wild type and walker a mutants confirm that b - adp binds selectively to d1 .
c , linear regression of the apparent kd of b - adp at different concentrations of adp allows calculation of the actual kd of b - adp and adp .
d , itc shows that adp binds to two types of sites with distinct affinities ( fitted to a two - site model ) .
e , adp binding to p97 pretreated with apyrase ( to remove prebound adp ) was measured by itc and fitted to a two - site model ( as in panel d ) .
the calculated kd ( , ) and h ( , ) of d1 and d2 do not vary significantly from untreated p97 ( shown in purple ) .
characterization of adp binding to p97other studies found that adp is prebound to recombinant preparations of p97 ( 1419 ) .
therefore , prior to carrying out ligand binding experiments , we made a detailed characterization of nucleotide already bound to p97 .
samples of p97 were heat - denatured , the precipitated protein was removed by centrifugation , and the resulting supernatant was examined spectroscopically .
the supernatant had an absorption maximum at 260 nm , consistent with adenine - based nucleotides .
further analysis by hplc showed co - elution with adp but not with atp controls ( data not shown ) , indicating that adp is prebound to our p97 preparations .
we quantified the amount of adp prebound to p97 and found that typically 0.9 0.2 molecules of adp were present per p97 monomer ( fig .
to determine which aaa domains adp was bound to , heat denaturation experiments were repeated using walker a mutants designed to specifically reduce the affinity of nucleotide in d1 and d2 .
however , p97 mutated in d2 alone ( k524a ) contained a similar amount of prebound adp as wild type , whereas p97 mutated in d1 , either alone ( k251a ) or in combination with d2 ( k251a , k524a ) , contained little prebound adp ( fig .
this indicates that adp is prebound solely to d1 as walker a mutation of this domain is sufficient to reduce prebound adp .
mutations of the srh motif of d1 and d2 ( r359a , r635a ) did not alter the amount of adp prebound to d1 consistent with structural predictions that arg-359 does not interact with adp ( 17 ) .
these experiments define the initial nucleotide - bound state of our recombinant p97 preparations , showing that adp is prebound to 90% of wild - type d1 domains ( 0.9 adp molecules per monomer ) . from these results
, we hypothesized that adp binds with highest affinity to d1 since it is retained throughout the purification and normal dialysis of recombinant p97 . to determine the affinity of adp for d1
, we carried out binding experiments using fluorescent bodipy - adp ( b - adp ) as a probe .
wild - type p97 was added incrementally to b - adp , and binding was detected by the resulting increase of fluorescence anisotropy ( fig .
binding experiments were then performed with d1 and d2 walker a mutants to identify which domain b - adp binds to .
the d2 mutant exhibited similar b - adp binding to the wild - type , suggesting that b - adp does not bind to d2 .
mutation of d1 ( k251a and k251a , k524a ) , however , disrupted normal b - adp binding ( 7-fold greater concentration necessary for half - maximal saturation ) , thus confirming that b - adp binds to d1 ( fig .
the dissociation constant ( kd ) of adp binding to d1 was determined by measuring the apparent kd of b - adp at four concentrations of competing unlabeled adp .
plotting the apparent kd of b - adp against the competing adp concentration allowed the kd of adp to be estimated at 1.4 m ( fig .
we titrated an excess of adp against wild - type p97 , yielding an exothermic binding profile ( fig .
fits of a single site binding model were inconsistent with the binding profile at higher adp concentrations , indicating that adp also binds to a second site .
other studies have demonstrated that d2 can also bind adp ( 14 , 20 ) and , in agreement with this , a two - site binding model fitted the experimental data well .
the two - site model predicts a higher affinity site with a kd of 1.1 0.8 m and apparent stoichiometry of 0.2 0.05 and a low affinity site with a calculated kd of 86 51 m but poorly defined stoichiometry due to the low affinity of this site ( stoichiometry was set to 1 for fitting ; see supplemental table 1 ) .
the apparent stoichiometry of the high affinity site ( 0.2 0.05 ) is consistent with the saturation of a small population of vacant d1 domains , as predicted by fig .
2a , and supporting this , the kd is the same as measured previously for d1 ( fig .
, we interpret that adp binds with significantly higher affinity to d1 ( kd 1 m ) than d2 ( kd 90 m ) .
we next asked whether adp prebound to d1 influenced the binding of adp to d2 .
p97 was therefore treated with apyrase for varying times to remove differing amounts of adp from d1 ( 19 ) , and adp binding was then measured by itc .
longer apyrase treatments led to higher measured stoichiometries of adp binding to d1 ; however , d1 and d2 adp binding parameters ( kd and h ) remained similar to untreated p97 ( fig .
thus , under these conditions , adp binding to d2 is not influenced by adp bound to d1 . supporting this , the kd of adp binding to d2 when d1 adp binding was disrupted ( k251a ) was in the same range as d2 in wild - type p97 as determined by itc ( data not shown ) . in summary , we have identified adp prebound to the d1 domain of our p97 preparations ( consistent with other studies ( 1419 ) ) and have quantified the amount adp bound to wild type and mutants .
measurements of the affinity of adp binding to d1 have revealed an apparent kd of 1 m . given that adp is found in all d1 domains of p97 crystal structures ( pdb code 1e32 ; 1yq0 ; 1yqi ; 1ypw ) ,
however , adp is bound in a deep pocket in the crystal structure , in between two monomers .
this suggests a slow off rate and a possibly even slower on rate , explaining the low kd .
furthermore , different buffer conditions used for crystallization versus enzymatic assays could also potentially contribute to this ( 1419 ) .
in contrast to d1 , the d2 ring has comparatively low affinity for adp , predicting a role as the major site of atp hydrolysis .
a , itc data of atps binding to d2 walker a mutant ( k524a ) .
b , itc data of atps binding to p97 mutated in the d1 walker a motif ( k251a , fitted to a two - site model ) . note the jagged profiles of raw data results from titration of different volumes of ligand .
a , itc data of atps binding to d2 walker a mutant ( k524a ) .
b , itc data of atps binding to p97 mutated in the d1 walker a motif ( k251a , fitted to a two - site model ) . note the jagged profiles of raw data results from titration of different volumes of ligand .
affinity of atps binding to d1 and d2we wanted to examine atp binding to p97 .
however , as we detected residual atpase activity in p97 mutants designed to knock out catalysis of atp hydrolysis ( data not shown ) , we instead used a hydrolysis - resistant atp analog . atp analog , amppnp , has much lower affinity for p97 than atp ( 21 ) , indicating that for the purposes of binding experiments , it does not accurately represent atp .
our preliminary experiments showed that atps bound to p97 with considerably higher affinity than amppnp ( data not shown ) , and so we therefore used atps to mimic atp .
for these and subsequent itc experiments , we did not remove adp from d1 by apyrase digest as the treatment did not provide the large amounts of adp - free p97 necessary .
however , we have carefully characterized adp already bound to p97 , allowing it to be accounted for in analysis .
we first measured atps binding to d2 walker a mutant , k524a , which is deficient in d2 nucleotide binding and has adp prebound to d1 ( fig .
the mid - point of the binding curve estimates that a small population of d1 sites ( 0.16 0.03 d1 sites per monomer ) is unoccupied with prebound adp and binds to atps early in the titration .
the data do not fit well to a single site model , implying that competition with prebound adp must also be modeled . a competition model assuming that 0.9 molecules of adp are present per monomer ( from fig .
this fit predicts that atps binds to d1 with a kd of 2.1 0.6 m and an overall stoichiometry of 1.05 0.04 molecules of atps per monomer ( 6.3 atps per hexamer ) ( fig .
the kd of adp for d1 calculated by the fit ( 0.6 0.15 m ) is in reasonably good agreement with independent itc measurements of adp binding to wild type ( fig .
figure 4.stoichiometry and cooperativity associated with atps binding p97 . a , itc profile of wild - type p97 binding to atps .
the data are fitted to a competition model calculating that p97 is saturated at 1.6 atps per monomer .
b , modified heat denaturation experiments in which atps was added to p97 ( with adp prebound or removed by apyrase digest ) .
results show that atps binding to p97 is saturated at 1.51.6 molecules of nucleotide per monomer ( 910 per hexamer ) and is independent of adp prebound to d1 .
c , tryptophan fluorescence changes of wild type and walker a mutants in response to atps binding show that the fluorescence change results from atps binding to d2 and is not altered by nucleotide bound to d1
d , tryptophan fluorescence of srh mutant ( r359a , r635a ) shows no positive cooperativity .
fitting to a single site model shows that atps is bound with similar affinity to itc measurements of wild - type p97 .
e , itc data of r359a , r635a binding to atps indicate that the stoichiometry of atps binding is 1.66 molecules of atps per monomer ( 10 per hexamer ) , similar to wild - type p97 .
stoichiometry and cooperativity associated with atps binding p97 . a , itc profile of wild - type p97 binding to atps .
the data are fitted to a competition model calculating that p97 is saturated at 1.6 atps per monomer .
b , modified heat denaturation experiments in which atps was added to p97 ( with adp prebound or removed by apyrase digest ) .
results show that atps binding to p97 is saturated at 1.51.6 molecules of nucleotide per monomer ( 910 per hexamer ) and is independent of adp prebound to d1 .
c , tryptophan fluorescence changes of wild type and walker a mutants in response to atps binding show that the fluorescence change results from atps binding to d2 and is not altered by nucleotide bound to d1
d , tryptophan fluorescence of srh mutant ( r359a , r635a ) shows no positive cooperativity .
fitting to a single site model shows that atps is bound with similar affinity to itc measurements of wild - type p97 .
e , itc data of r359a , r635a binding to atps indicate that the stoichiometry of atps binding is 1.66 molecules of atps per monomer ( 10 per hexamer ) , similar to wild - type p97 .
we next tested atps binding to d2 using the d1 walker a mutant , k251a , which has reduced affinity for nucleotide in d1 and so contains little prebound adp ( fig .
fits of a two - site model to itc binding profiles predicted a high affinity site with a kd of 3 2 m and a low affinity site with a kd of 74 34 m ( fig .
( the model fitted best when the stoichiometry of atps binding the high affinity site was 1 ; however , modified heat denaturation experiments ( described later ) did not confirm this , indicating a stoichiometry of 0.75 ( see supplemental fig .
we assigned the high affinity site as atps binding to d2 because additional mutation of d2 ( k251a , k524a ) disrupted high affinity atps binding ( see supplemental fig .
3b ) . the low affinity site could be a subset of low affinity d2 domains , or more likely , residual binding of atps to the mutated d1 domain .
in contrast to the different affinities of adp binding to d1 and d2 , atps binding to walker a mutants suggests that d1 and d2 have similar affinities for atps ( d1 kd = 2 m and d2 kd = 3 m ) .
additionally , our data are consistent with measurements of the affinity of adp binding ( fig .
2 , c and d ) , providing evidence that atps binding to d1 does not alter the affinity of adp bound to other d1 domains .
stoichiometry of atps binding to wild - type p97to investigate atps binding to wild - type p97 , we carried out further itc experiments .
the itc binding profile of atps titrated against wild - type p97 showed that atps binding was exothermic at lower atps concentrations but became endothermic at higher atps concentrations ( fig .
the mid - point of the exothermic portion indicated a stoichiometry of 0.66 0.125 atps molecules binding per p97 monomer .
this is consistent with atps initially binding to four vacant sites per hexamer , presumably from the empty d2 ring and the small population of adp - free d1 domains .
the later endothermic phase corresponds to atps competing with adp for binding sites in the d1 ring and represents the difference in enthalpy of adp dissociation and atps association . since prebound adp was also present , we fitted a competition model that accounts for 0.9 molecules of prebound adp per monomer ( from fig .
2a ) but assumes that d1 and d2 have the same affinity for atps ( fig .
3 ) . in addition , we fixed the kd of adp at 0.85 m and assumed that low affinity adp binding to d2 was negligible .
4a ) , and notably , the total stoichiometry of atps was 1.6 0.16 molecules per monomer ( 9.6 molecules atps per hexamer ) .
we confirmed this predicted stoichiometry by measuring the maximal amount of atps bound by p97 through a modified heat denaturation protocol .
an excess of atps was added to wild - type p97 , and the majority of unbound atps was removed before the sample was heat - denatured .
( the excess of atps was limited to 4-fold as experimental error increased substantially at higher concentrations . ) binding to p97 was close to saturation when 1.5 molecules of nucleotide were bound per p97 monomer ( 9 molecules per hexamer ) ( fig .
4b ) . repeating this experiment using apyrase - treated p97 gave a similar result ( fig .
4b ) , demonstrating that the maximal amount of atps bound is not influenced by the presence of adp in d1 .
together these results indicate that within a p97 hexamer , only 910 nucleotides fill the 12 possible binding sites .
given that the d1 and d2 rings have differing structural properties ( 6 ) and that crystallographic structures show full occupancy of d1 ( 1418 ) but partial occupancy of d2 in the case of adp - alf3 ( 14 ) , we speculate that one ring is fully occupied by atps , whereas the other ring is partially occupied .
the balance of evidence suggests that the d1 ring is fully occupied with 6 molecules of atps , whereas the d2 ring can only bind 34 molecules of atps .
cooperativity associated with atps binding to d2we next probed the mechanism of atps binding to the d2 ring by exploiting a trp residue positioned on the linker between d1 and d2 and close to the d2 active site ( trp-476 ) .
the fluorescence of this residue is reported to increase with d2 nucleotide binding ( 20 ) due to a change in the environment of the trp residue resulting from either the proximity of nucleotide to trp-476 or a conformational change in p97 .
titration of atps into wild - type p97 produced a sigmoidal increase in fluorescence that fitted to a cooperative binding equation with a hill coefficient ( nh ) of 2.8 0.3 ( fig .
we repeated the titration with the d1 and d2 walker a mutants to confirm that this fluorescence change was a consequence of atps binding to d2 . in the same atps concentration range , mutation of d2 ( k524a and k251a , k524a ) was sufficient to prevent any increase in trp fluorescence ( fig .
this clearly demonstrates that changes in trp fluorescence are exclusively associated with atps binding to the d2 ring alone .
the sigmoidal increase in trp fluorescence indicates positive cooperativity within the hexamer upon atps binding to d2 . the hill coefficient of 2.8 implies that three or more p97 monomers positively cooperate upon atps binding to the d2 ring .
a similar effect was observed in p97 purified from rat liver cytosol ( nh = 4 0.1 ) , in this case indicating cooperation of at least four p97 monomers ( see supplemental fig . 4 and table 2 ) .
although the significance in the slight difference in hill coefficient between recombinant and endogenous p97 is unclear , both results suggest a cooperative change involving at least 34 monomers .
we asked whether the nucleotide state of d1 alters the cooperative fluorescence changes by comparing atps titrations when d1 is in differing nucleotide - bound states .
we compared trp fluorescence profiles of wild - type p97 ( with adp prebound to d1 ; fig .
2a ) to the d1 k251a mutant ( d1 empty and reduced affinity for atps ; figs . 2a and 3b ) and apyrase - treated p97 ( d1 empty and atps binding to both domains ; fig .
4c ) , demonstrating that the nucleotide - bound state of d1 does not influence the cooperative trp-476 fluorescence changes associated with atps binding to d2 .
cooperative conformational change downstream of atps binding since it has been proposed that the activity of individual monomers in a hexameric ring are coordinated by arginine fingers ( from the srh motif ) protruding from one monomer to the next ( 13 ) , we asked whether these arginine residues in p97 ( arg-359 , arg-635 ) are involved in the cooperative fluorescence changes we observed on atps binding . significantly ,
atps binding to the double arginine mutant ( r359a , r635a ) resulted in a decrease in the magnitude of the fluorescence change ( reduced from 11.5 in wild type to 11.3 in r359a , r635a ) and a loss of the sigmoidal shape of the binding curve indicating no positive cooperativity ( fig .
fitting to a single site model predicted that atps binds with a kd of 1.9 0.6 m to r359a , r635a .
this is comparable with previous measurements for wild - type p97 and confirms that these mutations do not disrupt the affinity of atps binding ( fig .
the smaller increase in trp-476 fluorescence of r359a , r635a ( from 1 to 1.3 ) can be assigned directly to nucleotide binding alone , whereas the larger cooperative increase ( from 1 to 1.5 ) observed with wild - type p97 is consistent with nucleotide binding and an additional cooperative conformational change .
we also used itc experiments to measure the stoichiometry of atps binding to r359a , r635a , fitting a competition model to the data to account for prebound adp ( fig .
the total stoichiometry of atps binding derived from this fit was 1.66 0.07 ( fig .
4e ) , similar to that obtained for wild - type p97 ( 1.6 ; fig .
the kd for atps binding to d1 and d2 ( 7.8 1.6 m ) was slightly increased but of a similar magnitude to wild - type p97 , and the competition phase of the data was slightly more endothermic , which we attribute to altered conformational flexibility within the system .
these results show that mutation of this srh arginine finger does not significantly affect the affinity or stoichiometry of atps binding , but does lead to a loss of cooperativity .
the cooperativity observed for wild - type p97 therefore appears to be associated with a conformational change that occurs downstream of atps binding , and is mediated by the srh arginine finger .
since mutations of arginine fingers of the srh motif are also associated with loss of atpase activity ( see supplemental fig .
2b ) ( 13 ) , we conclude that this cooperative conformational change is required for atp hydrolysis .
this work presents the first quantitative study of the nucleotide binding properties of both d1 and d2 rings of a type ii aaa atpase and reveals striking differences between the homologous aaa domains of p97 . despite sharing over 40% sequence identity ,
we have determined markedly different dissociation constants of adp binding to the d1 and d2 rings : around 1 m for d1 and 90 m for the d2 ring ( summarized in supplemental table 3 ) .
in contrast , the affinity of atps binding to d1 and d2 is similar for both rings , with a dissociation constant of 2 m .
other work has suggested that the d2 ring is the major site of atp hydrolysis ( 27 ) , a finding supported by crystal structures that capture d2 ( pdb code 1e32 ; 1yq0 ; 1yqi ; 1ypw ) in multiple nucleotide - bound states , but d1 bound only to adp ( 1416 ) .
our results support these data and suggest that the difference in affinity of adp for d1 and d2 provides a plausible explanation for these observations .
although the affinity of atps for d1 and d2 is similar , our results show that the stoichiometry of binding is distinct ; atps binding is limited to a subset of d2 domains ( 34 out of 6 possible sites ) , whereas the d1 ring binds atps to full occupancy .
the stoichiometry of atps binding to d2 predicts that atps binding to the first 34 d2 sites leads to changes in the final 23 d2 sites to prevent atps binding .
5 summarizes possible symmetrical and random arrangements of atps binding to d2 consistent with our results .
i ) only 2.23 molecules of photoactivated atp derivative were cross - linked to endogenous p97 ( 21 ) , and ii ) only one d2 domain of the three monomers in the asymmetric unit of the p97-adp - alf3 crystal structure had full occupancy of alf3 ( 14 ) . in the aaa+ atpase superfamily , there is a precedent for partial occupancy of atp in homohexameric aaa proteins with single rings such as clpx , ruvb , and hs1u ( 2931 ) , but this is the first report for a tandem aaa protein and suggests a potential conservation of mechanism despite the wide variety of aaa+ atpase functions .
the complete d2 ring is shown uppermost with the d1 ring and n domains below ( partially obscured ) .
four of the possible arrangements of atp binding consistent with our measurements of stoichiometry and cooperativity of atps binding are shown .
3 or 4 molecules of atp could bind symmetrically as a trimer of dimers ( a ) or a dimer of trimers ( b ) , or alternatively , in an asymmetric organization ( c and d ) .
additionally , atp could bind to three or four neighboring d2 domains ( not shown ) .
the complete d2 ring is shown uppermost with the d1 ring and n domains below ( partially obscured ) .
four of the possible arrangements of atp binding consistent with our measurements of stoichiometry and cooperativity of atps binding are shown . 3 or 4 molecules of atp could bind symmetrically as a trimer of dimers ( a ) or a dimer of trimers ( b ) , or alternatively , in an asymmetric organization ( c and d ) .
additionally , atp could bind to three or four neighboring d2 domains ( not shown ) .
we have additionally tested in a limited set of situations whether the nucleotide - bound state of one ring alters the affinity of the other .
our results show that adp and atps binding in d2 is unchanged by the presence of adp bound in d1 and does not alter the affinity of adp bound to d1 .
furthermore , the stoichiometry of atps binding to d2 is unaffected by adp bound in d1 , and conformational changes in d2 are the same , regardless of the nucleotide - bound state of the d1 ring .
our results provide the first evidence that the nucleotide binding properties of d1 and d2 rings are independent .
this does not rule out an interdependence of d1 and d2 in other aspects of p97s mechanism , such as the detailed kinetics of atp hydrolysis or in the transfer of conformational energy to potential adaptors .
however , under the conditions of the experiments carried out here , both rings can act independently in terms of nucleotide binding . in this study , we carried out a detailed analysis of tryptophan fluorescence changes previously shown to arise from nucleotide binding ( 20 ) and have identified a positive cooperative conformational change associated with atps binding to d2 in both recombinant and endogenous p97 .
the hill coefficient of the conformational change predicts that this involves at least three monomers , and we have shown that the conserved arginine residues of the srh motif ( most likely arg-635 ) are key to mediating this change .
interestingly , this positive cooperative conformational change does not regulate atps binding because we find that mutation of the key srh residues ( r359a , r635a ) prevents cooperativity but does not significantly alter atps binding affinity or stoichiometry .
however , this mutation does decrease atp hydrolysis to low levels , in agreement with previous studies ( 13 ) , suggesting that the positive cooperative conformational change could be an essential component of the atp hydrolysis pathway .
studies of mixed mutant hexamers have also indicated that the potential intermonomer contacts made by these residues contribute to the atp hydrolysis pathway ( 13 ) .
interestingly , crystal structures of the d1 domain ( e.g. pdb code 1e32 ) unambiguously show arg-359 protruding into the active site of the neighboring monomer ( 17 ) , potentially giving directionality to any conformational change . in vivo , p97 is thought to separate or translocate protein complexes by using atp binding and hydrolysis to change conformation .
changes in conformation upon nucleotide binding ( 68 , 14 , 19 , 20 , 32 ) and also the nucleotide dependence of assembly of some p97-adaptor complexes has already been demonstrated ( 33 , 34 ) ; however , there is lack of detailed knowledge of the steps involved .
our identification of a 34 atp binding stoichiometry and cooperativity of at least three monomers suggests that the d2 ring of the p97 hexamer does not operate a concerted mechanism in which all d2 sites simultaneously bind atp , hydrolyze atp , and change conformation ( 10 ) .
instead , the stoichiometry and cooperativity are more consistent with a processive mechanism where multiple rounds of atp hydrolysis and conformational change form a full reaction cycle
. further experiments utilizing linked oligomers are required to investigate whether atp binding and hydrolysis follow a symmetrical and rotational mechanism or are a stochastic process as has been shown for clpx ( 31 ) . in comparison with other aaa+ enzymes , our results suggest that the relationship between d1 and d2 is similar to that of close homologue , n - ethylmaleimide - sensitive factor ( nsf ) .
the stability of adp bound to the d1 domain of p97 resembles one aaa ring of nsf ( nsf d2 ) , which binds atp with high affinity ( kd = 30 nm ) and has only been crystallized bound to atp or atp analogs ( 3537 ) . in common with the d2 ring of p97 , the other aaa ring of nsf ( nsf d1 ) shows higher atpase activity ( 38 , 39 ) . in another tandem aaa+ atpase , hsp104 , atp hydrolysis in one ring ( nbd1 ) is regulated by the nucleotide - bound state of the other ( nbd2 ) ( 40 ) , and substrate remodeling activity is linked to impaired hydrolysis at individual aaa domains ( 41 ) .
although our results suggest that nucleotide binding to the d1 and d2 rings of p97 is independent , we do not rule out more complex interdependence of atp hydrolysis in the presence of adaptor / substrate proteins similar to hsp104 . intriguingly , the p97 d2 ring shares some resemblance to a distant relative with a single aaa ring , clpx , which also binds 34 atp molecules per hexamer in a similar range of affinity ( kd = 0.5 m compared with 2 m for d2 binding atps ) and moreover displays a positive cooperative change accompanying atp binding ( 31 ) .
taken together , these similarities suggest that despite the presence of an additional aaa ring in p97 , combined with evolutionary and functional divergence , elements of the atp hydrolysis mechanism of homohexameric aaa+ proteins may be conserved .
| p97 , an essential chaperone in endoplasmic reticulum - associated degradation and organelle biogenesis , contains two aaa domains ( d1 and d2 ) and assembles as a stable hexamer .
we present a quantitative analysis of nucleotide binding to both d1 and d2 domains of p97 , the first detailed study of nucleotide binding to both aaa domains for this type of aaa+ atpase . we report that adenosine 5-o-(thiotriphosphate ) ( atps ) binds with similar affinity to d1 and d2 , but adp binds with higher affinity to d1 than d2 , offering an explanation for the higher atpase activity in d2 .
stoichiometric measurements suggest that although both adp and atps can saturate all 6 nucleotide binding sites in d1 , only 34 of the 6 d2 sites can bind atps simultaneously .
atps binding triggers a downstream cooperative conformational change of at least three monomers , which involves conserved arginine fingers and is necessary for atp hydrolysis . |
congenital anomalies of the pulmonary veins are rare.[13 ] pseudo - scimitar syndrome or meandering tortuous right superior pulmonary vein means an anomalous course of the vessel which may not be the sole draining vessel of the ipsilateral lung .
it is a very rare entity and can be wrongly labeled as scimitar syndrome or arteriovenous malformations , which can lead to unnecessary surgery .
computed tomographic angiography ( cta ) of chest is very useful imaging modality to establish the diagnosis of anomalous vein along with excellent depiction of the anatomy and unnecessary intervention or surgery can be avoided .
a 30-year - old man presented with a history of cough and chest pain for two months .
chest radiograph [ figure 1 ] showed a tortuous tubular structure in the right lung field which was seen reaching till the right hilum superiorly .
the scan was acquired dynamically with 100 ml of non ionic contrast with injection rate of 3.5 ml / sec using 2.5 mm slice thickness and a 0.75 mm reconstruction interval .
the cta showed it to be meandering tortuous right superior pulmonary vein [ figure 2 ] .
the vein was coursing inferiorly and superiorly in the right lower lobe and then coursing posteriorly .
instead , it was coursing posterior to the right main bronchus and was then draining into the left atrium in the region of the right inferior pulmonary vein .
volume rendered images ( vrt ) , maximum intensity projection ( mip ) and multiplanar reformatted images ( mpr ) were made to exactly delineate the course of this tortuous vessel [ figure 2a d ] .
no arterial feeders to this vessel or any nidus was seen on cta thus ruling out an arteriovenous malformation ( avm ) .
there was no lung hypoplasia ( scimitar syndrome ) . the bronchial anatomy and the situs were normal .
the chest radiograph showing a tortuous tubular radio - opaque structure ( arrows ) in the right lung field which was seen reaching till the right hilum superiorly coronal oblique ( a ) and sagittal ( b ) reformatted image of ct angiography showing anomalous tortuous vein ( arrows ) coursing in right upper and middle lobe and draining into left atrium .
it was coursing posterior to the right main bronchus and was then draining into the left atrium in the region of the right inferior pulmonary vein .
volume rendered technique ( vrt ) images , anterior ( c ) and posterior ( d ) view showing the anomalous right superior pulmonary vein ( arrows )
it is very difficult to differentiate and diagnose anomalous tortuous pulmonary veins on chest radiograph .
usually , most of these lesions are falsely labeled as scimitar syndrome or arteriovenous malformations .
the classification of congenital anomalies of pulmonary veins is complex and classified into three types ( a ) , normal or abnormal course with anomalous pulmonary venous drainage , ( b ) no abnormal venous connection with anomalous pulmonary venous drainage , or ( c ) abnormal venous diameters .
unilateral anomalous pulmonary vein with hypogenetic lung had been described on computed tomography ( ct ) .
multiplanar reconstructions of helical ct is used for diagnosing the cases of pulmonary vein abnormalities for establishing the diagnosis .
many theories have been postulated regarding the abnormal course of pulmonary veins and it has been suggested that delayed union of the common pulmonary vein with the pulmonary venous plexus leading to connections with both the umbilical vitelline veins and the left atrium causes it .
meandering vein is thought to be due to persistence of left atrial connection with obliteration of the splanchnic connection .
a meandering vein usually means an anomalous course of the vessel which may not be the sole draining vessel of the ipsilateral lung .
the scimitar syndrome also known as venolobar syndrome or hypogenetic lung syndrome comprises of partial anomalous pulmonary venous drainage of lung , lung hypoplasia and systemic arterial supply from aorta or one of its branches to the lung .
scimitar syndrome is characterized by the following features : ( a ) lung hypoplasia ( b ) dextroposition of the heart , ( c ) hypoplasia of the right pulmonary artery , ( d ) anomalous arterial supply of the right lower lobe from the abdominal aorta ( e ) an anomalous right pulmonary vein connection to the systemic venous circulation either below or above the diaphragm most commonly to the inferior vena cava .
it produces left to right shunt , which may be asymptomatic or can even lead to heart failure .
the cta is the investigation of choice for detecting the anomalous pulmonary venous connections and vascular lung lesions .
it is effective , fast , widely available and with isotropic voxel reconstructions of the arterial and venous supply of the lesion , which can be seen in great detail and in any plane after post processing at the work station .
the mpr , mip and vrt images are very helpful in depicting the vascular structures , as well as the blood supply of the lesions .
if there is an avm , the arterial feeder or nidus and the venous component can be easily seen and diagnosed .
earlier pulmonary angiography was the modality of choice for diagnosing this condition but now cta is the single most important investigation for diagnosing such pathologies and has many advantages .
it is non invasive , easy to perform , less time consuming and with lesser radiation exposure . nowadays with advanced multislice ct scanners
in contrast , scimitar syndrome and arteriovenous malformation usually require surgery or embolization for treatment as they have an abnormal vascular shunt
. computed tomographic angiography ( cta ) should be done in suspicious vascular lesions of the lung as it confirms the diagnosis along with excellent depiction of the anatomy using vrt , mpr and mip images .
we would also like to stress that the clinicians or surgeons should not try to operate or biopsy such lesions before doing ct of the patient as these vascular lesions can lead to life risking hemorrhages . | the pulmonary venous abnormalities are very rare and are difficult to diagnose .
we present a case of middle age male who presented with cough and chest pain .
chest radiograph showed abnormal tortuous vessel in right lung .
computed tomographic angiography of chest demonstrated meandering anomalous right superior pulmonary vein . |
two adjacent 14.5 ha fields of cotton were selected for the study near pantego , nc in 2011 ( dp 1028 b2rf ) and 2012 ( dg 2570 b2rf ) .
the fields were separated by a 0.9-m ditch and surrounded on three sides by cotton and on one side by a road , with more cotton across the road .
sampling grids were marked every 0.4 ha in both fields , so that 36 sampling points were defined in each field .
sampling points on field edges were located within 30 m from the field perimeter .
points were georeferenced using a trible geoxt ( trimble , sunnyvale , ca ) for later geographical information systems post - processing .
cotton was sampled at each point during the third , fourth , and fifth weeks of bloom ( 21 , 28 july , and 4 august 2011 and 27 july , 3 and 10 august 2012 ) by taking 25-sweep samples with a 41-cm - diameter sweep net and , two rows over from the area that was swept , 10 quarter - sized boll samples .
stink bugs were identified to species at each location , and bolls were assessed for internal boll injury following north carolina state university extension guidelines ( bacheler et al .
, cotton exceeded the recommended treatment threshold guidelines and was sprayed with dicrotophos and beta - cyfluthrin ( bidrin 8 at 560 g ai / ha and baythroid xl at 23.5 g ai / ha ) . stink bug injury was not detected in the field during the fifth week of bloom in 2011 , and data from this week were dropped from the study . after the third week of bloom in 2012 , cotton exceeded the recommended treatment threshold guidelines and was sprayed with dicrotophos and beta - cyfluthrin using the same rates as in 2011 . because stink bug injury was still present , the field was sprayed again after the fourth week of bloom in 2012 using the same chemicals and rates as before .
the 2012 field was sprayed using strips , because it was part of a separate study analyzing the effectiveness of strip sprays for stink bug management ; hence only the 36 sample points from 2012 with the spatially uniform spray were used for analysis from the fourth and fifth week of bloom in 2012 .
in contrast , the 2011 field was uniformly sprayed following common grower practices , so all 72 sampling points could be used .
remote sensing data were collected during the third week of bloom on 22 july 2011 using the rapideye satellite ( rapideye ag , berlin , germany ) and on 26 july 2012 using a digitalglobe satellite , quickbird ( digitalglobe inc . ,
longmont , co ) , both of which were equipped with a pushbroom multispectral satellite .
satellite providers calibrate to reflectance from the earth s surface using objects with known units of reflectance and a radiative transfer model with sensitivities for atmospheric scattering and absorption .
the frequency of the calibration is proprietary and truly calibrated reflectance values were not available .
satellite data were imported into arcmap 10.0 ( esri 2011 , redlands , ca ) , and regions of interest were drawn around the sampling area at each 0.4 ha .
this encompassed an area that was 3639 pixels in the rapideye image ( pixel 16.5 m ) and 204207 pixels ( pixel 2.4 m ) in the quickbird image .
not all the sampling points were included in the rapideye image , so only 66 sampling points could be analyzed .
values from these regions of interest were extracted by mask in arcmap and imported into an electronic spreadsheet .
an ndvi value for each location was calculated using the following equation ( rouse et al . 1974 ) :
( rnirrred)(rnir+rred )
where rnir = the value encompassed in the wavelengths from the near infrared region and rred = the value encompassed in the wavelengths from the red region .
the spatial analysis by distance indices ( sadie version 1.22 , perry et al .
1999 ) tool was used to determine aggregation of stink bug densities , boll injury , and ndvi .
the overall index of dispersion ( ia ) indicates either an aggregated ( > 1 ) , random ( = 1 ) , or uniform pattern ( < 1 ) . the probability ( p )
is derived after a large number of randomizations as a formal test of randomness : the null hypothesis of spatial randomness is rejected for p < 0.025 ( aggregation ) or p > 0.975 ( uniformity ) with a 5% error rate . in total , 6,000 randomizations were used for each test .
there were insufficient adult stink bug numbers and boll injury on 4 august 2011 and 3 august 2012 for sadie aggregation analysis .
the sadie association tool was one of two methods used to investigate the association between boll injury from stink bugs and the ndvi value from each location .
spatial associations were measured between ndvi values of each year and adult stink bug numbers at each week of bloom , as well as ndvi values and boll injury at each week of bloom .
an overall index of association ( x ) was determined between each of the paired datasets , with a positive association for x > 0 ( p < 0.025 ) and a negative association for x < 0 ( p < 0.975 ) .
mean x is calculated from the local spatial associations ( xk ) at each sampling location k. at the local scale , a positive association between two variables indicates the presence of either a patch or a gap for both variables ; a negative association indicates the presence of a patch for one variable and gap for the other variable at the same location .
local association and aggregation indices were interpolated using the inverse distance weighting spatial method in arcmap 10.0 ( environmental systems research institute 2011 ) .
correlation analysis ( proc corr , sas institute 2008 ) was conducted in 2011 for ndvi values and the numbers of stink bug adult and injured bolls for the third ( 21 july ) and fourth week of bloom ( 28 july ) .
not enough stink bugs were captured in the fifth week of bloom in 2011 for correlation analysis . the 2011 remote sensing image did not include all the sampling points , and six were dropped from the analysis .
separate correlation analyses for 2012 data were conducted for ndvi values and the numbers of stink bug adult and injured bolls in the third ( 27 july ) , fourth ( 3 august ) , and the fifth week of bloom ( 10 august ) .
similar correlation analyses were performed for total stink bug adult numbers at each week of bloom and ndvi values .
values were transformed ( (x + 1 ) ) , when needed , to satisfy the assumptions of the correlation ( zar 1999 ) .
a visual representation of the association between boll injury and ndvi values was generated by interpolating the percent injured boll values and both the local aggregation and association indices using inverse distance weighting in arcgis .
over the course of the 2011 sampling period , there were 11 , 7 , and 3 e. servus adults captured on the third , fourth , and fifth weeks of bloom , respectively , and 4 , 1 , and 0 e. servus nymphs captured on the third , fourth , and fifth weeks of bloom , respectively ; there were also five c. hilare say adults and one c. hilare nymph captured on the fourth week of bloom in 2011 . only e. servus was captured in 2012 , 17 , 0 , and 5 e. servus adults captured on the third , fourth , and fifth weeks of bloom , respectively , and three e. servus nymphs captured on the third weeks of bloom .
for example , 6.2% injured bolls were found during the third week of bloom in 2011 compared with 28.2% injured bolls in 2012 during the third week of bloom . even after insecticide treatment after the third week of bloom in 2012 , 10.8% of the bolls were injured in the fourth week of bloom . after the second insecticide treatment in 2012 , injured bolls were reduced to 2.5% , below the treatment threshold of 10% injured bolls . in contrast , boll injury was essentially undetectable after the insecticide treatment following the fourth week of bloom in 2011 . in 2011 , adult stink bugs showed a trend to being spatially aggregated on the third week of bloom ( i = 1.510 , p = 0.0402 ; fig .
1a ) but not on the fourth week of bloom ( i = 1.081 , p = 0.2693 ; fig .
insufficient numbers of stink bugs were captured for analysis during the fifth week of bloom . in 2012 ,
adult stink bugs were not spatially aggregated during the third ( i = 1.086 , p = 0.2618 ) and fifth week of bloom ( i = 0.692 , p = 0.8579 ) and not enough were captured for analysis during the fourth week of bloom . in 2011 ,
boll injury due to stink bugs was not spatially aggregated on the third ( i = 1.039 , p = 0.5934 ) or fourth ( i = 0.881 , p = 0.6404 ) week of bloom ( fig .
in contrast , the 2011 ndvi values were spatially aggregated ( i = 2.658 , p = 0.0002 ; fig .
in 2012 , boll injury due to stink bugs was not spatially aggregated on the third ( i = 0.903 , p = 0.5934 ) or fifth ( i = 0.789 , p
= 0.7161 ) weeks of bloom , but a trend for aggregated injury was observed on the fourth week of bloom ( i = 1.705 , p = 0.0409 ; fig .
the ndvi values were aggregated across all 72 locations used for associations with stink bug densities and boll injury on the third week of bloom ( i = 3.949 , p = 0.0002 ; fig .
2a ) , and across half ( 36 ) of the sampling locations used for associations with stink bug densities and boll injury on the fourth and fifth weeks of bloom ( i = 3.643 , p = 0.0002 ; fig .
1.2011 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values , stink bug adult numbers , and boll injury from the third week and fourth week of bloom ; ( b ) local sadie association indices between ndvi values from the third week of bloom and stink bug adult numbers and boll injury from the third and fourth weeks of bloom ; wob , week of bloom .
fig .
2.2012 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom ; ( b ) associations between ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom .
2011 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values , stink bug adult numbers , and boll injury from the third week and fourth week of bloom ; ( b ) local sadie association indices between ndvi values from the third week of bloom and stink bug adult numbers and boll injury from the third and fourth weeks of bloom ; wob , week of bloom .
2012 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom ; ( b ) associations between ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom .
total number of adult stink bugs during the third week of bloom were positively correlated ( r = 0.28 , df = 66 , p = 0.0220 ) and positively associated ( x = 0.5288 , p < 0.0001 ; fig .
1b ) with ndvi values in 2011 and negatively correlated ( r = 0.25 , df = 72 , p = 0.0356 ) and unassociated ( x = 0.3838 , p = 0.9996 ) with ndvi values in 2012 . in the fourth week of bloom ,
total number of adult stink bugs were positively associated ( x = 0.5918 , p
1b ) with ndvi values in 2011 , and no stink bugs were found in the fourth week of bloom in 2012 . finally , total number of adult stink bugs were uncorrelated ( r = 0.05 , df = 36 , p = 0.7548 ) and unassociated ( x = 0.1064 , p = 0.2735 ) with ndvi values in 2012 during the fifth week of bloom .
boll injury during the third week of bloom was positively correlated ( r = 0.29 , df = 66 , p = 0.0195 ) and positively associated ( x = 0.3000 , p = 0.0094 ; fig .
1b ) with ndvi values in 2011 and were uncorrelated ( r = 0.02 , df = 72 , p = 0.8570 ) and unassociated with ndvi values in 2012 ( x = 0.0655 , p = 0.7075 ) .
boll injury during the fourth week of bloom was positively correlated ( r = 0.45 , df = 66 , p < 0.0001 ) and positively associated ( x = 0.3853 , p = 0.0010 ) with ndvi values in 2011 but were negatively correlated ( r = 0.49 , df = 36 , p = 0.0023 ) and negatively associated ( x = 0.4495 , p = 0.9967 , fig .
1b ) in 2012 . finally , boll injury was uncorrelated ( r = 0.03 , df = 36 , p = 0.8824 ) and unassociated ( x = 0.1693 , p = 0.8341 ) with ndvi values in 2012 during the fifth week of bloom .
in 2011 , adult stink bugs showed a trend to being spatially aggregated on the third week of bloom ( i = 1.510 , p = 0.0402 ; fig .
1a ) but not on the fourth week of bloom ( i = 1.081 , p = 0.2693 ; fig .
insufficient numbers of stink bugs were captured for analysis during the fifth week of bloom . in 2012 ,
adult stink bugs were not spatially aggregated during the third ( i = 1.086 , p = 0.2618 ) and fifth week of bloom ( i = 0.692 , p = 0.8579 ) and not enough were captured for analysis during the fourth week of bloom . in 2011 , boll injury due to stink bugs was not spatially aggregated on the third ( i = 1.039 , p = 0.5934 ) or fourth ( i = 0.881 , p = 0.6404 ) week of bloom ( fig .
in contrast , the 2011 ndvi values were spatially aggregated ( i = 2.658 , p = 0.0002 ; fig .
in 2012 , boll injury due to stink bugs was not spatially aggregated on the third ( i = 0.903 , p = 0.5934 ) or fifth ( i = 0.789 , p = 0.7161 ) weeks of bloom , but a trend for aggregated injury was observed on the fourth week of bloom ( i = 1.705 , p = 0.0409 ; fig .
the ndvi values were aggregated across all 72 locations used for associations with stink bug densities and boll injury on the third week of bloom ( i = 3.949 , p = 0.0002 ; fig .
2a ) , and across half ( 36 ) of the sampling locations used for associations with stink bug densities and boll injury on the fourth and fifth weeks of bloom ( i = 3.643 , p = 0.0002 ; fig .
1.2011 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values , stink bug adult numbers , and boll injury from the third week and fourth week of bloom ; ( b ) local sadie association indices between ndvi values from the third week of bloom and stink bug adult numbers and boll injury from the third and fourth weeks of bloom ; wob , week of bloom .
fig .
2.2012 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom ; ( b ) associations between ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom .
2011 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values , stink bug adult numbers , and boll injury from the third week and fourth week of bloom ; ( b ) local sadie association indices between ndvi values from the third week of bloom and stink bug adult numbers and boll injury from the third and fourth weeks of bloom ; wob , week of bloom . 2012 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom ; ( b ) associations between ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom .
total number of adult stink bugs during the third week of bloom were positively correlated ( r = 0.28 , df = 66 , p = 0.0220 ) and positively associated ( x = 0.5288 , p < 0.0001 ; fig .
1b ) with ndvi values in 2011 and negatively correlated ( r = 0.25 , df = 72 , p = 0.0356 ) and unassociated ( x = 0.3838 , p = 0.9996 ) with ndvi values in 2012 . in the fourth week of bloom ,
total number of adult stink bugs were positively associated ( x = 0.5918 , p
1b ) with ndvi values in 2011 , and no stink bugs were found in the fourth week of bloom in 2012 . finally , total number of adult stink bugs were uncorrelated ( r = 0.05 , df = 36 , p = 0.7548 ) and unassociated ( x = 0.1064 , p = 0.2735 ) with ndvi values in 2012 during the fifth week of bloom .
boll injury during the third week of bloom was positively correlated ( r = 0.29 , df = 66 , p = 0.0195 ) and positively associated ( x = 0.3000 , p = 0.0094 ; fig .
1b ) with ndvi values in 2011 and were uncorrelated ( r = 0.02 , df = 72 , p = 0.8570 ) and unassociated with ndvi values in 2012 ( x = 0.0655 , p = 0.7075 ) .
boll injury during the fourth week of bloom was positively correlated ( r = 0.45 , df = 66 , p < 0.0001 ) and positively associated ( x = 0.3853 , p = 0.0010 ) with ndvi values in 2011 but were negatively correlated ( r = 0.49 , df = 36 , p = 0.0023 ) and negatively associated ( x = 0.4495 , p = 0.9967 , fig .
finally , boll injury was uncorrelated ( r = 0.03 , df = 36 , p = 0.8824 ) and unassociated ( x = 0.1693 , p = 0.8341 ) with ndvi values in 2012 during the fifth week of bloom .
in 2011 , adult stink bugs showed a trend to being spatially aggregated on the third week of bloom ( i = 1.510 , p = 0.0402 ; fig .
1a ) but not on the fourth week of bloom ( i = 1.081 , p = 0.2693 ; fig .
insufficient numbers of stink bugs were captured for analysis during the fifth week of bloom . in 2012 ,
adult stink bugs were not spatially aggregated during the third ( i = 1.086 , p = 0.2618 ) and fifth week of bloom ( i = 0.692 , p = 0.8579 ) and not enough were captured for analysis during the fourth week of bloom . in 2011 , boll injury due to stink bugs was not spatially aggregated on the third ( i = 1.039 , p = 0.5934 ) or fourth ( i = 0.881 , p = 0.6404 ) week of bloom ( fig .
in contrast , the 2011 ndvi values were spatially aggregated ( i = 2.658 , p = 0.0002 ; fig .
in 2012 , boll injury due to stink bugs was not spatially aggregated on the third ( i = 0.903 , p = 0.5934 ) or fifth ( i = 0.789 , p = 0.7161 ) weeks of bloom , but a trend for aggregated injury was observed on the fourth week of bloom ( i = 1.705 , p = 0.0409 ; fig .
the ndvi values were aggregated across all 72 locations used for associations with stink bug densities and boll injury on the third week of bloom ( i = 3.949 , p = 0.0002 ; fig .
2a ) , and across half ( 36 ) of the sampling locations used for associations with stink bug densities and boll injury on the fourth and fifth weeks of bloom ( i = 3.643 , p = 0.0002 ; fig .
1.2011 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values , stink bug adult numbers , and boll injury from the third week and fourth week of bloom ; ( b ) local sadie association indices between ndvi values from the third week of bloom and stink bug adult numbers and boll injury from the third and fourth weeks of bloom ; wob , week of bloom .
fig .
2.2012 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom ; ( b ) associations between ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom .
2011 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values , stink bug adult numbers , and boll injury from the third week and fourth week of bloom ; ( b ) local sadie association indices between ndvi values from the third week of bloom and stink bug adult numbers and boll injury from the third and fourth weeks of bloom ; wob , week of bloom . 2012 inverse distance weighted spatial interpolations of ( a ) local sadie aggregation indices of ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom ; ( b ) associations between ndvi values and stink bug adult numbers from the third week of bloom and boll injury from the fourth week of bloom .
total number of adult stink bugs during the third week of bloom were positively correlated ( r = 0.28 , df = 66 , p = 0.0220 ) and positively associated ( x = 0.5288 , p < 0.0001 ; fig .
1b ) with ndvi values in 2011 and negatively correlated ( r = 0.25 , df = 72 , p = 0.0356 ) and unassociated ( x = 0.3838 , p = 0.9996 ) with ndvi values in 2012 . in the fourth week of bloom ,
total number of adult stink bugs were positively associated ( x = 0.5918 , p < 0.0001 ; fig .
1b ) with ndvi values in 2011 , and no stink bugs were found in the fourth week of bloom in 2012 . finally , total number of adult stink bugs were uncorrelated ( r = 0.05 , df = 36 , p = 0.7548 ) and unassociated ( x = 0.1064 , p = 0.2735 ) with ndvi values in 2012 during the fifth week of bloom .
boll injury during the third week of bloom was positively correlated ( r = 0.29 , df = 66 , p = 0.0195 ) and positively associated ( x = 0.3000 , p = 0.0094 ; fig .
1b ) with ndvi values in 2011 and were uncorrelated ( r = 0.02 , df = 72 , p = 0.8570 ) and unassociated with ndvi values in 2012 ( x = 0.0655 , p = 0.7075 ) .
boll injury during the fourth week of bloom was positively correlated ( r = 0.45 , df = 66 , p < 0.0001 ) and positively associated ( x = 0.3853 ,
p = 0.0010 ) with ndvi values in 2011 but were negatively correlated ( r = 0.49 , df = 36 , p = 0.0023 ) and negatively associated ( x = 0.4495 , p = 0.9967 , fig .
finally , boll injury was uncorrelated ( r = 0.03 , df = 36 , p = 0.8824 ) and unassociated ( x = 0.1693 , p = 0.8341 ) with ndvi values in 2012 during the fifth week of bloom .
both stink bug adult number and stink bug boll injury were associated with ndvi values .
associations with stink bug numbers and ndvi were strongest in 2011 in the third and fourth week of bloom , within a week that the remote sensing image was captured .
associations with ndvi and boll injury were strongest in 2011 with boll injury in the fourth week of bloom . because the procedure for determining injured bolls is an indirect method based on the plant response to stink bug feeding a time - lag between the phenological stage of the plant and stink bug injury should be expected .
however , it is unknown why stink bug numbers and stink bug injury were more prevalent in areas with high ndvi values in 2011 but less prevalent in the same areas during 2012 .
one caveat is that , relatively low numbers if stink bugs were captured in the study .
hence , the sample size might have been small to explain meaningful phenomena , even though the numbers we captured paralleled similar studies using the sweep net ( reay - jones et al . 2009 , tillman and cottrell 2015 ) . furthermore , an early - maturing variety was used in 2011 , while an early to mid- maturing variety was used in 2012 .
finally , responses in plant growth likely differed in both years due to the interaction of the variety , weather ( e.g. , from 15 may to 15 august 2011 , 35 more centimeters of precipitation and 167 more heat units , calculated with 16c as a base temperature ( dd60 ) , were recorded compared with the same dates in 2012 ) , planting date and other unrecorded or unknown factors .
ndvi values between both years tended to be consistently lower or higher relative to the same parts of the field .
therefore , although the plant growth patterns were likely different between the 2 yr , there was a range of growth within the field during each year . however , our hypothesis that stink bugs would be associated with highly vigorous cotton , as expressed by high - relative ndvi values , regardless of the conditions was only true in 1 yr .
one striking difference between the 2 yr was the prevalence of stink bugs in 2012 compared with 2011 .
stink bugs may have been attracted to vigorously growing cotton in 2011 , similar to l. lineolaris ( willers et al .
1999 ) , but the effect of conspecific stink bug density was a stronger driver of spatial distribution than the effect of plant vigor in 2012 .
stink bug injury was much higher in 2012 than 2011 ; therefore , one would expect the injury to be more spatially widespread given the fact that stink bugs are thought to feed on individual locules from multiple bolls rather than remaining on individual bolls and feeding on multiple locules ( willrich et al .
, there was no association between injured bolls during the third week of bloom and the ndvi values in 2012 , but adult stink bug numbers were negatively associated with ndvi values in the third week of bloom . in contrast , there was a positive association in 2011 between injured bolls during the third week of bloom and ndvi values , in addition to a positive association with stink bug adult numbers and ndvi values .
as a result , the third week of bloom in 2012 could have been a transition period , where stink bugs had injured most of the bolls of a preferred size in vigorously growing cotton and were moving to less vigorous growing areas to feed on previously unfed upon bolls of the appropriate size . without multiple points of remote sensing data and more data from the crop , this is not possible to determine , especially given the indeterminate growth pattern of cotton .
future studies should focus on the association among other crop characteristics and stink bug injury for remote sensing to be useful .
the above hypothesis is contingent on the association of vigorously growing cotton and the presence of medium - sized bolls that e. servus prefers ( siebert et al .
cotton growth patterns and boll size can vary from year to year ( siebert et al . 2005 ) , among varieties , and within the field , as seen in this experiment . the ability of e. servus to distinguish among boll sizes combined with its mobility may render this insect one of the more difficult to predict using remote sensing . in both years , abundances were likely affected by insecticides applied to reduce infestation levels .
however , stink bug captures at the rate of 0.010 adults and 0.002 nymphs per sweep in 2011 , as well as 0.013 adults and 0.001 nymphs in 2012 were in - line with a previous published paper from south carolina ( reay - jones et al .
2009 ) finding 0.006 adults and 0.003 nymphs per sweep in cotton . similarly , although total stink bug numbers were not reported , based on extrapolation from interpolation maps , similar numbers of c. hilaris were also captured with a sweep net in cotton in a georgia study ( tillman and cottrell 2015 ) .
however , relatively low stink bug captures in this study were one reason that boll injury was also assessed and are another reason that boll injury , rather than direct sampling of stink bugs , is the basis of the threshold for stink bug treatment in cotton ( bacheler et al .
boll injury in this study also showed only a trend to being spatially aggregated once in 2012 but not in 2011 .
our results mirror previous findings where stink bug densities and boll injury were shown to be aggregated using sadie only 25% of the time in southeastern us cotton ( reay - jones et al .
the sadie analysis determined associations and aggregations , particularly in 2011 for boll injury , when stink bug injury was relatively low . during the third week of bloom , areas of the field with local association indices between 1.66 and 3.98 could be visually overlaid with areas of the field with higher relative boll injury levels to a high degree of accuracy . in 2012 , when stink bug injury was higher , association indexes were not significant and
rough estimate of association . in general , associations between areas of boll injury and ndvi values were difficult to visually match on an intrafield basis , although on some dates they were significantly associated on a whole field level . this study suggests the potential of remote sensing as a tool to assist with sampling stink bugs in cotton , although more research is needed using ndvi and other plant measurements to better predict the incidence of stink bug injury . | sampling of herbivorous stink bugs in southeastern u.s .
cotton remains problematic .
remote sensing was explored to improve sampling of these pests and associated boll injury .
two adjacent 14.5-ha cotton fields were grid sampled in 2011 and 2012 by collecting stink bug adults and bolls every week during the third , fourth , and fifth weeks of bloom .
satellite remote sensing data were collected during the third week of bloom during both years , and normalized difference vegetation index ( ndvi ) values were calculated .
stink bugs were spatially aggregated on the third week of bloom in 2011 .
boll injury from stink bugs was spatially aggregated during the fourth week of bloom in 2012 .
the ndvi values were aggregated during both years .
there was a positive association and correlation between stink bug numbers and ndvi values , as well as injured bolls and ndvi values , during the third week of bloom in 2011 . during the third week of bloom in 2012 , ndvi values
were negatively correlated with stink bug numbers . during the fourth week of bloom in 2011 ,
stink bug numbers and boll injury were both positively associated and correlated with ndvi values . during the fourth week of bloom in 2012 ,
stink bugs were negatively correlated with ndvi values , and boll injury was negatively associated and correlated with ndvi values .
this study suggests the potential of remote sensing as a tool to assist with sampling stink bugs in cotton , although more research is needed using ndvi and other plant measurements to predict stink bug injury . |
coronoid hyperplasia is a rare condition which is macroscopically characterized by an increase in the dimensions of the coronoid process , resulting from an abnormal bony elongation of histologically normal bone .
the most obvious clinical feature of this condition is reduced mouth opening associated with facial asymmetry .
in addition to restricted opening , protrusion and lateral excursions may also be affected . the cause of restricted mandibular movement is widely accepted to be an impingement of elongated coronoid process to the posterior aspect of the zygomatic bone , or to the inside of the zygomatic arch , at the mouth opening .
unilateral coronoid hyperplasia is usually due to a trauma or a pathologic condition and is associated with facial asymmetry , more frequently seen in women with histologic chondromatous or neoplastic changes most similar to ostechondroma .
however , others consider enlargement of the coronoid process to be a hyperplastic process rather than a true neoplasm . the primary clinical feature of coronoid enlargement is limitation of opening .
shira and lister reported the appearance of bony enlargement of the zygoma on the affected side as demonstrated on a submentovertex radiograph .
a thorough clinical history should include information about the onset and progression of pain and other subjective symptoms . in the case of coronoid hyperplasia ,
ct also allows surgical planning due to its accuracy to detect coronoid process volume and morphology .
the following is a report of a case of unilateral coronoid enlargement with restricted opening as well as an obvious zygomatic asymmetry .
a 15-year - old male with no medical history of interest reported to the out - patient department of government college of dentistry , indore , with the chief complaint of facial asymmetry [ figure 1 ] .
preoperative clinical photograph of the patient , revealing facial asymmetry the patient mentioned asymptomatic difficulty in opening his mouth . on examination
, he was found to have a reduced mouth opening , with limited lateral and protrusive jaw movements and no hypertrophy of the masseter muscles [ figure 2 ] .
reduced mouth opening of the patient a ct scan was carried out with three - dimensional reconstruction ( 3d ct ) , showing unilateral hyperplasia of the coronoid process and contact between said process and the zygomatic arch , limiting mouth opening- movement [ figures 3 and 4 ] .
3d ct reconstruction of face revealing facial asymmetry on left side of face large anteroposterior extent of left mandibular coronoid process as seen in three - dimensional ct reconstruction image due to massive anteroposterior extent of coronoid , transzygomatic approach was considered .
a hemicoronal incision ending in a preauricular extension up to the lobule of the ear was made through the skin , subcutaneous tissue , and galea . in the temporal region , this incision was up to superficial layer of the temporal fascia . at the root of the zygomatic arch ,
hemicoronal incision was taken and zygomatic arch was exposed two plates were adapted , bridging the osteotomy site on the zygomatic arch .
the arch was osteotomized , temporalis muscle was stripped off the coronoid , and the process exposed adequately .
an osteotomy cut was taken on the coronoid and the enlarged coronoid stump was removed [ figure 6 ] . enlarged coronoid process mass removed zygomatic arch was repositioned in its proper anatomic position and fixation was performed with titanium plates [ figure 7 ] .
repositioned zygomatic arch in its proper anatomic position and fixation done with titanium plates the wound was then closed in layers .
the patient reported after 6 months with adequate mouth opening and marked reduction in facial asymmetry [ figures 8 and 9 ] .
marked reduction in facial asymmetry seen after 6 months postoperatively increase in mouth opening appreciated 6 months postoperatively
elongation of the coronoid process of the mandible was described for the first time in 1853 by von langenbeckin . in 1899 , jacob described a synovial joint formation between an elongated coronoid process and the homolateral zygomatic bone .
several approaches are advised by different authors for performing coronoidectomy , such as intraoral approach , submandibular approach , and coronal approach . since the coronoid was very large anteroposteriorly , we planned a transzygomatic approach
. it can be difficult to establish the best time to administer treatment in infants or preadolescents , although most authors agree that except in patients with very severe limitation of the mouth opening , it is best to perform the operation once the growth process has finished in order to avoid recurrence , deformity , or even restricted movement .
active physiotherapy is to be commenced immediately after surgery and continued for at least 6 months for satisfactory results .
postoperative cases of limitation of mouth opening caused by fibrosis , secondary to incorrect reorganization of a hematoma at the site of operation , and even recurrence in the growth of the coronoid process have been described .
coronoid process hyperplasia as one the causes of mandibular hypomobility is largely underdiagnosed as it is a very rare entity , but a thorough clinical and radiological examination can help to rationalize the line of management and the ultimate clinical outcome .
in addition to limited mouth opening , facial asymmetry was also a clinical finding . in case of massive anteroposterior extension of the coronoid , coronoidectomy with transzygomatic approach followed by active immediate physiotherapy | coronoid hyperplasia is a rare disorder , which effectively is a mechanical problem associated with limited mouth opening . in some cases , enlarged
coronoid pushes the zygoma forward causing facial asymmetry .
this article looks at the case of a 15-year - old boy reporting in opd of government college of dentistry , indore , with chief complaint of facial asymmetry .
transzygomatic approach was taken to get proper access to the enlarged coronoid .
coronoidectomy was performed which significantly reduced facial asymmetry .
postoperative healing was uneventful and recurrence was not reported . |
odontogenic carcinosarcoma is an extremely rare malignant tumor that is a mixture of odontogenic carcinoma and sarcoma , such that both epithelial and mesenchymal components show malignant activity12345 .
either purely epithelial or mesenchymal or a mixture of both tissues may form neoplastic lesions . clinically , the tumor presents with aggressive , recurrent and metastasizing features12345 .
odontogenic carcinosarcoma is related with some tumors such as ameloblastoma , ameloblastic fibroma , ameloblastic fibrosarcoma , and osteosarcoma .
etiologically , the aforementioned lesions may affect the pathogenesis of odontogenic carcinosarcoma by transformation , but this is unclear .
moreover , owing to the scarcity of reported cases , its clinical significance remains unexplored13678 .
the aim of this report is to discuss the pathological and clinical features of odontogenic carcinosarcoma and increase our knowledge on this rare entity .
a 61-year - old male presented with discomfort , intermittent paresthesia and a slightly painful mass of the left mandible in july 2011 . physical examination revealed nonvital tooth number 37 was painful to percussion .
imaging studies showed a large radiolucent lesion of the left mandible which had a round shape and clear margins .
the mass included the root of the left 2nd molar extending to the anterior aspect of the ascending ramus.(fig .
1 ) in addition , it involved the left alveolar canal and destroyed the inner cortex of mandible.(fig .
2 ) magnetic resonance imaging ( mri ) identified invasion of the adjacent bone marrow , outer soft tissue ( submandibular area ) , and masseter muscle with no significant lymphadenopathy.(fig .
3 ) an incisional biopsy was performed , and the diagnosis was osteosarcoma of the jaw . on bone scan , another active bone lesion was detected in the proximal one third of the right tibia .
there was an intramedullary osteolytic lesion in the proximal diaphysis of the tibia with a thin sclerotic rim on mri images of the right tibia .
subsequently , computed tomography ( ct ) guided biopsy revealed a linear core of tissue consisting of delicate trabeculae of woven bone in fibrous stroma and no epithelial element.(fig .
4 ) the diagnosis of the tibial lesion was fibrous dysplasia , and seemed to be irrelevant to oral cancer .
nevertheless , the possibility of distant metastasis could not be completely excluded by ct guided biopsy .
therefore , surgery and excisional biopsy for the tibial lesion was recommended , but the patient refused the procedure . on september , 2011 , segmental resection including wide excision of the surrounding soft tissues
after evaluation of the panoramic view , oropharynx ct and oropharynx mri , a margin of more than 1.5 cm was decided for safety .
simultaneously , a left regional lymph nodes ( level i to partially level iii ) was removed in a mass by selective neck dissection.(fig .
5 ) the mandibular condyle was preserved and the position of the mandible was stabilized by a reconstruction plate with a right iliac bone graft.(fig .
a ) on histopathologic examination , all the margins were free of tumor infiltration with no metastatic tumor in the lymph nodes ; the final diagnosis was changed to odontogenic carcinosarcoma . after the operation , the patient had a favorable reconstruction result without significant complications . on march 2012 ,
b ) histopathologically , a few soft tissues around the fixation device were identified as inflammatory granulation tissue , but tumor recurrence was not present . up to now , the patient continues to do well postoperatively .
the h&e - stained sections disclosed an invasive tumor consisting of epithelial and mesenchymal neoplastic components .
although the epithelial neoplastic component showed nests and strands similar to ameloblastoma , the tumor cells were obviously malignant cells showing aberrant cytologic atypia , hyperchromasia , and abnormal mitoses.(fig .
a ) the mesenchymal component was morphologically consistent with sarcoma showing marked pleomorphism , enlarged hyperchromatic nuclei , and increased nuclear - to - cytoplasmic ratio .
b ) the tumor also invaded into surrounding skeletal muscle and connective tissue . as a whole
this diagnosis was supported by positive immunohistochemistry for cytokeratin and cam 5.2 of carcinomatous element and for vimentin of sarcomatous element.(fig
odontogenic carcinosarcoma was defined by the 1992 world health organization classification of odontogenic tumor as " a very rare neoplasm , similar in pattern to ameloblastic fibrosarcoma , but in which both the epithelial and the ectomesenchymal components show cytological features of malignancy"9 . in other words
, the tumor can architecturally resemble an ameloblastic fibroma in which both the epithelial and the mesenchymal components show cytologic evidence of malignancy .
extremely rare lesion and related neoplasms such as ameloblastoma , ameloblastic fibroma , ameloblastic fibrosarcoma , and osteosarcoma are summarized in table 1 to avoid confusion later on .
they occur mainly in the mandible , are seen equally in both sexes , and have high recurrence rates except for osteosarcoma .
de novo odontogenic tumors arise from remnants that originated in the embryologic process of odontogenesis .
the process of odontogenesis involves reciprocal inductive interaction and is thought to play a role in the development of odontogenic neoplasms . in pre - existing lesions ,
malignant transformation usually becomes clinically manifest as a recurrent tumor . in terms of origin and histopathology
, ameloblastoma demonstrates epithelial cytologic features of a benign tumor which grows locally invasive , while ameloblastic fibroma exhibits features of a benign mixed odontogenic tumor which consists of odontogenic epithelial and mesenchymal cells .
ameloblastic fibrosarcoma , a rare malignant odontogenic neoplasm consisting of epithelial and mesenchymal components , is characterized by malignant activity in the mesenchyme only .
osteosarcoma of the jaw is the most common malignant bone tumor arising from primitive transformed cells of the mesenchyme that exhibit osteoblastic differentiation and produce malignant osteoid .
finally , odontogenic carcinosarcoma has distinctively malignant activity in both components and high potential of metastasis . in general ,
in particular , radiation therapy carries the possibility of inducing oncogenesis , and ameloblastic series have potential capacity for the radiation resistance8 . in other words ,
given the scarcity of reported cases , odontogenic carcinosarcoma is difficult to characterize . in a retrospective study of a few cases ( table 2)12345 , the patients ' ages ranged from 9 to 63 years , and there was no gender preference .
it was more common in the mandible , and mainly believed to progress from related neoplasms .
the pathogenesis of malignant transformation is not completely understood , but it is considered to occur after multiple recurrences and surgical treatments . nevertheless , the tumor may arise de novo such as in the present case without previous disease and surgical intervention . in previously published cases , the tumor has a fatality rate of more than 50% with high rates of recurrence and metastasis . aside from two cases ,
however , delair et al.4 and present cases without evidence of recurrence or metastasis after the first operation without use of adjuvant therapy .
due to the short follow - up period in these 2 cases , these results were not able to be confirmed . in summary , this is a case report of a rare occurrence of odontogenic carcinosarcoma .
this malignant tumor shows mixed features of both carcinomatous and sarcomatous elements on histopathological study , and is clinically very aggressive features with high rates of recurrence and metastasis .
if the tumor is detected at an early stage and has mild features , then adequate surgical treatment alone may lead to good outcomes
. a larger number of cases and prolonged follow - up are need to further clarify the nature of odontogenic carcinosarcoma . | odontogenic carcinosarcoma is an extremely rare malignant odontogenic tumor with only a few reported cases .
it is characterized by a true mixed tumor showing malignant cytology of both epithelial and mesenchymal components .
it has been assumed to arise from pre - existing lesions such as ameloblastoma , ameloblastic fibroma , and ameloblastic fibrosarcoma . to date , the reported cases have exhibited considerably aggressive clinical behavior .
the case of an odontogenic carcinosarcoma in the mandible of a 61-year - old male is described herein .
the tumor destroyed the cortex of the mandible and invaded the adjacent tissues .
treatment was performed by surgical resection and reconstruction .
the purposes of this article are to introduce odontogenic carcinosarcoma through this case study , to distinguish it from related diseases and to discuss features of the tumor in the existing literature . |
it possesses zoonotic potential in ecological niches and spreads rapidly within populations of its natural hosts ; humans , birds , and pigs .
classical swine h1n1 influenza virus has been circulating in the usa pig populations and was the dominant strains with invariable gene content , only slightly affected by genetic drift from its first isolation in 1930 until 1998 .
in sharp contrast to the european pig populations , the isolation of human h3n2 influenza virus from pigs in north america prior to 1998 was sporadic , and
there was no evidence that these wholly human h3n2 viruses became established in the swine population in north america .
crossspecies infection of humans , pigs , and birds is not unique and has occurred in america , europe , and asia .
however , in 1998 , a novel triplereassortant h3n2 influenza virus was isolated from pigs in the usa .
this isolate had pb2 and pa of unknown avian origin , pb1 , ha , and na of human a / sydney/5/97 ( h3n2 ) like origin , and np , m , ns of classical swine a / swine / iowa/15/1930 ( h1n1)like origin .
,
triplereassortant swine h3n2 virus underwent further reassortment events in 1999 and 2001 resulting in new h1n2 and h1n1 reassortant subtypes , both of which continue to circulate in pig populations in the usa and have a propensity for reassortment .
h1n2 and h1n1 virus subtypes carrying swine ha ( h1 ) have reassorted with a seasonal human h1n1 a / new caledonia/20/99 ( h1n1)like virus .
finally , a virus related to the h1n1 and h1n2 virus subtypes , containing an n1 and m of eurasian swine lineage had assembled , acquiring epidemiologic features allowing sustained humantohuman transmission and caused the new pandemic ( h1n1 ) in 2009 .
in this study
, we attempted to find precursor viruses or genes of pandemic h1n1 influenza 2009 among 25 swine influenza viruses isolated the west central usa region between 2007 and 2009 .
samples ( nasal swabs and lung tissue lavage ) were taken from pigs with or without clinical signs of respiratory disease via farmerfunded syndromic surveillance .
darby canine kidney cell culture from the abovementioned samples according to standard protocols recommended for influenza virus isolation ( http://www.who.int/vaccine_research/diseases/influenza/who_manual_on_animaldiagnosis_and_surveillance_2002_5.pdf ) .
viral rna was isolated with total rna isolation kit ( qiagen , valencia , ca , usa ) .
segmentspecific pcr fragments were obtained with onestep rtpcr ( qiagen ) using influenza aspecific primers , described elsewhere .
two overlapping pcr products for polymerase genes pa , pb1 , and pb2 were obtained .
purified pcr products were sequenced with bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , foster city , ca , usa ) , primers available upon request .
sequence traces were assembled into contigs with lasergene seqman pro ( dnastar , madison , wi , usa ) .
alignments for phylogenetic studies were created with data provided by the influenza virus resource at the national center for biotechnology information .
,
phylogenetic trees were built with mega 4.0 software ( center for evolutionary medicine and informatics , tempe , az , usa ) using maximum composite likelihood algorithm and neighborjoining method of tree topology reconstruction .
values lower than 95% were considered to be unreliable .
for the phylogenetics study , we selected the genes that form the pstrig cassette ( pb2 , pb1 , pa , np , and ns ) . ha and
na genes were analyzed using a blast approach .
,
we conducted phylogenetic studies of the pstrig cassettes , which were similar in all triplereassortants including pandemic ( h1n1 ) 2009 and viruses that had been included in this study .
influenza viruses h3n2 , h1n2 ( 1999 ) , and h1n1 ( 2001 ) with ha of classical swine origin , h1n2 ( 2004 ) and h1n1 ( 2005 ) ha of human origin and h3n1 subtype viruses representing evolutionary stages of triplereassortants were selected .
for the outgroups , we used gene precursors from viruses isolated prior to triplereassortant emergence .
we sequenced entire genomes of 25 swine influenza viruses isolated during 20072009 from pigs located predominantly in oklahoma , but also from texas and kansas .
these states were selected for retrospective surveillance because of their close social and economic relationships , their proximity to the mexico border , and because of the constant exchange of animals between swine populations in oklahoma and kansas and those in texas . among the 25 viruses analyzed , four viruses were h3n2 and 21 were h1n2 subtype .
the ha ( h3 ) sequences of the viruses were closely related ( 9798% homology ) to a / swine / ontario/33853/2005 ( h3n2 ) . the na ( n2 ) sequence of a / swine / oklahoma/011506/07 ( h3n2 ) was closely related to a / swine / oklahoma/18089/99 ( h3n2 ) , while all other ( table 1 ) na ( n2 ) of h3n2 virus isolates were most closely related to a / swine / british columbia/28103/2005 ( h3n2 ) ( 98% homology ) .
this reflects heterogeneity among recently isolated h3n2 influenza viruses circulating in the us pig population .
the 21 ha ( h1 ) genes were closely related ( 97% homology ) to a / new york/209/2003 ( h1n2 ) .
the na ( n2 ) genes of these viruses were also phylogenetically closely related to a / swine / ontario/33853/2005 ( h3n2 ) ( 9798% homology ) ( with the exception of a / swine / texas/008648/08 ( h1n2 ) .
a / swine / texas/008648/08 contained a different ( n2 ) that is most closely related to a / swine / iowa/533/99 ( h3n2 ) ( 95% homology ) .
these data confirm that viruses isolated recently carry surface proteins from the different eras of triplereassortant development .
triplereassortant swine influenza viruses isolated from swine in 20072009 in united states we have not found any gene segments that cluster phylogenetically with those of eurasian swine or avian origin influenza . to estimate the phylogenetic relationships of the novel pandemic ( h1n1 ) 2009 virus and recently isolated us triplereassortant swine viruses , we conducted studies on pstrig cassettes of h3n2 and h1n2 viruses .
the main question was ` is the pstrig cassette of the novel pandemic ( h1n1 ) 2009 virus descended from the triplereassortant swine viruses that emerged in the united states in 1998 ? .
we have found that the pb2 , pb1 , ns , and np genes studied here form a phylogenetically indistinguishable and common cluster with triplereassortants isolated between 1998 to 2009 ( 1 , 2 , 3 , 4 ) .
surprisingly , the pa genes of pandemic ( h1n1 ) 2009 virus reliably ( bootstrap index = 100 ) segregate from the pool of triplereassortants isolated in 19982009 ( figure 5 ) .
these data suggest that the pstrig cassette of pandemic ( h1n1 ) 2009 was assembling in the same period as the triplereassortant viruses but underwent an independent evolution that has been happening in parallel with triplereassortants circulating in pig populations prior 2009 .
comparably long evolution distance between the pandemic ( h1n1 ) 2009 cluster and precursor viruses on all pstrig trees reflect this time of undetected circulation .
analogous sequences , among our 25 swine influenza isolates , have been removed for improved presentation .
analogous sequences , among our 25 swine influenza isolates , have been removed for improved presentation .
analogous sequences , among our 25 swine influenza isolates , have been removed for improved presentation .
analogous sequences , among our 25 swine influenza isolates , have been removed for improved presentation .
analogous sequences , among our 25 swine influenza isolates , have been removed for improved presentation .
three influenza virus subtypes , h5n1 , h7n7 , and h9n2 , were proposed as potential pandemic viruses during recent pandemic preparedness planning .
several mechanisms were suggested to explain the direct adaptation of avian viruses to a human host
,
,
but instead , a new pandemic virus most likely emerged according to classical mechanisms by coinfection of the host with two or more subtypes and highlighting swine as a potential mixing vessel for influenza viruses of human , swine , and avian origin .
,
emergence and spreading of the pandemic ( h1n1 ) 2009 influenza virus was fulminant and unpredictable .
preliminary molecular data suggested that this virus was related to the north american triplereassortant swine viruses .
among the 25 geographically restricted american triplereassortants
, we found that only pb2 , pb1 , ns , and np genes were phylogenetically related to pandemic ( h1n1 ) 2009 .
this suggests that h3n2 triplereassortants became established in the north american swine population in 1998 after multiple reassorment events and had no single common precursor . among isolated viruses ,
we have not found h1n1 or h1n2 triplereassortants carrying swine ha ( h1 ) , viruses originating in 1999 . in our study , we have shown that all h1n2 isolates have h1 , derived from a / new caledonia/20/99 ( h1n1 ) like human influenza .
viruses of this genotype have become dominant in west central us region since 2003
and remain dominant between 20072009 .
in addition to differences in the pstrig cassette , pandemic ( h1n1 ) 2009 influenza contains m and na genes of eurasian swine origin .
no eurasian swine lineage genes genetically related to a / swine / belgium / wvl1/1979 ( h1n1 ) or more recent eurasian swine influenza reassortants carrying genome segments of a / port chalmers/1/1973 ( h3n2 ) like were found in the swine population in the west central usa region .
in contrast , the circulation of swine influenza viruses of american origin in asia is widespread ,
,
most likely due to monopolarity of swine export / import relations ( http://www.aphis.usda.gov/publications/animal_health/content/printable_version/ahr_web_pdf_07/h_chapter_7.pdfs ) .
because of the small sample size , the presence of eurasian swine lineage genes in north american pigs can not be completely excluded .
our studies agree with surveillance results published earlier
,
,
,
,
,
( http://www.who.int/vaccine_research/diseases/influenza/who_manual_on_animaldiagnosis_and_surveillance_2002_5.pdf ) , and it is possible that there is no significant circulation of eurasian swine influenza virus in west central usa pig population . previously detected swine influenza cases in humans in the usa did not result in new pandemic viruses .
at least 11 sporadic cases of triplereassortant swine influenza a ( h1 ) in humans were reported in the period 20052009 in the usa .
detection of these cases among the myriad seasonal influenza cases almost excludes the possibility of hidden circulation of a novel h1n1 influenza virus during its evolution into a potentially pandemic virus .
this circumstantial evidence , along with our studies that show significant phylogenetic distance between pandemic ( h1n1 ) 2009 and triplereassortant swine viruses based on the analysis of the pstrig cassette , and the absence of eurasianorigin viruses and genes do not support a hypothesis of reassortment and emergence of pandemic ( h1n1 ) 2009 in the usa .
however , where these events actually took place remains unclear , but isolation in hong kong and characterization of the strain a / swine / hk/915/04 ( h1n2 ) , which has seven of eight gene segments , phylogenetically closely related to pandemic ( h1n1 ) 2009 influenza virus ,
suggests that the possibility of emergence in southeast asia before 2009 also could not be excluded .
triplereassortant swine influenza viruses have demonstrated ability to circulate in swine populations , exchange genome segments , and become endemic in the swine populations of north america and asia . even though the pandemic ( h1n1 ) 2009 precursor viruses have not been found in the swine or human populations yet , considerable effort should be made to isolate these precursors to better understand the key factors determining the ability of influenza viruses to cause pandemics . | please cite this paper as : evseenko et al .
( 2011 ) genetic composition of contemporary swine influenza viruses in the west central region of the united states of america .
influenza and other respiratory viruses doi : 10.1111/j.17502659.2010.00189.x .
background because of continuous circulation in different animal species and humans , influenza viruses have hostspecific phenotypic and genetic features .
reassortment of the genome segments can significantly change virus phenotype , potentially generating virus with pandemic potential . in 2009 ,
a new pandemic influenza virus emerged .
objectives in this study , we attempted to find precursor viruses or genes of pandemic h1n1 influenza 2009 among 25 swine influenza viruses , isolated in the west central region of the united states of america ( usa ) , between 2007 and 2009 .
the phylogenetically similar triplereassortant internal genes ( pstrig ) cassette of all the viruses studied here as well as the pstrig cassette of pandemic h1n1 viruses have close but equidistant phylogenetic relationships to the early triplereassortant swine h3n2 influenza a isolated in the usa in 1998 .
methods samples ( nasal swabs and lung tissue lavage ) were taken from swine with or without clinical signs of respiratory disease via farmerfunded syndromic surveillance .
all studied viruses were isolated in madin
darby canine kidney cell cultures from the abovementioned samples according to standard protocols recommended for influenza virus isolation .
sequences were obtained using bigdye terminator v3.1 cycle sequencing kit .
phylogenetic trees were built with mega 4.0 software using maximum composite likelihood algorithm and neighborjoining method for tree topology reconstruction .
results among the 25 viruses studied , we have not found any gene segments of eurasian origin .
our results suggest that pandemic h1n1 viruses diverged and are not directly descended from swine viruses that have been circulating in usa since 1998 . |
pulmonary arterial hypertension ( pah ) is a cardiovascular complication that causes death in thalassemic patients .
pah in thalassemia was reported with a high incidence in several studies.14 there was evidence that pah in thalassemia is associated with platelet activation.57 acetylsalicylic acid ( asa ) lowers the thromboxane
a previous cohort study found that the rise of arterial partial pressure of oxygen ( pao2 ) in ten out of the 12 -thalassemic patients after asa ten grains or persantin administration for 24 weeks indicates that the observed hypoxemia is due to reversible platelet aggregation.9 a recent study showed that asa decreased pulmonary artery pressure , reduced right ventricular hypertrophy , and improved survival in the monocrotaline animal model of pah.10 the erythrocytes and platelets of thalassemic patients contained higher levels of reactive oxygen and lower levels of intracellular glutathione than normal erythrocytes and platelets11 that play a role in oxidative stress , thereby leading to the proper therapy .
studies of antiplatelet therapy are required before there are conclusive treatment guidelines for thalassemic patients with pah . in clinical practice
the aim of the research was to compare after 1 year of follow - up the pulmonary artery systolic pressure ( pasp ) of patients with thalassemia and pah to whom asa was and was not prescribed .
a retrospective cohort study was conducted at the hematological outpatient clinic at chiang rai hospital , chiang rai , thailand .
all new adult ( aged 15 years ) cases of thalassemia with pah defined as pasp > 35 mmhg by doppler echocardiography ( echo ) from january 2007 to january 2012 were evaluated and followed from the first month to 12 months later .
81 mg was prescribed daily for 1 year , and in another group , no asa was prescribed , due to contraindications for asa ( including bleeding , gastrointestinal side effects , and thrombocytopenia [ platelets < 10010/l ] ) .
pasp , functional class status , clinical right heart failure , and oxygen saturation were measured at baseline and at the end of 12 months .
the functional class status was defined by the new york heart association ( nyha ) classification.12 patients with other causes of pah or who used other antiplatelets and/or anticoagulants were excluded .
the study protocol was approved by the institutional research ethics committee and was carried out in accordance with the declaration of helsinki .
complete two - dimensional , m - mode , and doppler ( pulsed wave , continuous wave , and color ) echo was performed at rest . in the study patients ,
a tricuspid regurgitation jet was sought from all available midprecordial and apical positions until a flow signal with the maximum spectral representation of the highest velocities was obtained .
peak velocity was recorded from a holosystolic regurgitant jet . from the maximum velocity ( v ) of the regurgitant jet
, the systolic pressure gradient ( p ) between the right ventricle and the right atrium was calculated by modified bernoulli equation ( p = 4v2).13 right atrial pressure was estimated by the response of the inferior vena cava diameter to inspiration .
right atrial pressure was assumed to be 5 mmhg if the inferior vena cava completely collapsed with inspiration , 10 mmhg if the inferior vena cava dia meter decreased > 50% during inspiration , and 15 mmhg if it decreased < 50% .
if the inferior vena cava diameter was > 2.5 cm and reduced by < 50% during inspiration , right atrial pressure was assumed to be 20 mmhg.14 adding the transtricuspid gradient to the mean right atrial pressure provided the right ventricular systolic pressure or peak systolic pulmonary arterial pressure in the absence of right ventricular outflow tract obstruction .
pah was defined as pasp > 35 mmhg.15 in cases with an absence of tricuspid regurgitation , at the end of the diastolic pulmonic regurgitant the velocity was calculated using the pressure gradient between the pulmonary artery and right ventricular end diastolic pressure : pulmonary artery pressure = 4 ( vpr)2 + right atrial pressure .
the baseline characteristics were compared using exact probability tests for categorical variables ; student s t - test or wilcoxon rank - sum test was used to compare the mean difference of continuous variables .
propensity scores for prescribing asa versus no asa were calculated from a logistic regression model that estimated the likelihood of prescribing asa based on the observed patient characteristics .
data are presented by frequency , percentage , mean , standard deviation ( sd ) , beta coefficient , 95% confidence interval [ ci ] , and p - value .
all statistical analyses were two - tailed . a p - value of < 0.05 was considered statistically significant .
complete two - dimensional , m - mode , and doppler ( pulsed wave , continuous wave , and color ) echo was performed at rest . in the study patients ,
a tricuspid regurgitation jet was sought from all available midprecordial and apical positions until a flow signal with the maximum spectral representation of the highest velocities was obtained .
peak velocity was recorded from a holosystolic regurgitant jet . from the maximum velocity ( v ) of the regurgitant jet ,
the systolic pressure gradient ( p ) between the right ventricle and the right atrium was calculated by modified bernoulli equation ( p = 4v2).13 right atrial pressure was estimated by the response of the inferior vena cava diameter to inspiration .
right atrial pressure was assumed to be 5 mmhg if the inferior vena cava completely collapsed with inspiration , 10 mmhg if the inferior vena cava dia meter decreased > 50% during inspiration , and 15 mmhg if it decreased < 50% .
if the inferior vena cava diameter was > 2.5 cm and reduced by < 50% during inspiration , right atrial pressure was assumed to be 20 mmhg.14 adding the transtricuspid gradient to the mean right atrial pressure provided the right ventricular systolic pressure or peak systolic pulmonary arterial pressure in the absence of right ventricular outflow tract obstruction .
pah was defined as pasp > 35 mmhg.15 in cases with an absence of tricuspid regurgitation , at the end of the diastolic pulmonic regurgitant the velocity was calculated using the pressure gradient between the pulmonary artery and right ventricular end diastolic pressure : pulmonary artery pressure = 4 ( vpr)2 + right atrial pressure .
the baseline characteristics were compared using exact probability tests for categorical variables ; student s t - test or wilcoxon rank - sum test was used to compare the mean difference of continuous variables .
propensity scores for prescribing asa versus no asa were calculated from a logistic regression model that estimated the likelihood of prescribing asa based on the observed patient characteristics .
data are presented by frequency , percentage , mean , standard deviation ( sd ) , beta coefficient , 95% confidence interval [ ci ] , and p - value .
a total of 63 thalassemia patients with pah , 53 with e/-thal , six with homozygous -thal , and four with hb h disease were evaluated .
there were 47 ( 74.6% ) in the asa group and 16 ( 25.4% ) in the no asa group .
patients in the second group had the following contraindications for asa : seven ( 43.7% ) bleeding , seven ( 43.7% ) gastrointestinal side effects , and two ( 12.5% ) thrombocytopenia .
the mean age was 35.916.8 years and 28.314.1 years , and the mean pasp was 51.913.7 mmhg and 45.69.9 mmhg in the asa group and the no asa group .
the baseline characteristics of the patients did not differ significantly except for red cell transfusion and right ventricular diameter ( table 1 ) .
the patients in the asa group received fewer blood transfusions during the follow - up time of 12 months than those without asa ( 6.24.8 vs 11.04.6 units , p=0.002 ) , and the asa group had wider right ventricular diameter than the no asa group ( 29.55.8 vs 25.54.2 mm , p=0.016 ) . during the follow - up period of 12 months , pasp increased in 12 of 47 ( 25.5% ) patients in the asa group and in three of 16 ( 18.7% ) in the no asa group .
echo findings showed that the patients in the asa group had a higher mean pasp than the no asa group , but there was no statistical difference . after adjusting for propensity score ( based on type of thalassemia , splenectomy , o2 saturation , nyha classification , red cell transfusion , clinical right heart failure , hemoglobin level , platelet count ,
nucleated red cell , serum ferritin level , baseline pasp , left ventricular ejection fraction , main pulmonary artery diameter , right ventricular diameter , right ventricular systolic function , and diastolic function ) , there were no statistically significant differences in the functional class status , clinical right heart failure , and oxygen saturation between the two groups ( table 3 ) .
asa , as compared with no asa , did not significantly reduce pasp ( adjusted difference 0.95 ; 95% ci 16.99 to 15.10 ; p=0.906 ) .
in the present study , asa did not improve the functional class status , clinical right heart failure , and oxygen saturation and did not have a significant effect on pasp after 12 months .
both the asa and the no asa groups had reduced pasp by 4.711.0 mmhg and 4.37.1 mmhg with no clinical or statistical significance .
the results of this study were similar to those of a randomized clinical trial of asa and simvastatin for pah , which concluded that the results did not support the routine treatment of patients with pah with these medications.16 and thalassemia patients who experienced a thromboembolic event and received asa afterwards had a lower recurrence of thromboembolic event compared with those who were not taking asa , although these differences were not statistically significant.17 subgroup analysis of e/-thal ; asa , as compared with no asa , did not significantly reduce the pasp ( adjusted difference 3.62 ; 95% ci 11.84 to 4.60 ; p=0.225 ) . for the transfusion- and nontransfusion - dependent patients ,
in contrast to the previous study that reported that there was a rise of arterial pao2 after high - dose asa administration in thalassemia patients with pah,9 we found that oxygen saturation rose in the patients who had received low - dose asa but without statistical significance ( adjusted difference 0.31 ; 95% ci 1.71 to 2.33 ; p=0.757 ) .
pah developed in thalassemic patients because of platelet activation and hypercoagulable state.18,19 therefore , it was reasonable that antiplatelet therapy alone may be less effective for pah in thalassemia .
further studies of pah management will be required before conclusive recommendations can be made for anti - platelet or anticoagulant therapy or prevention for high risk of pah in thalassemia ( postsplenectomy status3 ) .
the present study may have some limitations , as it was not a randomized controlled trial , the duration of treatment was only 12 months , and the 6-minute walk test was not used as a clinical outcome due to its retrospective nature .
the lack of cardiac catheterization to confirm pasp is a limitation for a therapeutic trial of pah.20 however , echo is more sensitive and is a commonly used noninvasive tool to screen pah .
more evidence from observational studies or preferably randomized clinical trials may be required before it can be concluded that asa would offer any beneficial effects to these patients .
the current therapies for pah in thalassemia include the use of sildenafil21,22 and bosentan.23 however , a large cohort of patients on the topic are needed before definite recommendations can be made .
the present findings suggested that low - dose asa may not have a beneficial effect on pasp after 1-year treatment of pah in thalassemia . | objectiveto compare pulmonary artery systolic pressure ( pasp ) between thalassemic patients with pulmonary arterial hypertension ( pah ) for whom acetylsalicylic acid ( asa ) was and
was not prescribed after 1 year.methodsa retrospective cohort study was conducted at the hematological outpatient clinic at chiang rai hospital , chiang rai , thailand .
all new cases of thalassemia with pah from january 2007 to january 2012 were studied at the first month and at 12 months .
the patients were classified into two groups . in one group , asa 81 mg daily was prescribed for 1 year , whereas in another group no asa was prescribed , due to its contraindications , which included bleeding , gastrointestinal side effects , and thrombocytopenia .
pasp , estimated by a doppler echocardiography , was measured by the same cardiologist .
propensity score adjustment was used to control confounding variables by indication and contraindication .
multivariable regression analysis was used to evaluate the effects of asa.resultsof the 63 thalassemia patients with pah , there were 47 ( 74.6% ) in the asa group and 16 ( 25.4% ) in the no asa group .
asa , as compared with no asa , did not significantly reduce pasp ( adjusted difference 0.95 ; 95% confidence interval 16.99 to 15.10 ; p=0.906).conclusionlow - dose asa may not have a beneficial effect on pasp after 1 year of treatment of pah in thalassemia . |
laparoscopic cholecystectomy ( lc ) has been a standard operation and replaced the open cholecystectomy ( oc ) rapidly because the technique resulted in less pain , smaller incision , and faster recovery . from 1987 ,
in which year mouret performed the first lc , the new technique developed fast . despite the benefits of lc
, there is still concern about some severe complications of this technique , especially bile duct injury ( bdi ) . in order to reduce bdi
, surgeons must expose calot 's triangle clearly without a sense of touch in lc .
one is energized dissection , such as electric coagulation , monopolar electrosurgery , and ultrasonic scalpel .
based on some reports of multicenter , large - sample trial studies , the rate of bdi in lc was 0.100.42% around the world .
our center firstly used blunt dissection by flushing and aspiration to expose calot 's triangle and had a satisfactory result . from 2003 to 2015 , lc was performed on 21,497 patients in 12 years .
the calot 's triangle was exposed by blunt dissection routinely before transecting the cystic duct .
above all cases , bdi was diagnosed in 20 patients ( 0.09% ) . in this study , we were to introduce the technique of blunt dissection and to show the bdi cases in our institution .
from 2003 to 2015 , 21,497 lcs were performed in department of general surgery , sir run run shaw hospital of zhejiang university .
there were 7470 males and 14,027 females , aged 50.3 years ( 1484 years ) .
in all the cases , 19,343 patients were diagnosed as cholecystolithiasis , including 16,754 chronic cholecystolithiasis and 2589 acute cholecystolithiasis ; 2154 patients were diagnosed as cholecystic polypus .
the technique of blunt dissection with flushing and aspiration was used in each operation to expose calot 's triangle .
patients were placed in supine position and 4 entries ( 1 umbilical , 1 epigastric , and 2 in the right flank ) were made .
a veress needle was inserted directly under the umbilicus to allow for the flow of carbon dioxide into the peritoneal cavity , and the pneumoperitoneum was established at 1214 mmhg .
the cystic artery and cystic duct were ligated with clips after the cystic duct , the common hepatic duct , and the common hepatic duct were identified [ figure 1 ] .
the main equipment of the technique is laparoscopic suction and irrigation tubes . in the picture
, we identified the cystic duct ( a ) , the common hepatic duct ( b ) and the common hepatic duct ( c ) .
prophylactic antibiotics were used in all operated patients ( half an hour before surgery , intravenously ) . in cases of conversion from lc to oc , the preferred incision was a subcostal incision .
as inflammation around the gallbladder , the laparoscopic cholecystitis grading ( lcg ) was classified into four grades ( g0g3 ) [ table 1 ] .
laparoscopic cholecystitis grading the strasberg classification was used to describe the type of bdi ( based on surgical and cholangiographic findings ) and the dindo - clavien classification was used to stratify the severity of complications [ tables 2 and 3 ] .
type of bile duct injury the dindo - clavien classification cns : central nervous system ; ic : intermediate care ; icu : intensive care unit .
patient demographics include age and gender ; operation time , rate of conversion , surgeon ; the hospitalization days , complications , and types and treatments of bdi .
from 2003 to 2015 , 21,497 lcs were performed in department of general surgery , sir run run shaw hospital of zhejiang university .
there were 7470 males and 14,027 females , aged 50.3 years ( 1484 years ) .
in all the cases , 19,343 patients were diagnosed as cholecystolithiasis , including 16,754 chronic cholecystolithiasis and 2589 acute cholecystolithiasis ; 2154 patients were diagnosed as cholecystic polypus .
the technique of blunt dissection with flushing and aspiration was used in each operation to expose calot 's triangle .
patients were placed in supine position and 4 entries ( 1 umbilical , 1 epigastric , and 2 in the right flank ) were made .
a veress needle was inserted directly under the umbilicus to allow for the flow of carbon dioxide into the peritoneal cavity , and the pneumoperitoneum was established at 1214 mmhg .
the cystic artery and cystic duct were ligated with clips after the cystic duct , the common hepatic duct , and the common hepatic duct were identified [ figure 1 ] .
the main equipment of the technique is laparoscopic suction and irrigation tubes . in the picture
, we identified the cystic duct ( a ) , the common hepatic duct ( b ) and the common hepatic duct ( c ) .
prophylactic antibiotics were used in all operated patients ( half an hour before surgery , intravenously ) . in cases of conversion from lc to oc , the preferred incision was a subcostal incision .
as inflammation around the gallbladder , the laparoscopic cholecystitis grading ( lcg ) was classified into four grades ( g0g3 ) [ table 1 ] .
the strasberg classification was used to describe the type of bdi ( based on surgical and cholangiographic findings ) and the dindo - clavien classification was used to stratify the severity of complications [ tables 2 and 3 ] .
type of bile duct injury the dindo - clavien classification cns : central nervous system ; ic : intermediate care ; icu : intensive care unit .
patient demographics include age and gender ; operation time , rate of conversion , surgeon ; the hospitalization days , complications , and types and treatments of bdi .
during the 12-year study period , 21,497 lcs were performed by blunt dissection with flushing and aspiration , operation time 45 ( range : 20300 minutes ) minutes , average bleeding 14 ml .
mean hospitalization days were 2.1 days , and about 46% had hospitalization days of 1 day or less .
two hundred and thirty - nine cases had conversion because of severe adhesion , carcinoma of the gallbladder , gallbladder - intestine fistula , and bdi .
twenty patients had diagnosis of bdi , representing an incidence of 0.09% . in 11 cases ( 55% ) ,
three patients were acute cholecystolithiasis , and 17 patients were chronic cholecystolithiasis . based on the lcg , there were 8 g1 , 7 g2 , and 5 g3 .
the operating surgeon was a senior hpb staff in 17 cases ( 85% ) , an hpb surgery fellow in 2 cases ( 10% ) , and a general surgery resident in 1 case ( 5% ) .
the bdis in these 20 cases were as follows : 6 cases of common bdi ( 4 cases of lateral injury and 2 circumferential injury ) , 2 of common hepatic duct injury , 1 of right hepatic duct injury , 1 of accessory right hepatic duct , 1 of aberrant bdi , 1 of biliary stricture , 1 of biliary duct perforation , 3 of hemobilia , and 4 of bile leakage .
one case of right hepatic duct injury and 1 case of common hepatic duct injury were caused by thermal damage of electric coagulation . according to strasberg - bismuth classification of bdi , there were 4 type a ( 20% ) , 1 type b ( 5% ) , 1 type c ( 5% ) , 10 type d ( 50% ) , and 1 type e2 ( 5% ) .
operative data and overall perioperative outcomes in patients with bdi after lc chd : common hepatic duct ; cbd : common bile duct ; bdi : bile duct injury ; lcg : laparoscopic cholecystitis grading ; lc : laparoscopic cholecystectomy ; ss : secondary surgery ; ryh : roux - en - y hepaticojejunostomy .
seven cases were diagnosed bdi after operations and had conservative treatment . above the 20 cases , 17 cases ( 85% ) were selective operations ( 8 cases [ 47% ] were converted to repair bdi , 2 cases received second surgeries ) and 3 cases were emergency operations ( 2 cases [ 67% ] were converted to repair bdi ) .
ten cases were converted to open surgery ( 6 cases of common bdi , 2 of common hepatic duct injury , 1 of right hepatic duct injury , and 1 of accessory right hepatic duct ) .
the most common procedure performed was a primary closure in 8 with t - tube placement , and 2 roux - en - y hepaticojejunostomies ( 2 with a circumferential injury of the common bile duct ) .
two cases ( 1 biliary duct perforation and 1 bile leakage ) received the second surgery : 1 case of biliary duct perforation was performed roux - en - y hepaticojejunostomy and stayed in intensive care unit ( icu ) for more than 2 weeks ; 1 case of bile leakage was performed peritoneal lavage and drainage .
one patient had jaundice after surgery and was diagnosed biliary stricture by magnetic resonance cholangiopancreatography .
the other 6 cases ( 3 bile duct bleeding and 3 bile leakage ) also had conservative treatment with ultrasound guided percutaneous puncture catheter drainage and achieved remission of symptoms [ tables 4 and 5 ] .
twelve cases ( 60% ) were grade ii and 6 cases ( 30% ) of leakage or bleeding required ultrasound guided percutaneous puncture catheter drainage ( grade iiia ) .
common bile duct perforation with bile leakage developed in 1 case ( iv b ) , which received the second surgery to do roux - en - y hepaticojejunostomies and stayed in icu for more than 1 month because of multiorgan dysfunction .
the other one had a bile leakage with severe infection and received the second surgery to lavage and drainage ( iiib ) .
mean hospitalization days was 21 days ( range : 5158 days ) , and all the patients were fully recovered .
three patients were acute cholecystolithiasis , and 17 patients were chronic cholecystolithiasis . based on the lcg , there were 8 g1 , 7 g2 , and 5 g3 .
the operating surgeon was a senior hpb staff in 17 cases ( 85% ) , an hpb surgery fellow in 2 cases ( 10% ) , and a general surgery resident in 1 case ( 5% ) .
the bdis in these 20 cases were as follows : 6 cases of common bdi ( 4 cases of lateral injury and 2 circumferential injury ) , 2 of common hepatic duct injury , 1 of right hepatic duct injury , 1 of accessory right hepatic duct , 1 of aberrant bdi , 1 of biliary stricture , 1 of biliary duct perforation , 3 of hemobilia , and 4 of bile leakage .
one case of right hepatic duct injury and 1 case of common hepatic duct injury were caused by thermal damage of electric coagulation . according to strasberg - bismuth classification of bdi
, there were 4 type a ( 20% ) , 1 type b ( 5% ) , 1 type c ( 5% ) , 10 type d ( 50% ) , and 1 type e2 ( 5% ) .
operative data and overall perioperative outcomes in patients with bdi after lc chd : common hepatic duct ; cbd : common bile duct ; bdi : bile duct injury ; lcg : laparoscopic cholecystitis grading ; lc : laparoscopic cholecystectomy ; ss : secondary surgery ; ryh : roux - en - y hepaticojejunostomy .
seven cases were diagnosed bdi after operations and had conservative treatment . above the 20 cases , 17 cases ( 85% ) were selective operations ( 8 cases [ 47% ] were converted to repair bdi , 2 cases received second surgeries ) and 3 cases were emergency operations ( 2 cases [ 67% ] were converted to repair bdi ) .
ten cases were converted to open surgery ( 6 cases of common bdi , 2 of common hepatic duct injury , 1 of right hepatic duct injury , and 1 of accessory right hepatic duct ) .
the most common procedure performed was a primary closure in 8 with t - tube placement , and 2 roux - en - y hepaticojejunostomies ( 2 with a circumferential injury of the common bile duct ) . in 1 case ( aberrant bdi ) , the injury was repaired laparoscopically .
nine cases were diagnosed after operations . two cases ( 1 biliary duct perforation and 1 bile leakage ) received the second surgery : 1 case of biliary duct perforation was performed roux - en - y hepaticojejunostomy and stayed in intensive care unit ( icu ) for more than 2 weeks ; 1 case of bile leakage was performed peritoneal lavage and drainage .
one patient had jaundice after surgery and was diagnosed biliary stricture by magnetic resonance cholangiopancreatography .
this patient received conservative treatment and achieved remission of symptoms in 1 week . the other 6 cases ( 3 bile duct bleeding and 3 bile leakage )
also had conservative treatment with ultrasound guided percutaneous puncture catheter drainage and achieved remission of symptoms [ tables 4 and 5 ] .
twelve cases ( 60% ) were grade ii and 6 cases ( 30% ) of leakage or bleeding required ultrasound guided percutaneous puncture catheter drainage ( grade iiia ) .
common bile duct perforation with bile leakage developed in 1 case ( iv b ) , which received the second surgery to do roux - en - y hepaticojejunostomies and stayed in icu for more than 1 month because of multiorgan dysfunction .
the other one had a bile leakage with severe infection and received the second surgery to lavage and drainage ( iiib ) .
mean hospitalization days was 21 days ( range : 5158 days ) , and all the patients were fully recovered .
the rate of bdi in lc was 0.100.42% , and common bdi and common hepatic duct injury were the most common types , ranged from 61.0% to 77.5% .
before the adoption of this technique , 14,302 cases of laparoscopic cholecystectomies were performed in our institute by the conventional procedure and the occurrence rate of biliary injury was 0.37% which was significant higher than that reported in the current study ( p < 0.05 , chi - square analysis ) , but we did not use this comparison to approve the advantage of the current procedure because of the potential impact of the learning curve .
we reviewed large - series reports of lc , which had more than 10,000 cases on pubmed [ table 6 ] .
the occurrence rate of biliary injury in our series is lower than that in these reports that might account for the potential advantage of this procedure . according to these reports ,
causes of bdi include : ( 1 ) severe adhesion , it is difficult to expose calot 's triangle clearly ; ( 2 ) abnormal anatomy of bile duct ; ( 3 ) improper operation , such as thermal injury by electric coagulation , monopolar electrosurgery , and ultrasonic scalpel , bile duct stricture caused by clips ; ( 4 ) surgeons are not trained well .
showed an rct study in 2014 , the use of energetic dissection in lc , like electric coagulation and ultrasonic scalpel , was associated with higher risk of complication .
reported that the rate of bdi in surgeon s first lc was 1.7% , but in one 's 50 lc the rate reduced to 0.17% .
so in lcs , experienced and well - trained surgeons are very important . besides , the most important measure to avoid bdi was exposing the calot 's triangle clearly . in conclusion ,
our center firstly used blunt dissection by flushing and aspiration to expose the calot 's triangle .
the technique has two advantages : ( 1 ) blunt dissection , which can avoid thermal injury by using energetic dissection and reduce injury by sharp dissection ; ( 2 ) the equipment is cheap , simple , and easy for young surgeons to learn .
surgeons use laparoscopic suction and irrigation tubes to flush and aspirate the calot 's triangle repeatedly .
two hundred and thirty - nine cases ( 1.1% ) were converted to open , 20 cases ( 0.09% ) were bdi and 11 cases of bdi ( 55% ) were diagnosed in operations . compared with other institution around the world ( over 10,000 lcs ) , we had a significantly lower incidence of bdi in lcs [ table 6 ] .
in the 20 cases , there were 17 senior surgeons , 2 fellows , and 1 resident .
above all , we found that the rate of bdi and converted using our blunt dissection were both less than other large sample trial studies around the world . according to the analysis of surgeons ,
85% cases were performed by senior surgeons and only 15% cases were performed by junior surgeons .
so we believed that the technique of blunt dissection with flushing and aspiration is the best and easiest solution for surgeons to expose the calot 's triangle and reduce the incidence of bdi .
it has three advantages : ( 1 ) laparoscopic suction and irrigation tubes are blunt tools and have a high safety in dissection ; ( 2 ) it is easy for surgeons to control and it can flush and aspirate repeatedly to keep operation field clear without changing equipment frequently ; and ( 3 ) laparoscopic suction and irrigation tubes are cheap and available equipments , it can reduce the cost of operations .
the technique of blunt dissection by flushing and aspiration to expose the calot 's triangle is safe and simple .
detailed operation procedures : first step is open a hole in the anterior sheath over the calot 's triangle wall by dissecting forceps , then using laparoscopic suction and irrigation tubes to flush and aspirate the calot 's triangle repeatedly through the anterior sheath hole until we see the cystic duct clearly .
second step is flushing and aspiration in a direction parallel to the cystic duct , from its length down toward the junction of the cystic duct , common bile duct , and common hepatic duct until exposing the bile duct clearly .
third , in order to expose the cystic artery and abnormal anatomy , we can use the same way to flush and aspirate the tissue in the calot 's triangle and identify all the location of main arteries and bile ducts .
if the calot 's triangle can not be dissected clearly with severe adhesion , in our experience , we can not cut - off the arteries and bile ducts blindly , and we can use intraoperative cholangiography to locate bile ducts .
so we believe that blunt dissection by flushing and aspiration is an effective solution to expose bile ducts and arteries , especially abnormal anatomy .
besides , based on our experience , there were some notices for prevention of bdi : ( 1 ) check carefully before operations and evaluate the abnormal anatomy ; ( 2 ) do not cut - off the ducts before the main bile ducts are not clear ; ( 3 ) reduce the use of energetic dissection ; ( 4 ) intraoperative cholangiography can show the bile ducts clearly and prevent bdi , but now many experts considered that it is unnecessary for surgeons to perform intraoperative cholangiography in all the lcs . in conclusion ,
blunt dissection by flushing and aspiration to expose calot 's triangle , and results proved it to be a valuable method to avoid bdi .
| background : laparoscopic cholecystectomy ( lc ) has been a standard operation and replaced the open cholecystectomy ( oc ) rapidly because the technique resulted in less pain , smaller incision , and faster recovery .
this study was to evaluate the value of blunt dissection in preventing bile duct injury ( bdi ) in laparoscopic cholecystectomy ( lc).methods : from 2003 to 2015 , lc was performed on 21,497 patients , 7470 males and 14,027 females , age 50.3 years ( 1484 years ) .
the calot 's triangle was bluntly dissected and each duct in calot 's triangle was identified before transecting the cystic duct.results:two hundred and thirty - nine patients ( 1.1% ) were converted to open procedures .
the postoperative hospital stay was 2.1 ( 0158 ) days , and cases ( 46% ) had hospitalization days of 1 day or less , and 92.8% had hospitalization days of 3 days or less ; bdi was occurred in 20 cases ( 0.09% ) including 6 cases of common bdi , 2 cases of common hepatic duct injury , 1 case of right hepatic duct injury , 1 case of accessory right hepatic duct , 1 case of aberrant bdi 1 case of biliary stricture , 1 case of biliary duct perforation , 3 cases of hemobilia , and 4 cases of bile leakage.conclusion:exposing calot 's triangle by blunt dissection in laparoscopic cholecystectomy could prevent intraoperative bdi . |
distal radius fractures are one of the the most common adult fractures encountered during the clinical practice of an orthopedic surgeon.12 although several methods of treatment are suggested for these fractures , there are still controversies about the best treatment approach in the literature .
currently , open reduction and internal fixation is indicated in active patients with normal demands for daily living ; the goal of such treatments is a mobile and pain free wrist.3 volar plating of distal radius fracture is a method of treatment which has become increasingly popular .
compared to dorsal plate fixation , the volar approach has a theoretical advantage in reducing complications of tendon irritation.4 nevertheless , tendon rupture , after volar plate fixation , has been as high as 17%.5 the flexor tendon most commonly involved is the flexor pollicis longus ( fpl ) , but there have been reports of other tendon ruptures or irritation after volar plate fixation.67 suspected causes of tendon rupture include improper plate positioning , prominent screws , plate design , steroid use , loss of reduction or fracture collapse and inadvertent retention of drill guides.6789 watershed line is a prominent ridge in the most volar portion of distal radius .
it is also well documented that plates distal to watershed line or prominent volarly have increased the possibility of contact with the flexor tendons than those proximal to the watershed line.1011 plates that lie proximal to the watershed line , nestled in the volar concavity , are at a further distance from the flexor tendons than those distal to the watershed line .
plates that only extend to the watershed line along both columns are more forgiving.10 orbay has advocated restoration of the pronator quadratus ( pq ) to its native position after volar plating of the radius , providing a layer of vascularized tissue between the plate and the flexor tendons , theoretically protecting the flexor tendons from friction and irritation that could lead to rupture.1213 pq repairs , after volar plate fracture fixation , are generally durable .
they withstand forces which are generated at the distal radius throughout the healing process with a 4% failure rate.13 the purpose of this study was to evaluate the protectiveness of a complete repair of pq against flexor tendon rupture .
this prospective study was approved by our institutional review board . from september 2010 to september 2012 , 157 consecutive patients ( aged between 18 and 60 years ) with unstable distal radius fractures were included in the study .
preoperative radiographs [ posteroanterior ( pa ) , lateral and oblique ] were evaluated to determine unstable fractures .
the criteria proposed by lafontaine et al.14 and altissimi et al.15 were used to determine unstable distal radius fractures . besides this the presence of three or more of the following parameters if associated fractures were considered unstable : radial dorsal angle more than 20 , dorsal fracture comminution , intraarticular fracture line , presence of ulnar fracture , patient 's age more than 60 years and radial shortening of more than 4 mm .
exclusion criteria included patients younger than 18 or older than 60 years , open fractures , previous surgery or fracture in the distal radius , patients who were unable to complete postoperative visits and patients with a history of traumatic brain injury .
summary of methodology for selecting patients all surgical procedures were performed by three orthopedic surgeons who were experienced in the treatment of distal radius fractures
. a standard volar approach to the distal radius , through the flexor carpi radialis sheath , was carried out under general anesthesia .
the radial and distal insertions of the pq with a small rim of brachioradialis and volar capsule , respectively , were sharply incised and elevated from the radius subperiosteally . after exposing and reducing the fracture ,
the fragments were provisionally fixed with two or more k - wires and then volar plate was used for definite fixation . for definite fixation ,
the position of plates was determined by fracture fragments , although the trend was to fit the plate proximal to the watershed line , if possible .
peroperative clinical photographs showing ( a ) exposure of pq muscle ( b ) detachment of pq muscle with a rim of brachioradialis tendon ( c ) detachment of pq from distal insertion peroperative clinical photograph showing ( a ) placement of volar plate ( b ) reattachment of radial border of pq to brachioradialis ( c ) complete coverage of the plate at the radial side ( d ) reattachment of pq distal to a rim of volar capsule radiograph of wrist with forearm anteroposterior ( a ) and lateral ( b ) views showing fracture has united and implant
in situ since the pq with preserved peripheral rim of connective tissues allows sufficient purchase of sutures for full coverage of the plate , the pq muscle was sutured with absorbable materials ( vicryl ) with the forearm in supination position in a tension free manner . the distal margin of the pq repair is the most important and usually easy to do due to thickness of the tissue .
care must be taken to make the repair at this location continuous and tight and to avoid going too distally so as not to compromise the volar ligaments and wrist extension .
the corner of the pq incision is the second most important part of the repair and is most difficult because the tissue over the radial styloid is thin but is the key to closing the corner .
patients were followed up weekly during their postoperative course for the first 3 weeks and then 6 and 12 weekly and at 6 , 12 and 18 months after surgery , soong et al .
's grading system was used to evaluate plate position relative to the watershed line.9 for this classification , on the postoperative lateral radiographs , a line was drawn tangential to the most volar part of the volar rim , parallel to the volar cortex of radial diaphysis .
plates volar to the line , but proximal to the volar rim , were recorded as grade i. plates directly on or distal to the volar rim were recorded as grade ii .
all surgical procedures were performed by three orthopedic surgeons who were experienced in the treatment of distal radius fractures . a standard volar approach to the distal radius , through the flexor carpi radialis sheath , was carried out under general anesthesia .
the radial and distal insertions of the pq with a small rim of brachioradialis and volar capsule , respectively , were sharply incised and elevated from the radius subperiosteally . after exposing and reducing the fracture ,
the fragments were provisionally fixed with two or more k - wires and then volar plate was used for definite fixation . for definite fixation ,
the position of plates was determined by fracture fragments , although the trend was to fit the plate proximal to the watershed line , if possible .
peroperative clinical photographs showing ( a ) exposure of pq muscle ( b ) detachment of pq muscle with a rim of brachioradialis tendon ( c ) detachment of pq from distal insertion peroperative clinical photograph showing ( a ) placement of volar plate ( b ) reattachment of radial border of pq to brachioradialis ( c ) complete coverage of the plate at the radial side ( d ) reattachment of pq distal to a rim of volar capsule radiograph of wrist with forearm anteroposterior ( a ) and lateral ( b ) views showing fracture has united and implant
in situ since the pq with preserved peripheral rim of connective tissues allows sufficient purchase of sutures for full coverage of the plate , the pq muscle was sutured with absorbable materials ( vicryl ) with the forearm in supination position in a tension free manner . the distal margin of the pq repair is the most important and usually easy to do due to thickness of the tissue .
care must be taken to make the repair at this location continuous and tight and to avoid going too distally so as not to compromise the volar ligaments and wrist extension .
the corner of the pq incision is the second most important part of the repair and is most difficult because the tissue over the radial styloid is thin but is the key to closing the corner .
patients were followed up weekly during their postoperative course for the first 3 weeks and then 6 and 12 weekly and at 6 , 12 and 18 months after surgery , soong et al .
's grading system was used to evaluate plate position relative to the watershed line.9 for this classification , on the postoperative lateral radiographs , a line was drawn tangential to the most volar part of the volar rim , parallel to the volar cortex of radial diaphysis .
plates volar to the line , but proximal to the volar rim , were recorded as grade i. plates directly on or distal to the volar rim were recorded as grade ii .
the mean age in our series was 34 10 years ( range 20 - 60 years ) .
the fractures were classified according to the ao classification , volar locking plate was used in 56 ( 41.5% ) patients compared to 79 ( 58.5% ) patients in whom nonlocking plates were used .
independent t - test revealed that the average age of patients with locking and nonlocking plates was not statistically significant ( p = 0.829 ) .
the authors identified 41 ( 30.4% ) smokers , 4 ( 3% ) patients with diabetes mellitus ( dm ) and 1 ( 0.7% ) patient was on corticosteroid .
chi - square tests showed that in this study , according to soong et al .
's classification , the difference in the plate position between patients with or without locking plate was not statistically significant ( p = 0.46 ) .
the mean followup of the patients was 18.4 3.3 months ( range 12 - 24 months ) .
this period of followup of the patients with grade 0 , i and ii plates was not statistically significant by the spearman 's rank correlation coefficient ( r = 0.06 and p = 0.51 ) .
one 55-year - old diabetic female patient with flexor tendon rupture with ao type c fracture pattern was identified , whose fpl tendon had ruptured 16 months after surgery . in this patient ,
nonlocking plate with a grade i volar prominence with respect to the watershed line had been used .
almost all orthopedic surgeons , particularly hand specialists , believe that the pq muscle has a role in forearm stability and strength161718 and repair of this muscle after volar plating , according to swigart et al .
's findings , is reliable . in other words , restoring this muscle after volar plating can bear usual physiological forces with low probability of failure.13 therefore , it can be expected that after complete coverage of the plate by this muscle , the friction between flexor tendons and the plate and the consequent rupture of flexor tendon can be avoided .
although in most recent studies , proper fitting of the plate proximal to the watershed line is considered the most important factor in prevention of flexor tendon rupture.119 special attention must also be paid to other factors such as plate coverage with pq muscle and medical comorbidities , which must not be neglected .
having examined seven cases of cadaveric fresh frozen upper extremities , tanaca suggested that the probability of damage to the flexor tendon will be reduced if the plate is fitted proximal to the watershed line.11 however , it must be noted that in the present study , by excision of the pq muscle , its protective role against flexor tendon rupture has been ignored . in a retrospective study of 165 patients conducted by soong et al.,7
the pq muscle had been restored in all patients and the authors had thus concluded that placing the plate on the distal radius plays a significant role in creating flexor tendon rupture ; nevertheless , it must be noted that in their study , factors such as the quality and technique of restoring the pq muscle and the accompanying medical risk factors ( like smoking , steroids , or dm ) have not been explained . on the other hand ,
white et al.2 could not find a significant relationship between volar plate position and flexor tendon rupture , which is similar to our study . in our study ,
flexor tendon rupture occurred in the elderly patients and it thus appears that further research is essential to study the impact of age and other risk factors ( such as taking corticosteroid and dm ) on the quality of pq muscle because it may affect the quality and process of restoring pq muscle for plate coverage .
these results are similar to those shown by soong et al.7 and brown.9 in the present study , we only had one case of flexor tendon rupture in which the patient was 55 years old and diabetic and attrition of fpl tendon was observable at the time of surgical exploration and correlated to the plate edge .
's grading system , plate location related to the watershed line was in grade i. pq muscle seemed to have been damaged and did not cover the plate , which might probably be due to improper quality of the pq muscle at the time of restoration .
the true incidence of flexor tendon rupture may be higher , as in some reports , fpl tendon rupture as been reported after 6 years.20 the short followup period of patients was one of the limitations of our study . another limitation of our study was lack of a control group .
further research is recommended on more patients and especially a randomized clinical trial , to not only better reveal the impact of pq repair in preventing fpl ruptures but also identify the risk factors that might affect the quality of pq muscle repair . | background : distal radius fractures are one of the the most common adult fractures encountered during the clinical practice of an orthopedic surgeon.12 although several methods of treatment are suggested for these fractures , there are still controversies about the best treatment approach in the literature .
volar plating of distal radius fracture is a method of treatment which has become increasingly popular .
one of the complications of this technique is flexor tendon rupture .
the purpose of this study was to evaluate the protectiveness of complete repair of pronator quadratus muscle against flexor tendon rupture.materials and methods : from september 2010 to september 2012 , a consecutive series of 157 patients who were younger than 60 years with unstable distal radius fractures were included in the study .
a standard volar approach to the distal radius was carried out .
the radial and distal ends of pronator quadratus muscle were meticulously elevated from the radius and after volar plate fixation of the fracture , pronator quadratus muscle was restored to its normal insertion .
we achieved full coverage of the plate with this muscle and followed the patients postoperatively.results:a total of 135 patients were studied .
the mean age of patients was 34 10 years ( range 20 - 60 years ) .
one 55-year - old diabetic female patient with flexor tendon rupture was identified .
the flexor pollicis longus tendon had ruptured 16 months after surgery.conclusions:pronator quadratus repair should be done in distal radius fracture to protect flexor tendons . |
progress has been made in understanding the nature of root canal infection and periapical diseases .
advances in techniques and materials increase the success of surgical intervention in cases of failure of conventional root canal treatment or retreatment6 . during periradicular surgery
, residual soft tissues surrounding the root apex are removed , and root end cavity is prepared subsequently to the apex resection .
dental materials used in root end fillings must achieve perfect isolation of root canal in order to prevent the leakage of fluids arising from periradicular tissues that might support the growth of viable microorganisms eventually present in endodontic microniches .
ideal retrofilling materials must be present : 1- adhesion ability and three - dimensional sealing of the root canal system ; 2- be non - toxic ; 3-dimensional stability even in the present of humidity ; 4-not be absorbed and 5-present radiopacity2,10 .
in addition , antimicrobial , allied to biocompatibility , are properties that clearly contribute to the success of the surgical procedure4,12,15 .
mineral trioxide aggregate consists of an aggregate mainly composed by tricalcium silicate , dicalcium silicate , tricalcium aluminate , tetracalcium aluminum ferrite , dehydrated calcium sulfate and bismuth oxide16 . due to its compatibility and sealer properties
, mta is indicated for retrofillings in periapical surgeries , in sealing root drilling , in the protection of pulp tissue , in apical sealing in cases of incomplete root formation , and in treatment of root resorption17 .
although portland cement shows similarities with mta in its main components ( tricalcium silicate , dicalcium silicate , tricalcium aluminate , and tetracalcium aluminum ferrite ) , few reports have dealt with biocompatibility or antimicrobial aspects of this cement .
recently , the antimicrobial property of portland cement was investigated along with other endodontic filling materials against gram - positive and gram - negative bacteria .
nevertheless , portland cement and mta formulations was investigated with microorganisms grown aerobically , and did not include strictly anaerobic gram - negative bacteria14 .
thus , the aim of this study was to assess the antimicrobial properties of portland cement and mta against four bacterial species grown in anaerobic environment .
test materials : mineral trioxide aggregate ( mta dentsply , tulsa dental - usa , and mta angelus , londrina pr , brazil ) was manipulated strictly following the manufacturer 's instructions .
mo , usa in sterile saline solution ) , and portland cement ( votoran , votorantim , so paulo , brazil ) were manipulated as described elsewhere9 .
antimicrobial activity of endodontic materials were evaluated by agar diffusion method against four reference strains : enterococcus faecalis ( atcc 29212 ) , pseudomonas aeruginosa ( atcc 27853 ) , bacteroides fragilis ( atcc 23745 ) , and escherichia coli ( atcc 33780 ) .
bacteroides fragilis strain was incubated under anaerobic conditions or cultivated in pre - reduced anaerobic sterilized trypticase soy broth ( tsb - pras , difco
bacteria were diluted to obtain a suspension of approximately 5 x 10 colony forming units ml(0.5 in mcfarland 's nephelometer ) in sterile tsb .
bacterial suspensions were inoculated with sterile cotton swabs onto muller - hinton agar plates ( mha difco , detroit , usa ) supplemented with sheep blood ( 5% v / v ) , hemin ( 0.1 mg ml ) , and menadione ( 0.001 mg ml ) .
wells were prepared on mha with a copper puncher of 3 mm diameter and 4 mm depth , and immediately filled with freshly manipulated test materials .
after prediffusion of test materials for 2h at room temperature , agar plates were subsequently incubated at 37c/48h .
controls of contamination of dental materials , and agar medium sterility were performed by incubation of agar plates without bacterial inoculation .
all strains , except p. aeruginosa were incubated in anaerobic atmosphere ( 80% n2 , 10% co2 , and 10%h2 ) in a gaspack jar . to assess the effect of aerobic atmosphere on antimicrobial activity of mta formulations and ca(oh)2 , facultative microorganisms : e. coli and e. faecalis
microbial inhibition zones were measured with a 0.5 mm precision rule , and results were expressed as a mean ( and standard deviation ) of three independent experiments .
data were submitted to statistical analysis by anova and tukey tests with level of significance to compare the differences among two mta compositions , portland cement and calcium hydroxide .
mta formulations of angelus and dentsply , portland cement , and calcium hydroxide were showed to inhibit p. aeruginosa . nevertheless , mta formulations and portland cement were incapable to inhibit e. coli , e. faecalis and b. fragilis .
the calcium hydroxide also showed inhibitory activity , with larger inhibition zones against p. aeruginosa and b. fragilis .
the table 2 expresses the effect of aerobic atmosphere on antimicrobial activity of calcium hydroxide , mta angelus and mta pro - root against e. coli and e. faecalis .
ca(oh)2 showed larger inhibition zones against p aeruginosa than b. fragilis ( p<0.01 ) by tukey test .
values represent the diameter ( in mm ) of zones of inhibition in mean of three experiments and standard deviation .
although used by many authors1,8,10 differences in agar medium , diffusion capacity of inhibitory agents , bacterial strains and cellular density , as well as anaerobic atmosphere may interfere with formation of inhibition zones around materials used in antimicrobial testing9 .
however , there is not a consensus regarding to a gold standard test for the appraisal of antimicrobial testing of cements and other solutions used in dental therapy .
the results of inhibitory activity of both portland cement and mta were similar to those observed in the literature related to p. aeruginosa and e. coli strains , which demonstrated sensitivity and resistance to both materials , respectively8 .
in contrast to the results presented in this investigation , portland cement and mta angelus were recently shown to inhibit a diversity of aerobic and facultative microorganisms , including e. faecalis by agar diffusion method14 .
the incubation of the plates into anaerobic atmosphere aimed to reproduce the environment observed within the apical segment of infected root canal system .
differences in sensitivity among studies , in part , may be due to the anaerobic atmosphere during incubation procedure , since both mta and portland cement are rich in oxides .
subsequent to reaction with water on oxygen - rich environments , these compounds might generate reactive oxygen species ( ros ) , such as hydroxyl and hydroperoxyl radicals which exhibit antimicrobial activity3 .
growth of anaerobes requires an appropriate environment to reduce the intracellular generation of ros ; favoring the growth of anaerobic microorganisms in anaerobic environments , the formation of toxic oxygen radicals is likely to be reduced in intracellular location5 .
moreover , antimicrobial activity of ros is usually impaired by the presence of antioxidants and other reducing molecules such as quinones13
. variation on the concentration of reagents and/or molecules that may act as antioxidants present in culture medium might support the differences in results observed by many authors , who found sensitivity14 and resistance16 of e. faecalis strains to mta . in this study e. faecalis strain became susceptible to both mta formulations and ca(oh)2 after incubation in aerobic atmosphere , suggesting that oxygen - rich environments favor the antimicrobial activity of mta .
the e. coli strain remained resistant to mta and susceptible to ca(oh)2 after incubation in aerobic atmosphere .
studies have demonstrated that e. coli wild strains are relatively resistant to ros generated after incubation with metal ions such as sn+ , requiring the utilization of mutants deficient in sos system repair for the observation of the lethality of chemical agents7 .
calcium hydroxide usually increases ph and allowed an unfavorable microenvironment to the growth of p. aeruginosa and b. fragilis strains .
however , the participation of ros generation in antimicrobial activity of ca(oh)2 pastes may also be emphasized since e. coli and e. facealis presented susceptibility after incubation in aerobic environments .
it is important to mention that the results presented in this study were obtained in vitro and performed with planktonic cells grown in culture medium .
the materials tested could reveal different activities when used in vivo or in vitro with microorganisms grown in biofilms .
further studies are necessary to investigate the effect of these materials on bacteria commonly found in infected root canals .
according to the results obtained it is possible to conclude that :
mta dentsply , mta angelus and portland cement showed similar antimicrobial properties against p. aeruginosa.calcium hydroxide paste showed an antimicrobial activity higher than mta dentsply , mta angelus and portland cement.aerobic environment interfered with antimicrobial activity of mta formulations and ca(oh)2 against e. faecalis and of ca(oh)2 against e. coli .
mta dentsply , mta angelus and portland cement showed similar antimicrobial properties against p. aeruginosa .
calcium hydroxide paste showed an antimicrobial activity higher than mta dentsply , mta angelus and portland cement .
aerobic environment interfered with antimicrobial activity of mta formulations and ca(oh)2 against e. faecalis and of ca(oh)2 against e. coli . | the present study aimed to evaluate and compare the antimicrobial effect of mta dentsply , mta angelus , calcium hydroxide and portland cement .
four reference bacterial strains were used : pseudomonas aeruginosa , escherichia coli , bacteroides fragilis , and enterococcus faecalis .
plates containing mueller - hinton agar supplemented with 5% sheep blood , hemin , and menadione were inoculated with the bacterial suspensions .
subsequently , wells were prepared and immediately filled with materials and incubated at 37c for 48 hours under anaerobic conditions , except p. aeruginosa .
the diameters of inhibition zones were measured , and data analyzed using anova and the tukey test with 1% level of significance .
mta dentsply , mta angelus and portland cement inhibited the growth of p. aeruginosa .
calcium hydroxide was effective against p. aeruginosa and b. fragillis . under anaerobic conditions , which may hamper the formation of reactive oxygen species , the materials failed to inhibit e. faecalis , and e. coli . |
almost two hundred years after edward jenner 's landmark publication showing the efficacy of prophylactic immunization for smallpox1 , a worldwide campaign successfully eradicated this dread disease that produced a 30 per cent mortality after infection and resulted in 10 per cent of the world 's blind .
much of the success of the worldwide eradication programme is attributable to donald s. henderson , the leader of the world health organization ( who)-sponsored effort .
he has very succinctly summarized the eradication programme after a symposium on smallpox eradication that was held to commemorate the 30 anniversary of the historic declaration2 : the world and all its peoples have won freedom from smallpox a most devastating disease since earliest time , leaving death , blindness and disfigurement in its wake and which only a decade ago was rampant in africa , asia and south america .
henderson also has chronicled the eradication effort in a recent book3 , which details the roles of all of those who contributed , and where he tells of the hurdles and hardships that had to be overcome in each country as the ten year campaign unfolded between 1967 and 1977 .
accordingly , those who are interested , especially in the public health aspects of this campaign , are referred to these excellent first - hand accounts by those who were there . at this time
, one would like to delve into the immunology of this eradication effort , to ascertain why it was possible to rid the planet of this virus by vaccination . in this regard
, it must be recalled that in the decade between 1967 - 1977 , the science of immunology was still in its infancy .
it was a time before the revolution of molecular immunology was ushered in after 1980 .
the events leading up to this revolution in our understanding of the workings of the immune system are reviewed elsewhere45 .
thus , this eradication effort required a great deal of chutzpah , and also a great deal of luck , in addition to very very hard work .
the question now before us is how was it possible , and can we reproduce this feat with all of the infectious diseases known to be due to microbes in our environment ?
since smallpox was eradicated from the globe more than thirty years ago , very few people are alive today who have experienced the actual disease , so that it is worth recounting just how devastating the infection actually was .
henderson wrote in his book3 : in the last hundred years of its existence , smallpox is thought to have killed at least half a billion people .
all of the wars on the planet during that time killed perhaps 150 million . in the contest of smallpox vs. war , war lost .
smallpox killed roughly one - third of the unimmunized people it infected , and the disease was grisly . once a person was infected with smallpox ( which generally occurred via inhalation )
, there was an incubation period of around ten days before the person became noticeably sick .
the rash appeared first on the face , hands , and feet , and quickly rose into pustules .
smallpox pustules were hard , pressurized blisters filled with a clear , faintly opalescent pus .
the pain of the smallpox pustular rash was virtually unbearable . if the pustules merged into .
they died of shock . accordingly , now with the hindsight of thirty years of molecular immunology , it is obvious that this was an immunopathological disease of disseminated inflammation , i.e. systemic rubor , calor , tumor , and dolor , caused by the adaptive immune response to the virus , which evidently replicated unchecked by the innate response during the prodromal period . as we now know
that all of these cardinal signs of inflammation are ascribable to the production and action of pro - inflammatory cytokines ( e.g. il-1 , il-6 , tnf ) , primarily by antigen - presenting cells and t cells , it is quite possible that armed with our new anti - cytokine therapies , it might be possible to mitigate both the severe signs and symptoms of this syndrome , and also to ameliorate the huge mortality rate .
variola has a large , double - stranded dna genome with approximately 200 genes , and humans are the only known hosts susceptible to infection , which has been attributed to the species specificity of the viral gene products that circumvent innate immunity .
recent data indicate that the severity of poxvirus disease correlates with the lack of host control of viral replication within the 10 - 14 day incubation phase . in this regard ,
poxviruses devote a considerable proportion ( ~ 50% ) of their large genomes manipulating the host innate immune defenses6 .
there is a remarkable story of how edward jenner came to create the world 's first effective vaccine7 .
one hundred years before pasteur introduced the concept that all microbial diseases might be prevented via prophylactic vaccination or cured via therapeutic vaccination89 , jenner relied on his uncanny powers of observation and logic to come up with the methods to produce a vaccine for smallpox using the cowpox virus .
he also showed many others how to reproduce his results , and furnished all of those in the world a seed source of cowpox virus that could be used for immunizations .
as noted in the jenner article , what was originally thought to have originated from cows , the strain of poxvirus that came to called vaccinia in the 19 and 20 centuries was probably actually indigenous to horses10 .
new data from molecular genetics indicate that the vaccinia virus is most closely homologous to an equine poxvirus .
thus , instead of vaccination , we probably should speak of equination when referring to immunization .
henderson highlighted three parameters that allowed the eradication of smallpox :
( i)an inexpensive , heat - stable effective vaccine and a simple bifurcated needle used to deliver it reproducibly.(ii)an easily diagnosed disease by virtue of the characteristic rash.(iii)no animal reservoir or asymptomatic carriers , i.e. 100 per cent of those infected contracted the clinical disease .
an inexpensive , heat - stable effective vaccine and a simple bifurcated needle used to deliver it reproducibly .
an easily diagnosed disease by virtue of the characteristic rash . no animal reservoir or asymptomatic carriers , i.e. 100 per cent of those infected contracted the clinical disease .
all of these characteristics made feasible an effective surveillance - containment strategy that was instrumental in identifying and containing any outbreaks .
thus , non - medical personnel in the community could be trained to readily identify and report cases of characteristic smallpox , so that all contacts could be traced and vaccinated .
this surveillance - containment effort made possible the eradication of smallpox from each community and country without attaining 100 per cent vaccination rates . however , the goal of vaccinating at least 80 per cent of susceptible individuals was a necessary counterpart to the surveillance - containment policy producing
dryvax was the inexpensive , heat - stable , effective vaccine that was developed from vaccinia in the 1 half of the 20 century . during most of the 19 century ,
jenner 's live cowpox virus was transmitted from arm to arm in humans all over the world .
one can imagine how this practice could lead to problems before microbiology became appreciated in the late 19 and early 20 century .
for example , one egregious episode recorded was the transmission of syphilis when pus from an individual with secondary syphilis was transferred to children during a vaccination programme3 .
thus , in the 20 century the preparation of vaccine using infected calfskin was a significant step forward , in that large amounts of live virus could be prepared .
however , it was not until the 1950s that an eradication programme could be made feasible , when the cell - free filtrate of the calfskin vaccine preps were lyophilized ( hence the name dryvax ) , so that the vaccine could be transported and stored at ambient temperatures3 . also , arrangements were made with connaught laboratories in toronto to serve as a quality - control laboratory , testing batches of vaccine prepared all around the world so that reproducible vaccine could be made available .
according to henderson3 , only variola , vaccinia and monkeypox viruses are transmissible to humans , and only the variola viruses cause widespread , disseminated infections .
consequently , in recent times vaccinia has been enlisted as a vector in the creation of vaccines for other microbes , such as hiv . however , to generate vaccines that can be delivered safely to normal individuals , the one characteristic of vaccinia that made it such an effective vaccine for smallpox , i.e. its ability to replicate in human cells , was crippled via genetic engineering .
thus , gene - deleted modified vaccinia strains unable to replicate have been developed , e.g. modified vaccinia ankara ( mva ) and new york vaccinia ( nyvac ) , as well as avian pox ( canarypox and fowlpox ) strains unable to replicate in mammalian cells are under development for use as vectors to deliver genes from other microbes . in this regard
it is noteworthy that louis pasteur introduced the concept of attenuating virulent microbes to create live vaccines811 .
pasteur hypothesized that the live , replication - competent microbes depleted the host of vital trace nutrients , thereby rendering the host incapable of supporting the viability and reproduction of the virulent pathogenic organisms811 .
accordingly , pasteur was totally nave of how the immune system functions , by actively recognizing and engaging pathogenic microbes , as well as attenuated vaccines .
pasteur was correct that live attenuated microbes made for the best vaccines , but he was right for the wrong reasons .
live , replication - competent vaccines are more effective than replication - incompetent vaccines , because these establish a self - limited local infection that efficiently activates all of the immune cells and molecules involved in immunity , both innate as well as adaptive immunity .
thus , a self - limited infection ensures microbial peptide presentation via molecules encoded by both class i and class ii major histocompatibility complex ( mhc ) genes , thereby promoting the activation of both class i and class ii restricted t cell receptors ( tcrs ) .
also , a replication competent , attenuated microbe ensures the generation of a high enough concentration of microbial peptides , so that there will be a
strong tcr activation , enough to overcome negative feedback loops in place that attenuate a meaningful immune response12 .
in essence , a successful adaptive immune response is one that leads to the production of antibodies , as well as all of the cytokines that promote maximal cellular immune responses , mediated by both cytolytic and helper t cells .
moreover , a replication - competent attenuated vaccine can be delivered in smaller doses than a replication - incompetent vaccine , an important point when a worldwide vaccination programme is under consideration and billions of people must be vaccinated .
thus , jenner 's observation was that cowpox virus resulted in an attenuated local infection , and not a systemic infection .
with regard to smallpox , the hallmark of the disease is the 1 - 2 wk latent period followed by a generalized type iv delayed type hypersensitivity ( dth ) reaction to a disseminated very high viral load .
apparently , the virus goes undetected by the first line of defense , i.e. the innate immune system613 , so that when the adaptive immune system finally reacts the viral load is huge , so that the immunopathology is also huge . from the virus viewpoint , this sort of an infectious cycle ensures that enough progeny will be produced and disseminated that ensures sustained viability . by comparison ,
vaccinia infection remains localized to the site of inoculation , so that this virus is not disseminated systemically .
however , the local infection does result in viral replication , albeit limited to the local tissues .
one needs to identify how vaccinia differs from variola , and logic dictates that variola has genes encoding molecules that circumvent innate immunity , thereby allowing rapid viral replication and dissemination before the adaptive immune response takes hold . by comparison , probably due to species differences of gene products
, vaccinia can not effectively circumvent innate immune responses , which makes for a locally contained infection , and not widespread dissemination .
the $ 64 question is whether vaccinia itself can be used as a vector to deliver the gene products of other organisms , instead of genetically modified vaccinia , which circumvents the capacity of the virus to replicate in human cells .
experience with the modified vaccinias as well as the avian poxviruses indicates that although these are safe , these are relatively weak immunogens14 . because of the recent threat of bioterrorism and the news that the soviets had a large bioterrorist research and development programme focused on variola , the u.s .
the previously licensed smallpox vaccine in the u.s . , dryvax ( wyeth laboratories inc . ) had a questionable safety profile , because it consisted of a pool of vaccinia virus strains with varying degrees of virulence .
thus , six individual clones of viruses were isolated by plaque purification in tissue culture from a pool of 30 vials ( 3,000 doses ) of dryvax , nycbh , and were tested for immugenicity and virulence by comparison with dryvax. a test for immunogenicity included the diameter of erythema and lesions on day eight after scarification of rabbit skin .
the rabbit scarification model mimics the vaccine take observed following human vaccination with dryvax because of local replication of vaccinia , which activates a typical adaptive dth cellular immune response45 . the virulence test measured survival time and viral replication in brain tissue after intracerebral injection of suckling mice .
one of the six clones selected for further testing was found to be less virulent than dryvax and just as immunogenic .
a pilot lot of 750,000 doses of the vaccinia clone propagated on a human lung fibroblast cell line was prepared and tested in 100 volunteers compared with 30 subjects who received dryvax. the tissue culture derived clone elicited a 100 per cent take - rate , 100 per cent seroconversion , and a comparable t cell response , monitored via cytotoxic t lymphocyte ( ctl ) , proliferation , and -interferon ( ifn ) elispot16 . of note ,
further testing of this clone showed that a full - length copy of the ifn- receptor and the tnf receptor were both absent whereas these virulence factors were intact in other isolates16 . to enhance preparedness in the event of a possible variola terrorist attack , a 2002 presidential initiative recommended vaccination of enlisted military personnel and civilan health care workers who might become first responders . by june 2004 ,
cases of progressive vaccinia , eczema vaccinatum , and foetal vaccinia were completely avoided by careful screening of potential vaccinees .
one serious ae noted in dryvax vaccinations was a myopericarditis that appeared within 1 - 2 wk post vaccination .
when carefully monitored with ekg and cardiac enzyme tests , the cloned vaccine gave an incidence of 0.5 per cent ( seven cases in 1307 subjects ) , compared with 0.8 per cent of individuals who received dryvax ( three cases in 363 subjects ) , a difference not statistically significant16 .
however , most of these cases were subclinical or asymptomatic . moreover , no cases of myopericarditis occurred among 1819 vaccinia - experienced subjects vaccinated with either vaccine16 .
accordingly , given these data , which represent an extensive testing of the safety and efficacy of a vaccine in humans , it can be concluded that this strain of plaque - purified vaccinia could serve as a safe and effective vector for many other dangerous microbes , such as hiv .
the regulatory authorities , especially of those countries where hiv is endemic at a high prevalence , should be advised to carefully consider the risk / benefit ratio of replication - competent vaccinia as a vector to reduce and perhaps eradicate hiv and other intractable infectious diseases .
smallpox virus was eradicated from the globe because of its unique virological and immunological characteristics .
whether a similar eradication effort can have similar results with other microbes remains to be seen .
malaria is still endemic in many developing countries despite all efforts , due in large part to the lack of an effective vaccine .
polio incidence has been reduced considerably worldwide thanks to an effective vaccine combination , but vaccination efforts have been thwarted in countries like pakistan and nigeria due to civil unrest and the execution of vaccinators by factions distrustful of modern medicine .
even so , the characteristics of variola vs. vaccinia infections may well point the way to the development of a vaccinia - based replication - competent vector that could be used to engineer vaccines useful for the prevention of many of the diseases that plague us today , including hiv infection . | one of the most celebrated achievements of immunology and modern medicine is the eradication of the dreaded plague smallpox . from the introduction of smallpox vaccination by edward jenner , to its popularization by louis pasteur , to the eradication effort led by donald henderson , this story has many lessons for us today , including the characteristics of the disease and vaccine that permitted its eradication , and the obviousness of the vaccine as a vector for other intractable infectious diseases .
the disease itself , interpreted in the light of modern molecular immunology , is an obvious immunopathological disease , which occurs after a latent interval of 1 - 2 weeks , and manifests as a systemic cell - mediated delayed type hypersensitivity ( dth ) syndrome .
the vaccine that slayed this dragon was given the name vaccinia , and was thought to have evolved from cowpox virus , but is now known to be most closely related to a poxvirus isolated from a horse .
of interest is the fact that of the various isolates of orthopox viruses , only variola , vaccinia and monkeypox viruses can infect humans .
in contrast to the systemic disease of variola , vaccinia only replicates locally at the site of inoculation , and causes a localized dth response that usually peaks after 7 - 10 days .
this difference in the pathogenicity of variola vs. vaccinia is thought to be due to the capacity of variola to circumvent innate immunity , which allows it to disseminate widely before the adaptive immune response occurs .
thus , the fact that vaccinia virus is attenuated compared to variola , but is still replication competent , makes for its remarkable efficacy as a vaccine , as the localized infection activates all of the cells and molecules of both innate and adaptive immunity . accordingly
vaccinia itself , and not modified replication incompetent vaccina , is the hope for use as a vector in the eradication of additional pathogenic microbes from the globe . |
hematopoietic cell transplantation ( hct ) is a well - documented and proven life - saving treatment for patients with malignant and nonmalignant disorders in need of cellular replacement therapy with healthy autologous or allogeneic hematopoietic stem cell ( hsc ) and hematopoietic progenitor cell ( hpc ) .
hscs and hpcs are tissue - specific stem cells that are found in and collected from the bone marrow ( bm ) or cytokine - mobilized peripheral blood ( mpb ) of adults and children , or umbilical cord blood ( cb ) .
other types of cellular replacement therapy have the potential to be options in the context of the new and rapidly emerging field of regenerative medicine .
cells considered of value in regenerative medicine include embryonic stem cells , induced pluripotent stem cells , and various other tissue - specific stem and progenitor cell populations , such as those from nonhematopoietic tissue such as muscle and nerves . while it is not yet clear whether cellular replacement therapies using populations other than hscs and hpcs for hct have resulted in cures or significant health benefits , there is optimism that continued scientific and clinical efforts in the area of regenerative medicine will demonstrate such clinical benefits in the future .
many investigators worldwide are devoting efforts to this endeavor . the functional characteristics and capacities of the different stem and progenitor cell populations , which include self - renewal ( the capacity of these early immature cells to make more of themselves , a function usually reserved for and linked to stem cells rather than progenitor cells ) , survival , proliferation , differentiation , and movement ( migration , homing ) , are in part regulated by proteins such as cytokines and chemokines ( a subset of cytokines ) as well as other growth - modulating biomolecules such as hormones .
most studies of protein regulation in stem and progenitor cell functions focus on specific protein - receptor interactions at the surface of the cells , and the subsequent intracellular signaling transduction events and cell biological effects triggered by the interaction .
however , little previous effort has gone into how modification of a protein itself can influence cell regulation , and what role specific enzymes may play in the resulting protein - cell signaling events .
this review focuses on the enzyme dipeptidylpeptidase 4 ( dpp4 ) ; how it modifies the functional effects of proteins with demonstrated or putative dpp4 truncation sites in the regulation of hscs , hpcs , and hematopoiesis ; and how this modification may influence other cellular systems , in the context of regenerative medicine .
another focus is on cb hcts , and on the potential role for dpp4 inhibition in enhancing engraftment of cb hscs and hpcs in the clinical setting , a possible paradigm for also enhancing the engraftment of other cell types .
cb is now a well - accepted source of transplantable hscs and hpcs and has been used to treat more than 30,000 patients with a wide assortment of malignant and nonmalignant disorders .
it has been and can be used to treat largely the same array of disorders treated by bm or mpb hct .
it was first used in a setting of human leukocyte antigen ( hla)-matched sibling cb hct , and subsequently for partial hla - matched siblings , but most cb transplants are now done in a partially hla - matched unrelated allogeneic setting . the concept and scientific basis for cb hct
are based on laboratory studies that have show in detail the biological properties of these cells , and their efficient collection , transportation , processing , and cryopreservation as well as the recovery of thawed cells stored in a frozen state . since these laboratory studies ,
there have been follow - up studies on the biology of cb hscs and hpcs [ 10 - 16 ] , and the efficient recovery of these cells after 5 , 10 , 15 , and 23.5 years of storage in the author 's laboratory , which has served as the first proof of principle cb bank from which came the first five , and two of the next five , cb collections used for hla - matched sibling cb hct .
there are advantages to using cb as a source of hscs and hpcs for hct , compared to bm or mpb , including ease of collection , with no damage or harm to the baby or mother , the long - term storage of these cells in cb banks and thus rapid availability of these collections for patients , especially when such cells are needed in a quick and timely fashion for hct because of the progressive nature of the disease and the more nave status of the immune cells within cb that has allowed for less rigorous hla - matching and less graft - versus - host disease than that elicited by bm or mpb .
one such concern includes the slower time to recover neutrophils , platelets , and immune cells that may result from the finite number of cells one can collect at the birth of the baby , compared to what can be collected from bm or mpb .
this problem is also a concern for use of cb hct in adult patients who require more cells than children for engraftment .
this includes the use of two collections of cb , ex vivo expansion of cb hscs and hpcs , intrafemoral injection of cb , and the enhancement of the homing and engrafting capability of cb hscs and hpcs for more efficient cb hct . towards this aim , for over 10 years
, we have been investigating a role for modulating dpp4 activation and expression in the homing and engrafting capabilities of hscs and hpcs .
dpp4 , a member of the family of prolyl oligopeptidases , functions as a serine protease that can selectively cleave at the penultimate amino acid from the n - terminus of a protein , with degrees of specificity inherent in this truncation depending on whether that amino acid is proline or alanine or perhaps some other amino acid such as serine .
dpp4 can be found on the surface of certain cells , where it is also referred to as cd26 , but it is also found within cells and as soluble enzyme in serum , plasma , and other body fluids . the chemoattractant chemokine stromal cell - derived factor ( sdf)-1/cxcl12 has a dpp4 truncation site , and we demonstrated that dpp4-truncated sdf-1/cxcl12 lost its significant chemotactic ( directed cell movement ) effect on hpcs .
in addition , the dpp4-truncated inactive sdf-1/cxcl12 blocked the chemotactic activity of the full - length form of sdf-1/cxcl12 .
moreover , we were able to enhance the chemotaxis of hpc to sdf-1/cxcl12 by pretreating target cells with small peptide inhibitors of dpp4 ( e.g. , the tripeptide diprotin a ile - pro - ile or the dipeptide val - pyr ) or using cd26 knock - out ( -/- ) mouse bm cells before adding the sdf-1/cxcl12 to the target cells .
this led to our studies demonstrating that cd26 mice or mice pretreated with the dpp4 inhibitor diprotin a were less responsive than control mice to the hpc - mobilizing effects of granulocyte ( g)-colony stimulating factor ( csf ) .
the next studies from our group demonstrated that cd26 mouse bm cells , or bm cells pretreated with diprotin a or val - pyr , had increased homing efficiency and engrafting capability in lethally irradiated primary recipient congenic mice in both competitive and noncompetitive transplantation assays , and greater secondary repopulating capacity in a noncompetitive assay of lethally irradiated mice , than that of control bm cells .
this was associated with enhanced engraftment at both higher and limited doses of donor cells .
thus , deletion of cd26 in cd26 mice or inhibition of dpp4 ( with two separate dpp4 inhibitors ) demonstrated increased homing of a long - term bm repopulating and self - renewing hscs .
this work was quickly confirmed and extended by others [ 31 - 33 ] , followed by reports [ 34 - 36 ] demonstrating that inhibition of dpp4 on cd34 human cb or human mpb enhanced their engrafting capability in sublethally irradiated mice with nonobese diabetic severe combined immunodeficiency ( scid ) .
this mouse serves as an in vivo model for detecting human hscs , which are quantitated as scid - repopulating cells .
thus , the engrafting capability of mouse bm and human cb or mpb cells could be enhanced significantly by deletion or inhibition of dpp4 [ 34 - 36 ] . these data , plus information suggesting that inhibition of dpp4 activity in recipient mice could also enhance the engrafting capability of mouse bm cells in a lethally irradiated congenic mouse model of hct , led our group to conduct a pilot study that evaluated the role of dpp4 inhibition in patients receiving cb hct .
this pilot study evaluated the impact of an orally active small molecule inhibitor of dpp4 ( sitagliptin ; u.s .
food and drug administration - approved for use in treating type ii diabetes ) on the engraftment of single - unit cb in adult patients with end - stage hematological malignancies ( leukemia and lymphoma ) . while most studies on cb hct in adults have used double cb hct , we felt that this was not necessary because we were using single cb units that met the estimated requirement for numbers of nucleated cells per kg body weight needed for engraftment of adults , and the use of single cb hct would not complicate the interpretation of results inherent to the use of double cb hct , in which case only one of the two cb units usually wins out for long - term engraftment , especially because the characteristics of the winning cb unit can not yet be predicted .
we chose to assess the effects of engraftment using treatment of the recipient rather than the donor cells because sitagliptin is orally active and could be administered as a pill .
based on the reported safety of sitagliptin administered in this fashion to healthy adults , the kinetics of inhibition of dpp4 in these adults , which resulted in rapid and near - complete dpp4 inhibition that lasted up to 24 hours , and our belief at the time that the results of our enhanced mouse bm and human cd34 cb engraftment in mouse studies were due to inhibiting dpp4 , which prevented the truncation and inactivation of the homing / chemotactic chemokine sdf-1/cxcl12 , we chose to administer the pill once a day for only 4 days , starting 1 day prior to the cb unit , and on the day of the cb unit infusion , as well as for 2 days after the infusion .
we felt that this should be sufficient time for the sdf-1/cxcl12 homing and initial engrafting process to occur .
for the 17 patients who received red blood cell - depleted cb units and sitagliptin in this dosing regimen , the median time to neutrophil engraftment was 21 days . while we do not have proof that this was a significantly improved time for neutrophil engraftment for cb hct , the results were encouraging compared to the engraftment reported by others for single - unit cb hct in context of the numbers of cells used , hla disparity , and the extent of disease and clinical status of recipients .
we do believe that the engraftment capacity of single - unit cb hct can be improved to a much greater extent than we have reported by modifying the dose schedule sitagliptin , for the following reasons .
first , it became apparent during the clinical study that sitagliptin given once a day was not as effective for reducing the dpp4 enzyme activity in our patients as it was in normal volunteers , in that the inhibition of dpp4 activity in our patients was only apparent for the first 4 to 8 hours after the administration of sitagliptin , compared to more than 24 hours inhibition seen in the normal volunteers .
this decrease and rapid return of dpp4 activity was similar for each of the 4 days of sitagliptin administration .
thus , we have begun to administer sitagliptin every 12 hours for 4 days and are beginning a multicenter trial using this dosing schedule , because preliminary studies demonstrated more prolonged dpp4 activity inhibition with the twice versus once a day administration of sitagliptin .
it may be that the chemotherapy and radiation given to our patients caused increased cell release of dpp4 because of cell death , thus requiring a more frequent dosing schedule to provide more effective dpp4 inhibition .
our more recent studies , discussed below , also alerted us to the fact that dpp4 could truncate and decrease the activities of a number of csf molecules , which may be involved in nurturing the engrafting and blood cell - repopulating capacity of donor cb cells .
thus , future clinical studies may need to assess sitagliptin administration for longer than 4 days , in addition to the increased frequency of administration of this dpp4 inhibitor .
such studies must be performed with a focus on the safety of patients . as discussed in more detail later in this review ,
there are likely going to be uses for dpp4 modulators in other clinical situations involving different stem and progenitor cells , and more mature cells of different tissues and organs .
in addition to the effects of inhibition of dpp4 on sdf-1/cxcl12 functions of chemotaxis and homing , we recently reported similar effects on other functions of sdf-1/cxcl12 , including enhanced survival of hpcs and enhanced cytokine - induced ex vivo expansion of hpcs .
dpp4-truncated sdf-1/cxcl12 had no significant effect on these functions , and inhibition of dpp4 enhanced them .
these effects were extended to members of the csf family , including g - csf , granulocyte macrophage ( gm)-csf , interleukin ( il)-3 , and erythropoietin ( epo ) .
treatment of target cells with inhibitors of dpp4 ( e.g. , diprotin a ) greatly enhanced the csf activities of g - csf , gm - csf , il-3 , and epo on colony formation by human cb and mouse bm cells in vitro as well as the in vivo activities of gm - csf or epo .
inhibitors of dpp4 had no significant effect on the activities of other hematopoietically active cytokines such as macrophage ( m)-csf or on those of the potent costimulating cytokine stem cell factors or flt3-ligand , which do not have putative dpp4 truncation sites .
because members of the csf family that have dpp4 truncation sites may be involved in the cytokine storm elicited during recovery of mice after stresses such as chemotherapy or radiation , we evaluated whether increased dpp4 , which we noticed after mice were given radiation , might dampen the rebound in hematopoiesis seen after low and higher nonlethal doses of irradiation , and also after administration of 5-flurouracil ( 5fu ) or other chemotherapeutic drugs . in this context
, we found that cd26 or inhibition of dpp4 was associated with a faster and higher level of hematopoietic recovery after 5fu and 400 or 650 cgy gamma irradiation .
this , and the enhanced effectiveness of in vivo administration of gm - csf or epo to mice with decreased or absent dpp4 activity , plus the enhanced engrafting capabilities of mouse bm cells in lethally irradiated cd26 congenic mice or congenic mice pretreated with sitagliptin demonstrated how down - modulation of dpp4 can have a positive effect on both preclinical and clinical models of hscs . a search for other regulatory proteins with putative truncation sites identified numerous proteins that are known to have significant influences in many different cell , tissue , and organ systems . an extensive but certainly not an all inclusive list of such proteins can be found in our recent review article . this list suggests how dpp4 and its up- or down - modulation may be useful for better understanding protein regulation in many different systems , as well as for possible preclinical and subsequent clinical advantage .
as our previous reviews point out , the identification of putative dpp4 truncation sites in proteins does not prove that these are true truncation sites , and even if these are shown to be true truncation sites , the consequences of dpp4 truncation on the functional capacity of each individual protein will need to be determined experimentally , because there are many different possible scenarios for dpp4 truncation of proteins , with inhibition or enhancement of function . once these different actions are worked out for each protein , it will be necessary to determine how the truncated molecule is acting at a mechanistic level , and how modulation of this may influence the different possible functional activities of these proteins , and then whether and how this information may be useful for potential therapeutic benefit .
a recent paper reported that sitagliptin enhanced the numbers of circulating angiogenic cells and angiogenesis , although the exact protein player(s ) and mechanism(s ) involved in this have not yet been identified .
dpp4 has been shown to directly influence the actions and potency of a number of different proteins that have regulatory activities in hscs , hpcs , and hematopoiesis .
inhibition or functional deletion of dpp4 has resulted in enhanced protein activities in vitro and in vivo in preclinical models , and has been used with encouraging although not yet definitive results for enhancing the engrafting capability of limited numbers of cb cells in the context of a pilot study that evaluated cb hct in adult patients with end - stage hematological malignancies .
it is clear that dpp4 will likely have far - reaching effects on our improved understanding of the activities of a plethora of proteins and their regulation in different cell , tissue , and organ types .
understanding the roles of dpp4 in protein actions in these different systems may prove to be of use and benefit in hct and in ongoing efforts to more clearly define the fields of non - hct and regenerative medicine , and the possible clinical benefits of modulating dpp4 .
it is also likely that other enzymes will be found to be important for modulating protein function , information that would be of academic and potentially clinical utility . | dipeptidylpeptidase ( dpp ) 4 , also known as cd26 , is an enzyme present on the surface of a number of different cell types .
it is also found within cells and as a soluble protein in body fluids .
it can specifically truncate proteins at the penultimate n - terminus residue for some amino acids , such as alanine , proline , serine , and perhaps others .
dpp4 has been implicated in regulating the in vitro and in vivo functional activities of a number of hematopoietically active molecules , and this information , along with that on inhibition of dpp4 , has been studied in efforts to enhance hematopoietic cell transplantation ( hct ) , hematopoiesis after stress in mouse models , and in the clinical setting of single - unit cord blood ( cb ) hct .
this article reviews the current status of this compound 's effects on regulatory proteins , the field of cb hct , a potential role for modulating dpp4 activity in enhancing single - unit cb hct in adults , and future aspects in context of other cellular therapies and the area of regenerative medicine . |
mantle cell lymphoma ( mcl ) is a mature neoplasm of the b lymphocytes classified under the category of indolent nonhodgkin lymphomas ( nhl ) . however , unlike other indolent forms of nhl , mcl behaves more aggressively despite treatment 1 .
mcl comprises about 67 percent of adult nhls in the united states and europe with an incidence rate of 48 cases per million per year 1 , 2 , 3 .
the median age of diagnosis is 68 years with more frequent occurrence in caucasian males than females 4 , 5 , 6 , 7 .
the cell of origin for mcl is a naive b cell in 80% of the cases , but the remaining cases are derived from antigenstimulated b cells .
the translocation t(11,14 ) ( q13;q32 ) is universally present in mcl and is the primary genetic defect that results in unsuppressed expression of the protooncogene ccnd1 .
there are a variety of other genetic defects also involved , including loss of tumor suppressor genes and activation of other oncogenes 8 .
around 70% of cases of mcl present for the first time with advancedstage disease . around threequarters of patients
most common extranodal sites appear to be that of the bone marrow ( bm ) , spleen , liver , gastrointestinal ( gi ) tract , and the waldeyer 's ring 9 , 10 , 11 . in terms of gi tract involvement
, ascites has been described alongside lesions such as gastric ulcers , lymphomatous intestinal polyposis or peritoneal lymphomatosis . aside from ascites ,
serosal lymphomatosis with pleural and pericardial effusions has been described in later stages in the setting of known mcl with positive lymph node status or bm .
development of serosal effusion in the course of malignant lymphomas , either primary or otherwise , is rare and is considered as one of the adverse factors affecting overall survival 12 . however , the presence of peritoneal lymphomatosis with abdominal ascites as the first and primary presentation is rare and only few cases have been reported in the literature 13 .
flow cytometry ( fcm ) and immunohistochemical staining of the ascitic fluid have helped clinicians identify a diagnosis in such cases .
we found the fcm results of samples from different sites could be concordant or discordant within the same patient .
as in our case , mcl cell markers in the peritoneal fluid may be able to serve as surrogate for conventional bm aspirate and biopsy .
we are presenting a case of a 46yearold el salvadorian male with a past medical history of rheumatoid arthritis , dyslipidemia , and type 2 diabetes mellitus , who presented to the hospital with the complaints of sore throat , shortness of breath , cough , and chills .
he also mentioned increasing abdominal distension over the last 3 months associated with significant unintentional weight loss of approximately one hundred pounds .
his family history was significant for a son that was diagnosed with acute lymphoblastic leukemia at the age of 14 , who has since been in remission . on examination ,
generalized wasting was noted . he was noted to be febrile at 102f , tachycardic , tachypneic , and hypotensive in the emergency room .
initial laboratory workup showed marked leukocytosis of 71.6 k / ul with absolute lymphocytosis of 61.2 k / ui .
laboratory evaluation also revealed a hemoglobin at 4.9 gm / dl and platelet count of 111 k. a peripheral smear showed more than 50% atypical lymphocytes , multiple bands , and multisegmented neutrophils without evidence of blasts . with the presumptive diagnosis of septic shock from pneumonia , he was admitted to intensive care unit .
hemodynamic support was provided in the form of vasopressors , mechanical ventilation , and transfusion of blood products .
further workup revealed elevated uric acid of 10 mg / dl and lactate dehydrogenase ( ldh ) of 278 u / l .
serum protein electrophoresis , histoplasma antigen , aspergillus antigen , hiv , and hepatitis panel , epstein
ct scan of head , neck , chest , abdomen , and pelvis was performed and showed significant pelvic , retroperitoneal , and axillary lymphadenopathy .
peritoneal carcinomatosis with marked hepatosplenomegaly , ascites and opacification of maxillary and ethmoid sinuses were also noted .
peripheral blood fcm and cytogenetics were subsequently performed showing t(11:14)+ , cyclin d1 + , cd5 + , cd10 , cd23. with this data , the diagnosis of mcl was made ( fig .
as mentioned before , blood smear showed atypical lymphocytes which , in the settings of leukocytosis , could not be explained merely by infection but also secondary to a neoplastic process .
flow cytometry graphs of the peripheral blood [ left ] and peritoneal fluid [ right ] . following stabilization from septic shock
, he underwent a bm biopsy which showed extensive involvement by the mcl ( figs . 2 and 3 ) .
he presented with ann arbor stage 4 disease , hemoglobin less than 12 , an elevated ldh , and an ecog performance status of 2 . with this data , he was stratified as a highrisk stage 4 mcl .
cytological analysis of which was consistent with mcl by fcm ( figs . 1 and 4 ) .
h / e stain of the bone marrow showing predominant lymphocytes infiltration . from left to right showing the ihc stains of bone marrow cells positive for cyclin d1 , cd 20 , and cd 3 markers .
thereafter , he was started on chemotherapy with cyclophosphamide , doxorubicin , vincristine , and dexamethasone and received two cycles of therapy . despite discharge after further stabilization , he had multiple admissions from progression of his symptoms and recurrence of ascites that required multiple paracentesis .
due to inadequate response to the therapy , he was then switched to palliative chemotherapy with the use of bortezomib for seven cycles .
ascites , as described in our patient , with the finding of peritoneal carcinomatosis is considered an unusual presentation for mcl and has been rarely described in literature .
this case reflects the importance of cytological analysis of peritoneal fluid in a patient with intractable ascites not contributing from other comorbidities . in the event a bm analysis can not be made , this may serve as an alternative method of diagnosis .
throughout history , mcl has carried different names such as intermediate lymphocytic lymphoma , centrocytic lymphoma , mantle zone lymphoma , and lymphocytic lymphoma of intermediate differentiation 2 , 3 . since the 1990s
, all these names were categorized under mcl after finding that all described cases carried the t(11 , 14 ) ( q13 ; q32 ) 8 .
being a tumor rising from b cells , the surface markers that are detected by immunohistochemistry include panb cell antigens
sox11 could be a useful marker in cyclin d1negative mcl 12 , 13 , 14 .
though the translocation t(11 , 14 ) ( q13;q32 ) is the primary genetic defect , other genetic defects include loss of tumor suppressor genes like atm , cdkn2a , and tp53 with the concurrent activation of some oncogenes like myc , syk , bcl2 8 , 14 .
the presentation of the mcl can vary from chronic / indolent form to a more fulminant course resulting in shortened overall survival 1 .
diffuse large bcell lymphoma being the most common of all nhl has been associated with ascites . in a case series of 101 cytologypositive cases of malignant ascites , only 8% were shown to be lymphoma 15 . in another case
we find that gi tract involvement with or without ascites have been seen in indolent lymphoma to aggressive mcl 12 , 13 , 17 , 18 , 19 , 20 .
infiltration due to tumor mass or vascular leakage due to stimulation by the vascular endothelial growth factor contributes to the pathogenesis of the ascites .
ascites , usually described as bloody in nature , can be present concurrently with gi tract involvement , peritoneal lymphomatosis , or multiple lymphomatous polyposis of the intestine .
gastric mcl is more likely to occur with other gi diseases such as crohn 's disease and adenocarcinoma .
( table 1 ) 12 , 13 , 17 , 18 , 19 . illustrating five cases of mantle cell lymphoma with ascites on presentation lap , lymphadenopathy .
flow cytometric analysis was performed in four of the five cases and the expression of cytological markers in both ascitic fluid and bm or peripheral blood .
one of the cases presented as postsurgical seeding following splenectomy resulting in peritoneal involvement 19 . in the cases without bm involvement ,
cytological markers were consistent with mcl and patients were treated as such 12 , 13 , 17 , 18 .
two previous studies showed that different specimens from different sites in the same patient with nhl carry the same set of cytological markers most of the time 21 , 22 , 23 .
mentioned in his study of 29 patients with nhl that if there is discordance in results , then it is either secondary to two different primary lymphomas in the same patient or a result of modulation of antigen expression of the tumor , in relation to the host environment 22 . in one study , it was observed that discordant antigen expression was associated with a more aggressive course of disease 23 .
so far , the data are very limited as there are few cases that are able to be studied .
treatment for this particular disease is quite complex as very few treatment options provided a complete and longlasting response .
depending on the performance status , comorbidities , and age of the patient , current guidelines suggest that those with an improved performance status are more likely to be treated with combination treatment regimens such as rhypercvad / rhdmtxarac regimen .
those with a poorer performance status are suggested to be treated with rchop or rcvp based regimens 8 .
intraperitoneal application of rituximab was shown by martina chrysandt et al . to be effective in local control of the disease process in one of the case
though we remain unsure if we could use mcl cytological markers in the peritoneal fluid as a surrogate to conventional bm biopsy and testing , the data are promising in terms of concordance .
if bm testing is not able to be performed or testing has shown a lack of involvement of the bm by mcl , peritoneal sampling may be able to confirm a diagnosis .
| key clinical messageascites with the finding of peritoneal carcinomatosis is considered an unusual presentation for mantle cell lymphoma ( mcl ) and has been rarely described in literature .
this case reflects the importance of cytological analysis of peritoneal fluid in a patient with intractable ascites not contributing from other comorbidities . in the event
a bone marrow ( bm ) analysis can not be made , this may serve as an alternative method for diagnosing mcl taking into consideration the good concordance between peritoneal fluid and bm cytological markers . |
thyroid carcinoma usually presents as a palpable thyroid nodule or a nodule that is discovered incidentally on neck imaging for evaluation because of another reason .
the rate of malignancy for a thyroid nodule is of only 4 - 5% [ 2 , 3 ] .
the prevalence of thyroid nodules increases with age and is estimated to occur in roughly 50% of the us population 50 years of age and older .
thyroid cancer may present as a dominant nodule in a multinodular gland or as a solitary nodule .
a careful history is essential because it can reveal information that immediately stratifies a patient into a high risk category .
high risk factors include a prior history of head and neck radiation including radiation for hodgkin 's disease , radiation exposure from the chernobyl accident and a family history of thyroid cancer .
the risk of thyroid cancer in first degree relatives of patients with differentiated thyroid cancer has been shown to be six times that of the general population .
other important high risk factors include a history of rapid growth of a solid nodule , pain , dysphagia , or dysphonia .
although less than 20% of patients with thyroid carcinoma will present with a regional metastasis , a neck mass in the presence of a thyroid nodule raises the likelihood of malignancy .
young age ( < 20 years ) , older age ( > 70 years ) , and male gender may also represent an increased risk . to most
accurately determine the risk of malignancy , it is essential to consider a variety of factors ( table 1 ) .
there is no single laboratory study that is able to determine the presence of thyroid malignancy .
although elevated baseline calcitonin levels represent occult medullary carcinoma in 1040% of cases , the presence of false positive elevations , the low incidence of medullary carcinoma , and the high cost of testing have led to recommendations against routine testing in the united states .
calcitonin levels are usually reserved for high risk patients and those with a family history of medullary thyroid carcinoma or multiple endocrine neoplasia ( men ) .
thyroglobulin is not useful in the work up because there are a variety of conditions that can lead to elevated levels including thyroid inflammation , glandular stimulation or injury following radiation , surgery , or even a biopsy .
tsh should be measured to exclude a toxic nodule , as toxic nodules are almost never malignant and generally do not require fine needle aspiration . if the tsh is suppressed , then radioisotope imaging should be performed to confirm and localize the toxic nodule(s ) . clinical staging and
risk group classification is particularly important in the management of thyroid cancer because it often will direct management , in particular the decision to treat the patient with radioactive iodine , external beam radiation , or combination therapy .
the lack of prospective randomized studies assessing the relationships between tumor stage , treatment , and outcome , means that the majority of data that is used to create risk - group classifications is derived from retrospective reviews . as a result , there are variety of classification systems that are based on factors such as age , tumor size , gender , tumor grade , multicentricity , metastatic disease , and other variables . the ages system is based on factors including age , grade , extent , and size of the tumor ( table 2 ) . in the ages system , those patients with an aggregate score 4 + are high risk and those with a score less than 4 are low risk .
the ames system considers age , distant metastasis , extent , and size of tumor ( table 3 ) . additionally , there are systems devised by the european organization for research on treatment of cancer ( eortc ) , the national thyroid cancer treatment cooperative study , and others [ 13 , 16 ] .
irrespective of the classification system that is used , it is important to consider risk group stratification during the management of a patient with thyroid cancer .
separate from these stratification systems is the tnm tumor staging system ( table 4 ) which serves to provide a uniform language when evaluating management and outcome .
papillary thyroid carcinoma ( ptc ) is the most common form of the follicular cell derived carcinomas and comprises three quarters of all newly diagnosed thyroid cancers .
papillary thyroid cancers usually carry mutations and rearrangements that activates the mitogen activated protein kinase ( mapk ) .
rearrangements of ret and ntrk1 tyrosine kinases , activating mutations of braf and activating mutations of ras are sequential components of the mapk activation .
these rearrangements result in the production of chimeric proteins , named as ret / ptc and trk .
consequently , patients with men2 syndromes do not have an increased incidence of papillary thyroid cancer . in adults ,
close to 40 percent of sporadic papillary cancers have these rearrangements , with those in ret being about three times more common .
the incidence of ret rearrangements in papillary cancers is higher in children , affecting up the 80 percent in the case of children exposed to external x - irradiation and those exposed to the chernobyl nuclear accident .
the braf proteins are serine - threonine kinases that activate the raf / mek / mapk signaling pathway .
the t1799a mutation of the braf gene , affects up to 69 percent of papillary thyroid cancers [ 18 , 2224 ] . in one report
, braf mutations were found in 44 percent of 500 patients with papillary thyroid cancer .
braf mutations are associated with extrathyroidal tissue invasion , lymph node metastases and advanced tumor stage .
braf point mutations are less frequent in childhood thyroid cancers , including those children affected by the chernobyl nuclear calamity .
although activating ras mutations are found in follicular adenomas , they have also been reported in follicular variant of papillary thyroid cancers .
there may be an increased incidence of papillary cancer in patients with familial adenomatous polyposis .
the papillary thyroid cancers of these group patients with familial adenomatous polyposis have frequent rearrangements of ret .
other family studies have indicated a familial predisposition at 2q21 to ptc , as well as a familial predisposition at 8q24 to ptc with melanoma .
surgery is the primary treatment for papillary thyroid cancer and a complete resection offers the best chance of cure [ 10 , 34 ] .
while the role of a complete surgical resection is established , the extent of the initial surgical resection has been topic of controversy .
primary management of thyroid malignancy has included nodulectomy , hemithyroidectomy with and without isthmusectomy , subtotal thyroidectomy , and total thyroidectomy .
the high rate of recurrence associated with nodulectomy has confirmed that this approach is inadequate . while there has been a trend toward more aggressive surgical resection based on the well - documented multicentric nature of the disease ,
there are still some who insist that a partial thyroidectomy is sufficient proponents of total thyroidectomy point to data that demonstrates a rate of multicentric bilateral disease ranges from 18% to 46% [ 36 , 37 ] .
opponents of total thyroidectomy suggest that multicentric bilateral disease is often microscopic and not clinically significant .
this is further supported by the observation that several large studies have failed to demonstrate a survival advantage to total thyroidectomy when compared with hemithyroidectomy [ 38 , 39 ] . in spite of these findings , most clinicians agree that there is a subset of patients who benefit from total thyroidectomy while there is another distinct subset of patients that may be treated more conservatively .
the concern regarding over - treating a patient by performing a total thyroidectomy instead of a hemithyroidectomy relates to the surgical risk including an increased risk of vocal cord paralysis and a risk of permanent hypoparathyroidism . in an effort to guide surgical management ,
the ages and the ames classification systems represent two commonly used systems that are used to classify patients into either a high risk group or a low risk group .
it has been suggested that low risk patients can be managed with a thyroid lobectomy and isthmusectomy while high risk patients are better managed with a total thyroidectomy .
most surgeons agree with the recommendation to perform a total thyroidectomy when faced with a high risk patient .
several studies have failed to demonstrate that low risk patients experience an improvement in cause - specific mortality after undergoing a total thyroidectomy [ 12 , 40 ] while several large studies have demonstrated that there is a benefit to total thyroidectomy [ 39 , 41 ] .
the conflicting data can make the decision - making process a difficult one . as a result ,
not all surgeons favor such guidelines and many prefer to perform a total thyroidectomy in spite of the risk stratification suggesting that the rate of morbidity in the hands of an experienced surgeon is exceedingly low .
a recent study found that surgeons who perform more than 100 thyroidectomies per year reported complication rates of 4.3% while those surgeons who performed less than 10 thyroidectomies per year reported a four - fold increase in complication rate . in spite of the complication rate , when a subtotal thyroidectomy is performed for the management of thyroid carcinoma
finally , the american thyroid association ( ata ) recently recommended that a total thyroidectomy should be performed for the management of well - differentiated thyroid carcinoma .
whereas one recent expert consensus guideline from the ata [ 4 , 44 ] , recommended consideration of routine central neck dissection for most patients with papillary thyroid cancer , the guideline from the national comprehensive cancer network only recommends central neck dissection in the presence of grossly positive metastasis .
follicular carcinoma is less common than papillary thyroid carcinoma and typically behaves more aggressively than papillary thyroid carcinoma .
the aggressive nature of follicular thyroid carcinoma is largely a result of its propensity for hematogenous dissemination .
unlike papillary thyroid carcinoma , which spreads through lymphatics , follicular carcinoma has a tendency to metastasize hematogenously to the lung and bones .
the incidence of distant metastasis at presentation is not uncommon occurring between 10 and 20% of new cases [ 4547 ] . as a result ,
the key to effective management of follicular carcinoma is making an early diagnosis and total thyroidectomy .
the genetics aberrations of the follicular thyroid cancer includes the translocation ( t[q13;p25 ] ) that results in fusion of part of the dna - binding segment of the pax8 gene and the peroxisome proliferator - activated receptor gamma 1 ( ppar - gamma-1 ) gene ; pax8 is a thyroid transcription factor and ppar - gamma-1 is a transcription factor that stimulates cell differentiation and inhibits cell growth .
overexpression of normal c - myc and c - fos genes , as well as mutations of h - ras , n - ras , and k - ras protooncogenes , is found in follicular adenomas , follicular cancers , and occasionally papillary cancers [ 49 , 50 ] .
hypermethylation of rassf1a , a known tumor suppressor gene , has been described in one study , in 9 of 12 follicular thyroid cancers and in 44% of benign adenomas , and in 20% of papillary thyroid cancers reflecting and early step in thyroid carcinogenesis .
in contrast to papillary thyroid cancer , the frozen section pathological analysis is not helpful in making the diagnosis of follicular carcinoma because capsular and vascular invasion must be identified and this requires fine section analysis that can not be completed with fna or at the time of frozen section [ 53 , 54 ] .
once the diagnosis of follicular carcinoma has been confirmed , the decision to proceed with a completion thyroidectomy or follow the patient is based on a variety of factors including risk group analysis .
not only does a total thyroidectomy improve prognosis but it is very helpful in achieving accurate surveillance by facilitating rai administration and thyroglobulin monitoring .
there is controversy regarding the low risk group patients who present with minimally invasive lesions .
the decision to perform a completion thyroidectomy is often based on age of the patient and invasiveness of the tumor . in patients greater than 50 years old or those with extensive invasion , we recommend a completion thyroidectomy .
patients with low grade tumors , tumors that demonstrate minimal invasion , can be managed conservatively with observation and serial ultrasound examination .
there is a very low incidence of metastasis in low grade follicular carcinoma [ 55 , 56 ] and unlike papillary thyroid carcinoma , minimally invasive lesions are rarely multifocal .
however , as follicular lesions grow larger than 3 cm , nearly 30% will demonstrate malignant conversion .
while some have advocated suppressive therapy for low risk patients , this approach has failed to demonstrate the benefit of suppressive therapy .
unlike squamous cell carcinoma , the impact of lymph node metastasis from thyroid cancer on survival is negligible , at least in the younger population .
several studies have suggested that lymph node metastasis may not impact survival [ 58 , 59 ] however when adjusted for age , it is clear that although regional disease does not significantly impact on survival in young patients , it does decrease survival in patients over the age of 45 .
long term studies have confirmed these observations and identified that in the population of patients over 45 years old , regional metastasis does impact survival . as a result , most experts agree that clinically involved regional lymph nodes should be managed with a lymph node dissection
. there is , however , controversy regarding the impact of elective neck dissection on survival .
thyroid cancer commonly progresses in a defined pathway from the first echelon lymph nodes of the paratracheal region to the lateral compartment ( levels iii and iv ) , however it has been documented that regional disease may occur in the jugular chain without evidence of disease in the paratracheal basin in as many as 18% of cases . in spite of this rather high rate of skip metastasis , most surgeons do not advocate a lateral neck dissection unless there is clinical evidence of disease in the lateral compartment .
the rate of occult regional metastasis in papillary thyroid carcinoma is relatively high however occult metastasis in follicular carcinoma is rare .
hence , in patients with follicular carcinoma , an elective lymph node dissection is not commonly performed in the n0 neck . if lymphadenopathy is detected , a paratracheal and pretracheal central compartment neck dissection should be performed . in papillary thyroid carcinoma
, the high rate of paratracheal disease constitutes an ipsilateral paratracheal neck dissection when there is clinical evidence of disease .
the approach to management of the n0 neck in papillary carcinoma has been a topic of controversy largely because there are few randomized , controlled , long term studies that demonstrate the impact of surgical treatment of the n0 neck .
currently , most surgeons perform a pretracheal and ipsilateral paratracheal lymph node dissection on high risk patients ( those patients aged 45 years or older , or patients with tumors greater than 4 cm , or invasive disease ) .
only when the thyroid tumor crosses the midline is a contralateral lymph node dissection warranted .
when a contralateral lymph node dissection is performed , the patient must be informed of the increased risk related to transient and permanent hypoparathyroidism .
as discussed earlier , in spite of the relatively high rate of documented skip metastasis , most surgeons do not advocate a lateral neck dissection .
the rationale for this approach stems from the lack of data demonstrating the benefit of a lateral neck dissection for subclinical disease and the prevailing belief that i131 can be used to manage microscopic lateral metastasis . unlike follicular carcinoma
in which lymph nodes metastases are rare , metastasis in papillary thyroid carcinoma is common . while only a small proportion of patients will initially present with a neck mass , a significant number of patients will harbor subclinical paratracheal disease at the time of diagnosis .
the size of the primary thyroid tumor has little bearing on the extent or location of regional disease .
several groups including our own , have found that while large cancers of the thyroid may remain localized , microcarcinoma may present with extensive regional disease . as a result
, it is difficult to predict the risk of regional disease based on the size of the tumor and therefore every patient must be evaluated for regional metastasis .
i131 has not been demonstrated effective for management of gross regional disease . in the past
, there was controversy regarding the extent of the dissection however most surgeons now advocate a selective neck dissection ( levels iii , iv , and vi ) in which the jugular vein , sternocleidomastoid muscle , and the accessory nerve are preserved .
although rare , when invasion of the surrounding structures occurs , a modified neck dissection may be warranted .
remnant ablation with 131i is routinely performed in all patients except for very low risk patients after total thyroidectomy to decrease the risk of recurrence and facilitate monitoring of thyroglobulin .
usually thyroid replacement is discontinued and a low iodine diet instituted in an effort to increase the avidity of the residual thyroid tissue .
mu / l , the patient is given radioiodine in doses ranging 30 mci to 100 mci .
patients in whom residual microscopic disease is suspected and those with more aggressive tumor histology or known distant metastasis are administered higher doses of i131 in the range of 100 to 300 mci .
hypothyroidism can be avoided by administering recombinant human tsh ( rhtsh , thyrotropin alfa ) before administration of 131-i for remnant ablation [ 66 , 67 ] .
short - term results of radioiodine ablation appears to be similar after rhtsh administration or thyroid hormone withdrawal . in one study
the radiation dose to the thyroid bed of a tracer amount of radioiodine was similar after two injections of thyrotropin alfa or thyroid hormone withdrawal .
in retrospective studies , the percentage of patients with successful ablation was similar in patients treated with rhtsh compared to withdrawal [ 66 , 67 ] .
pacini et al . , performed an study , with remnant ablation with 30 mci ( 1110 mbq ) after withdrawal of thyroid hormone , and after rhtsh aloneand after combining withdrawal and rhtsh , resulted in successful ablation in 84% , 54% percent , and 79% of patients , respectively .
the rate was significantly lower in patients prepared with rhtsh alone . in another trial , remnant ablation with 54 mmi ( 2000 mbq ) after withdrawal of thyroid hormone or after rhtsh resulted in similar rates of successful ablation .
although one retrospective study found similar short - term recurrence rates in patients prepared with traditional thyroid hormone withdrawal , no long - term randomized study has been published comparing both approaches .
because of the lack of long - term data on recurrence and disease - specific survival , some centers are offering rhtsh ablation to only low risk patients preferring traditional hypothyroid withdrawal for patients at higher risk of recurrence or death from disease .
the response of pulmonary metastasis to i131 is predicated on the size of the lesion , the age of the patient , and its ability to concentrate i131 [ 73 , 74 ] .
in contrast , bone metastases , which occur more commonly in follicular thyroid carcinoma , are less responsive to therapy .
the use of recombinant human tsh ( rhtsh ) for remnant ablation and in the treatment of metastatic disease is now being studied and has the advantage of avoiding symptomatic hypothyroidism .
although randomized controlled trials are lacking , benefit was suggested in a large meta - analysis .
higher risk patients are initially treated with a greater degree of suppression . due to the risk of osteoporosis
, many clinicians will now reduce the levothyroxine dose once the patient has been free of disease for several years .
although prospective controlled trials are lacking , several studies have shown excellent local - regional control of disease with external beam radiotherapy ( ebrt ) in patients with locally advanced differentiated thyroid cancer after the primary surgery and before or after radioiodine [ 7779 ] .
several groups have suggested that ebrt be considered in patients over 45 years with suspect persistent local disease after surgery which is unlikely to respond to radioiodine [ 78 , 80 ] .
following total thyroidectomy , as many as 15% of patients will develop neck disease 0 to 5 years after initial therapy [ 64 , 81 ] .
since the volume of recurrent or distant metastatic disease correlates relatively well with the prognosis , early diagnosis and management is essential .
whole body i131 scanning and should be performed in high risk patients 6 to 12 months after the primary surgery .
low risk patients can usually be evaluated by recombinant tsh ( thyrogen ) stimulated thyroglobulin levels avoiding the need for an iodine scan .
since the majority of recurrences occur in the thyroid bed , neck ultrasonography represents the most sensitive means for detecting recurrence in the presence of elevated thyroglobulin . for those patients with elevated thyroglobulin and a negative iodine scan , pet /
ct is exceptionally helpful in identifying recurrent and distant disease and has been shown to provide important prognostic information .
evaluated 10 patients with suspected recurrent papillary thyroid cancer using pet and concluded in this small series that pet was useful in the evaluation of patients with suspected recurrent papillary thyroid cancer when the i131 scan is negative . in a more recent study , eight patients underwent combined pet / ct scanning for suspected recurrence .
four ( 50% ) of 8 patients underwent pet / ct indicating recurrence in the head and neck
. a total of 11 lesions in these 4 patients were suspicious for recurrence on combined pet / ct imaging .
three patients with 8 lesions suspicious for recurrence on pet / ct underwent surgical removal of disease .
all 3 patients had pathologic confirmation of recurrence , with 75.0% of the 8 lesions being positive .
the authors concluded that combined pet / ct imaging is a valuable tool for the diagnosis and localization of recurrent thyroid cancer .
recent evidence suggests that pet - positive disease is unlikely to i131 and may be associated with a more biologically aggressive tumor .
the management of recurrent or metastatic disease largely depends on the extent of the disease . when there is microscopic persistent or recurrent disease , i131 dosing should be repeated .
however when palpable gross tumor is identified , surgical management is the treatment of choice . when distant disease is identified , adjuvant i131 is usually the preferred .
noniodine avid tumors represent a challenge and may indicate a poorly differentiated tumor that is often associated with a more aggressive behavior and a poor prognosis .
ebrt may be used for gross residual disease unresponsive to radioiodine and not amenable to further surgery .
external beam radiation or surgical resection can also be used for the management of pain or to decompress nerve root compression in the vertebrae however neither therapy has an impact on prognosis .
there are few studies demonstrating the efficacy of systemic chemotherapy however there is data to suggest that doxorubicin may be effective in some patients ( 6075 mg / m every 3 weeks ) .
hrthle cell carcinoma ( hcc ) is a relatively rare form of thyroid cancer that is generally considered to be a more aggressive variant of follicular carcinoma .
similar to follicular carcinoma , the distinction between a benign hrthle cell adenoma and a hcc is determined on the presence of vascular or capsular invasion .
similar to follicular cell carcinoma , the diagnosis can not be made on fine needle aspiration or frozen section analysis .
, it was found that the median age of presentation for patients with follicular cell carcinoma was 55 years while the hcc patients presented at a mean age of 62 years .
patients presenting with hcc have a lower risk of regional metastasis at presentation when compared with follicular carcinoma , but a slightly increased risk of distant metastasis .
when followed long term , the number of patients that eventually develop distant metastasis is higher in hcc patients ( 30% ) than papillary or follicular thyroid carcinoma patients .
when distant metastasis occurs , 40% occur in the bone and as many as 30% occur in the lung .
hcc with minimal capsular invasion ( < 1 mm ) can be safely managed with a thyroid lobectomy and isthmusectomy . however , because it has been demonstrated that patients treated with a total thyroidectomy have a lower risk of recurrence [ 84 , 88 ] , recommend a total thyroidectomy .
all high risk patients or patients with more extensive capsular invasion are managed with a total thyroidectomy .
less that 200 new cases of hcc carcinoma are diagnosed in the united states each year . as a result , there are few long term outcome studies however , evans and vassilopoulou - sellin found in 1998 no difference in outcome between hcc and follicular cell carcinoma when cases are stratified by extent of invasion .
like follicular cell carcinoma , the most important factor in assessing the prognosis seems to be the extent of capsular invasion . when tumors invade less than 1.0 mm , the rate of recurrence in exceedingly small , however as the extent of capsular invasion increases , the likelihood of regional and distant metastasis increases .
medullary thyroid carcinoma ( mtc ) is derived from the nonepithelial parafollicular cells ( c - cells ) which produce the peptide calcitonin . since the parafollicular cells
embryo logically develop from the neural crest or diffuse neuroendocrine system , they commonly produce neuropeptides and catecholamines . tumors deriving from diffuse neuroendocrine system , such as carcinoid tumors , pancreatic islet tumors , and pheochromocytomas , are cytologically and functionally similar .
while the gender distribution is roughly equivalent , the sporadic form of the disease occurs in 80 to 90% of newly diagnosed cases .
the familial form of the disease occurs in an autosomal dominant trait as either isolated familial medullary thyroid carcinoma ( fmtc ) , multiple endocrine neoplasia type iia ( men iia ) , or multiple endocrine neoplasia type iib ( men iib ) ( table 5 ) .
each of these familial forms of the disease is associated with a ret oncogene mutation located on chromosome 10 .
since the penetrance of medullary thyroid cancer is > 90% in people with the ret gene mutation , prophylactic thyroidectomy is recommended at a young age .
not unlike other forms of thyroid carcinoma , sporadic mtc typically presents as a thyroid nodule with or without an associated neck mass .
since mtc is derived from the diffuse neuroendocrine system , patients may present with a paraneoplastic syndrome resulting in diarrhea and abdominal cramping as a result of prostaglandin and vasoactive peptide release .
more aggressive tumors may present with a neck mass or in rare cases distant metastasis to the lungs , liver , or adrenal glands . in some cases , tumors may remain stable or quiescent while other tumors may progress rapidly . there has been little data to help predict the biological aggressiveness of mtc .
the most accurate method for diagnosis of mtc is an fna of the thyroid nodule or neck mass .
while cytodiagnostic features are recognized , immunostaining for calcitonin in the presence of negative thyroglobulin staining , is the most accurate method of diagnosis .
however , it is interesting to note that familial mtc has a predilection for presenting as a thyroid nodule at the junction of the upper third and lower two - thirds of the thyroid gland because this is the area of the gland in which the density of c cells is the highest .
sporadic disease usually presents as a unilateral nodule while familial disease is more commonly bilateral and multifocal .
familial disease is considered more aggressive and commonly presents in the second or third decades while sporadic disease presents later , in the sixth or seventh decades .
measurements of serum calcitonin and carcinoembryonic antigen ( cea ) , to determine whether they are produced by the tumor and , if so , as a baseline for comparison with results obtained before and after surgery .
patients with higher preoperative serum calcitonin concentrations have larger tumors and are less likely to achieve normal concentrations after .
patients who have normal serum cea and serum calcitonin , are considered biochemically cured and have the best prognosiswith a five recurrence rate of only 5% .
the serum calcitonin concentration falls slowly in some patients , with the nadir not being reached for several months . a high basal serum calcitonin value six or more months after surgery is presumptive evidence of residual disease .
subsequent follow - up should include periodic physical examination and measurements of serum calcitonin and cea .
larger thyroid tumors are associated with an increased risk of neck disease and therefore tumors greater than 2 cm should be treated with an ipsilateral selective neck dissection in addition to a bilateral paratracheal lymph node dissection .
some have suggested that all patients should be treated with bilateral selective neck dissections because patients with unilateral intrathyroidal tumors , as many as 81% of patients have lymph node metastasis in levels ii v and 44% have disease in the contralateral lymph nodes level ii v .
aggressive surgical therapy is warranted because occult metastases are common and surgery is the only effective method of therapy .
there is no evidence that medical therapy has any role in the primary management of mtc and there is little evidence to suggest that adjuvant medical therapy will impact outcome .
there is no role for radioiodine or thyroid hormone suppression in the treatment of medullary cancer .
brierley et al . treated a series of 40 patients at high risk for recurrence with external beam radiation and demonstrated an improvement in local - regional control ( 86% versus 52% ) at 10 years [ 80 , 97 ] however several other studies have failed to demonstrate an improvement in survival with external beam radiotherapy [ 98 , 99 ] .
systemic chemotherapy has little impact on the natural course of the disease , and while schlumberger et al .
demonstrated 20% response rate in patients treated with doxorubicin , there is little data to support the use of systemic chemotherapy in mtc .
a new therapy using pretargeted anticarcinoembryonic antigen radioimmune therapy ( rit ) has shown a survival benefit in a small clinical trial . when faced with regional recurrence , surgery is the treatment of choice .
radioactive iodine uptake is negligible in mtc and therefore aggressive surgical management of regional disease is justified .
if a bilateral neck dissection was not performed at the time of initial therapy , bilateral neck dissections should be considered , even in the face of unilateral recurrence .
as discussed , conventional adjuvant therapy offers little for patients with regional or distant disease .
when patients demonstrate a persistently elevated calcitonin level following surgery , it is because there is residual or recurrent disease .
ct , mri , and pet have all been used with little success unless there is gross disease remaining in the neck or thyroid bed .
the management of a patient with a persistently elevated calcitonin level and no radiographic evidence of disease is controversial .
this is because there is evidence to suggest that this patient group does not necessarily have a compromised outcome and when reoperation is pursued , the rate of success is low .
we recommend bilateral selective neck dissections for those patients that have not already undergone a neck dissection .
prognosis is better in younger patients , female gender , familial disease , and those with disease confined to the thyroid gland .
the overall 5-year survival rate ranges from 25% to 75% [ 104 , 105 ] . when nodal disease is present at the time of diagnosis , which occurs in 50% of patients , the 5-year survival rate drops to less than 50% .
a recent series suggests that the 5 and 10 year survival rates are as high as 90% and 80% .
are anaplastic thyroid carcinoma ( atc ) , it accounts for 1439% of thyroid carcinoma deaths .
mutations of the p53 tumor suppressor gene that lead to production of an inactive p53 protein occur in most anaplastic thyroid cancers , and not in other thyroid .
mutations of the beta - catenin gene occur in up to 65 percent of anaplastic thyroid carcinomas .
it has been suggested that differentiated thyroid cancers may deteriorate into anaplastic thyroid cancers , it has been proposed that several different gene mutations provide an early growth advantage and that p53 mutations are the final step in the development of an anaplastic cancer .
evidence comes from studies describing well differentiated regions inside anaplastic cancers and the consequent mutation of braf , acquiring p53 mutation in a second step [ 111 , 112 ] .
however , in one study of 17 anaplastic cancers , no ret gene rearrangements were detected , providing no evidence for the evolution of papillary into anaplastic thyroid cancer .
somatic mutations of the catalytic subunit of the phosphatidylinositol 3-kinase ( pik3ca ) have been identified in about 23 percent of anaplastic thyroid cancers . in anaplastic thyroid cancers with
well differentiated components , the pik3ca mutations are restricted to the anaplastic component . the diagnosis of anaplastic thyroid carcinoma can be elusive . not uncommonly , an fna will be repeatedly interpreted as undifferentiated carcinoma until an open biopsy provides enough tissue to reveal the anaplastic nature of the carcinoma . unlike other forms of thyroid cancer , anaplastic is characteristically aggressive and unrelenting .
accounting for less than 5% of malignant thyroid cancers , anaplastic thyroid cancer is often heralded by a rapidly growing thyroid mass with destruction of the adjacent cartilage , nerve and infiltrating muscles .
the group of chan et al . in hong kong recently analyzed the clinico - pathological features , treatment and outcome of all patients with atc treated over the past four decades in their institution .
the results confirmed the dismal outlook noted in previous studies , with a median survival of only 97 days , with 1- and 3-year survival of 14% and 8% respectively .
age 65 years or younger , lack of metastatic disease , surgical resection , and radiation to the neck were associated with better prognosis on univariate analysis .
cytotoxic chemotherapy was not associated with improved survival and disappointingly , no change in survival was seen over the 40-year period of the study .
this and other studies emphasize the need for new treatment strategies for atc [ 115117 ] .
anaplastic thyroid carcinoma typically presents in the elderly population with a history of thyroid goiter . up to 20% of patients
will have a history of well - differentiated thyroid cancer or co - existing well - differentiated thyroid cancer present within the thyroid specimen [ 119 , 120 ] .
patients usually present with a rapidly growing neck mass , true vocal cord paralysis , and or dysphagia .
anaplastic carcinoma may arise de novo or as a conversion tumor arising in the presence of a preexisting papillary or follicular carcinoma .
when anaplastic carcinoma arises de novo , the tumor may progress rapidly metastasizing to the lungs , liver , and bones within weeks of presentation .
a fna is often the first approach to diagnosis however necrosis and degeneration of the tumor may occur as the tumor rapidly out grows its blood supply .
alternatively , the fna may yield poorly differentiated cells suggestive of either lymphoma , anaplastic thyroid cancer , or poorly differentiated carcinoma of unknown origin .
flow cytometry is useful to rule out lymphoma , however immunohistochemistry and electron microscopy may be necessary to confirm the diagnosis of anaplastic thyroid carcinoma .
there is controversy regarding the ideal management of anaplastic carcinoma largely because it is rapidly progressing and poorly responsive to therapy .
the options for therapy range from multimodal therapy , surgical resection , debulking , radiotherapy , chemotherapy , and palliative therapy [ 118 , 122 , 123 ] .
early disease , disease confined to the thyroid gland , is poorly responsive to unimodality therapy , however in a recent study , investigators reviewed a cohort of 516 patients with anaplastic thyroid carcinoma reported to 12 population based cancer registries between 1973 and 2000 .
they found that patients less than 60 years old with intrathyroidal anaplastic thyroid cancer appear to have a better prognosis than older patients with extra - thyroidal spread following total thyroidectomy and external beam radiation .
long term survivors are few however in those that do survive the disease , they commonly have only a small focus of anaplastic tumor arising within a preexisting well - differentiated thyroid cancer [ 17 , 125 ] .
hyperfractionated therapy has resulted in a marginal improvement in response rates however the side effects , including esophagitis , can be debilitating .
tennvall et al . reported using preoperative hyper fractionated radiation ( 30 gy ) , doxyrubicin , and surgical resection followed by postoperative radiation ( 46 gy ) .
they demonstrated promising results with this regimen , controlling local recurrence in 48% of patients and disease free survival in 12% .
in advanced disease , the goals include establishing an airway , a source of nutrition , and an acceptable quality of life .
surgery is warranted for the control or prevention of airway obstruction or extensive skin ulceration .
patient age , gender , tumor size , extent of disease , leukocytosis , presence of acute local symptoms , coexisting multinodular goiter and well differentiated thyroid carcinoma , surgical resection , and multimodal therapy all reportedly influence patient survival according to some studies [ 130 , 131 ]
axitinib ( ag-013736 ) is a potent small molecule inhibitor of vegf receptors 1 , 2 , and 3 .
a single arm multicenter study evaluated the efficacy of this novel compound in advanced thyroid cancers .
sixty patients with metastatic or unresectable locally advanced thyroid cancer refractory to or not suitable candidates for radioiodine treatment , received axitinib at a starting dose of 5 mg orally twice daily . the majority of the patients had differentiated thyroid cancer and two had anaplastic cancer .
there were 18 partial responses and 30 patients had stable disease . the median progression free survival had not been reached at a median follow up of 273 days .
importantly the study was a proof of principle as levels of soluble vegf receptor were consistently decreased and levels of vegf in the blood were increased demonstrating pharmacodynamic activity against targeted vegf receptors [ 132 , 133 ] .
sorafenib , a multitargeted small molecule kinase inhibitor , including the vegf receptor and braf kinase , has also been evaluated in patients with thyroid cancer .
of 19 evaluable patients with metastatic , iodine refractory , papillary thyroid cancer enrolled in a phase ii trial of sorafenib , five patients achieved a partial response and eight had stable disease with sorafenib .
median response duration was 14.2 months ( 1215 months ) and median time to progression of all patients was 4 months ( 4 days to 14.5 months ) .
pharmacodymanic analysis of tumor tissue before and after sorafenib treatment ( at 1 and 2 weeks ) in two patients revealed reduction in perk ( downstream of vegfr and braf ) and pakt ( downstream of vegfr ) .
sorafenib is currently us food and drug administration approved for the treatment of renal cell cancer and hepatocellular carcinoma .
it has side effects including a desquamating rash often on the hands and can cause gastrointestinal symptoms .
it is currently being used in many trials across the world and in many malignancies and its role in the treatment of thyroid cancer is likely to increase . in the phase ii trial of gupta - abramson et al .
, thirty patients were treated for a minimum of 16 weeks with sorafenib 400 mg twice daily .
seventeen ( 95% ) of 19 patients for whom serial thyroglobulin levels were available showed a marked and rapid response in thyroglobulin levels with a mean decrease of 70% .
toxicity was consistent with other sorafenib trials , although a single patient died of liver failure that was likely treatment related .
the overall clinical benefit rate ( partial response stable disease ) in this study was of of 77% , median pfs of 79 weeks , and an overall acceptable safety profile .
amg-706 finally , in a third abstract reported at the 2007 annual meeting of the american society of clinical oncology , amg-706 , another multikinase inhibitor ( vegf / pdgf receptors , kit and ret ) , was studied in a phase ii trial of patients with advanced differentiated thyroid cancer or medullary thyroid cancer .
the results for the differentiated group with median follow up of 32 weeks were presented at the meeting .
side effects were tolerable with diarrhea and hypertension being the most common severe toxicities ( 11 and 22% , resp . ) .
these three studies show the renewed interest in refractory thyroid cancer management and that small molecule inhibitors offer one of the most exciting prospects for the treatment of these patients .
cyclo - oxygenase 2 ( cox-2 ) is an enzyme overexpressed in many thyroid cancers .
inhibition of this enzyme has been shown to reduce progression of tumors in animal models and humans .
two common oncogenes in papillary thyroid cancer , ret / ptc1 and ret / ptc 2 , induce expression of cox2 .
celecoxib , a widely available selective cox-2 inhibitor , seemed therefore to be an attractive potential treatment for this disease .
unfortunately an early study using this drug failed to show a benefit at a dose of 400 mg twice daily .
gefitinib ( iressa ) is a tyrosine kinase inhibitor of the epidermal growth factor receptor ( egfr ) .
this receptor is highly expressed on normal and malignant thyroid tissue , and has been associated with a worse prognosis in well differentiated thyroid cancer .
although a phase ii clinical trial with gefitinib reported no objective responses by recist criteria in 27 patients with radioiodine resistant thyroid cancer , reduction in tumor volume was noted in 32% of the patients , and 12% of patients had no evidence of progression after one year of treatment .
overall survival was 17.5 months in the cohort [ 138 , 139 ] . some of the multikinase inhibitors mentioned above have shown hints of activity in the few patients enrolled with atc .
another antiangiogenesis compound , combrestatin a4 phosphate ( ca4p ) , has further been evaluated in a cohort of patients with atc . unlike the other angiogenesis inhibitors such as bevacizumab , an antibody against vegf , or the vegf receptor kinase inhibitors ,
ca4p is a tubulin - binding vascular disrupting agent that stops blood flow through existing blood vessels , depriving the tumor of its nutrients and oxygen .
a phase ii study with this agent in 18 patients with atc was reported last year .
although there were no objective responses , six patients had stable disease and 28% had no progression of disease for greater than 3 months [ 28 , 140 ] .
based on these results , an international phase iii study has been planned evaluating the use of ca4p in combination with the widely used chemotherapy pair of paclitaxel and carboplatin .
the study is a huge undertaking given the rare and aggressive nature of the disease , and offers some hope to a population of patients with no other effective treatments .
one of the hallmarks of mtc is the association with the rearranged during transfection protooncogene ( ret ) ( figure 1 ) . in normal states ,
this gene is involved in cell signaling , and regulates the production of proteins that are essential for spermatogenesis and the development of the autonomic nervous system and kidneys .
ret is a transmembrane protein that can be activated by extracellular signals and initiate a complex cascade of intracellular reactions regulating cell growth .
a tyrosine kinase enzyme located on the intracellular domain of ret activates this cell signaling cascade .
mutations in specific regions of the ret gene have been described in mtc , and these occur in both the sporadic and familial form of the disease [ 141 , 142 ] .
these mutations have led to fascinating experiments looking at corrective gene therapy as a treatment strategy .
zd6474 ( zactima , vandetanib ) is a novel anilinoquinazoline compound with a molecular weight of 475 daltons .
it was considered worthy of further development after demonstrating potent inhibition of vegfr-2 tyrosine kinase in recombinant enzyme assays as well as additional activity against vegfr-3 , egfr and ret tyrosine kinases .
vandetanib showed excellent selectivity for these kinases compared with related receptor tyrosine kinases such as platelet - derived growth factor receptor ( pdgfr)-b and c - kit [ 143145 ] .
its activity in hereditary mtc has been established in a single arm phase ii study , which reported a 17% confirmed and 7% unconfirmed partial response rate in 30 patients , with a 50% stable disease rate . in 23 patients there was a decrease in calcitonin levels by at least 50% which was maintained for 4 weeks or longer .
toxicities were manageable , with the most common adverse events being diarrhea , rash and asymptomatic qtc prolongation .
the encouraging results of this trial have spurred accrual onto an ongoing international randomized phase ii trial comparing zd6474 to placebo .
once the results from this trial are available , the authors feel that there will probably be enough evidence for the approval of zd6474 in the treatment of metastatic or unresectable mtc , a true landmark in this disease . | thyroid cancer is the endocrine tumor that bears the highest incidence with 33 550 new cases per year .
it bears an excellent prognosis with a mortality of 1530 patients per year ( jemal et al . ;
2007 ) .
we have been treating patients with thyroid carcinoma during many years without many innovations .
recently , we have assisted to the development of new agents for the treatment of this disease with unexpected good results . here
we present a review with the old and new methods for the treatment of this disease . |
berberine , one of the main constituents of rhizoma coptidis , is usually used to treat bacterial diarrhea in china , and it has also been used to treat diabetes for thousands of years in traditional chinese medicine . in 1988 , the glucose - lowering effect of berberine was reported when 60 diabetic patients were treated with berberine for diarrhea in china . since then , there have been many studies that confirmed berberine s antidiabetic effect . in a pilot study , yin j et al . found that berberine significantly decreased hba1c levels in diabetic patients who took 500 mg berberine 3 times daily for 13 weeks ( from 9.5% to 7.5% ) and the effect of decreasing hba1c was comparable with that of metformin .
in addition , a randomized , double - blind , placebo - controlled and multiple - center trial showed that treatment with berberine ( 0.5 g , twice daily ) for 3 months in 57 type 2 diabetic patients significantly reduced levels of fasting and postprandial plasma glucose and hba1c by 1.4 , 3.1 mmol / liter , and 0.9% , respectively , which was accompanied with decreasing triglyceride and total cholesterol concentrations by 35.9% and 18% , respectively . the mechanisms through which berberine reduces blood glucose are not entirely clear at present .
a previous study showed that berberine reduced body weight and improved insulin resistance in db / db mice and high fat - fed wistar rats .
berberine upregulated amp - activated protein kinase ( ampk ) activity in adipocytes and myotubes , and facilitated glut4 translocation in myotubes .
kong wj et al . reported that berberine reduced levels of fasting blood glucose and fasting serum insulin , increased insulin sensitivity , and elevated insulin receptor ( insr ) mrna in the liver of type 2 diabetes mellitus rats .
berberine also improved glucolipid metabolism through modulating metabolic - related pparalpha / delta / gamma protein expression .
in addition , berberine decreased gluconeogenesis through upregulation of the glucokinase and glucose-6-phosphate dehydrogenase activities and downregulation of glucose-6-phosphatase activity .
yin j et al . reported that berberine enhanced glucose metabolism by stimulation of glycolysis .
berberine significantly inhibited the activities of intestinal disaccharidases and beta - glucuronidase in diabetic rats , so it could significantly lower postprandial blood glucose levels induced by sucrose or maltose loading in normal rats .
recent studies indicated that berberine enhanced glucagon - like peptide-1 ( glp-1 ) secretion in streptozotocin - induced diabetic rats and normal rats .
glp-1 is secreted by the intestinal l cell and induces glucose - dependent stimulation of insulin secretion .
berberine could also regulate glucose homeostasis through decreased oxidative stress , and many studies have shown that diabetes mellitus is associated with increased oxidative stress .
the adult human intestine is colonized by approximately 100 trillion bacteria , which is about 10 times the number of total cells of the human body .
recent evidence suggests that the gut microbiota composition is associated with obesity and type 2 diabetes .
analyzed 5088 bacterial 16s rrna gene sequences from the gut microbiota of genetically obese ob / ob mice and their lean counterparts .
they found that ob / ob mice had a 50% reduction in the abundance of bacteroidetes and a proportional increase in firmicutes .
furthermore , they found that the gut microbiota in the ob / ob mice had a more effective capacity to harvest energy from the diet than the lean littermates , and that this capacity could be transmitted to germ - free mice who were transplanted with caecal microbiota from ob / ob mice .
researchers found that the proportions of firmicutes and clostridia were significantly reduced , while the relative abundance of bacteroidetes and betaproteobacteria was increased in the diabetic group compared to the control group .
recently , obesity , insulin resistance and type 2 diabetes have been closely associated with a low - grade inflammatory state characterized by abnormal cytokine production , increased acute - phase reactants and activation of a network of inflammatory signal pathways .
the low - grade inflammation was induced by the change of gut microbiota , which promoted metabolic endotoxemia and triggered the development of inflammation via the lps and cd14/tlr4-dependent mechanism .
the protective effect of gut microbiota modulation against diabetes had been confirmed in recent observations .
probiotics are nonpathogenic live microorganisms that , when ingested , confer health benefits to the host .
investigated the effects of oral administration of the probiotics compound on the occurrence of diabetes in 4-week - old non - obese diabetic ( nod ) mice . at 32 weeks of age , 21% ( 4/19 ) of mice in the probiotics - treated nod group were diabetic , in comparison with 81% ( 17/21 ) of the phosphate buffered saline ( pbs)-treated group ( p<0.001 ) .
protected mice showed reduced insulitis and a decreased rate of beta cell destruction . in another animal study
, researchers observed that a fermented milk product , dahi , which contains probiotic bacteria , significantly delayed the onset of glucose intolerance , hyperglycemia , hyperinsulinemia , dyslipidemia , and oxidative stress in high fructose induced diabetic rats .
the ob / ob mouse is a mutant mouse that eats excessively and becomes profoundly obese with hyperinsulinemia and dyslipidemia .
given norfloxacin and ampicillin in drinking water ( 1g / l each ) for 2 weeks , with free access to sterile food and water , the numbers of cecal aerobic and anaerobic bacteria in ob / ob mice were maximally suppressed .
the enhanced insulin sensitivity was independent of food intake , weigh loss or adiposity , which was not due to a defect in glucose absorption , as the expression of sodium glucose cotransporter 1 ( sglt-1 ) and glucose transporter 2 ( glut2 ) in jejunum was not affected by gut microbiota modulation .
endotoxins such as lps from gram - negative bacteria in the gut have been shown to play an important role in the development of insulin resistance . in this study , both plasma lps levels and the expression of jejunal tnf- level were significantly lower in the antibiotic - treated than the control and pair - fed mice , suggesting that modulating gut microbiota by norfloxacin and ampicillin ameliorated the inflammatory status in the intestine of ob / ob mice . when the diet - induced obese and insulin resistance ( dio ) mice were treated with non - absorbable antibiotics polymyxin b and neomycin , the mice demonstrated a gradual reduction in glycemia during a washout period , associated with modified cecal microbiota profile . in brief , modulation of gut microbiota ameliorated glucose intolerance in mice and altered the inflammatory and metabolic status of the host .
berberine has been shown to have significant antimicrobial activity against bacteria , fungi , parasites , worms , and viruses . in term of bacteria ,
berberine has demonstrated highly significant activity against staphylococcus , streptococcus , salmonella , klebsiella , clostridium , pseudomonas , proteus , shigella , vibrio , and cryptococcus species .
moreover , berberine inhibited the overgrowth organisms such as staphylococci and coliforms , while having no effect on indigenous lactobacilli and bifidobacteria .
berberine is thought to act topically in the gastrointestinal tract , and it is poorly absorbed , so berberine might modulate gut microbiota without systemic anti - infective activity .
berberine blocks adhesion of bacteria to epithelial cells , inhibits the intestinal secretory response of cholera and e. coli toxins , and normalizes mucosal histology following cholera toxin damage .
berberine also inhibits growth of salmonella typhi through decrease arylamine n - acetyltransferases activity and gene expression .
domadia et al . reported that berberine inhibited the assembly function of ftsz and halted escherichia coli cell division .
ftsz is an essential cell division protein in bacteria , which is well conserved across bacterial species .
to date there has been no experimental or clinical investigation that can reveal the modulating gut microbiota property of berberine in antidiabetic treatment .
it is thereby necessary to introduce animal experiments to investigate the antidiabetic mechanism of berberine .
male db / db diabetic mice , ages 58 months , can be treated with different doses of berberine or placebo in their drinking water for a certain period . at the end of the treatment
, an ogtt can be performed , and plasma triglyceride , insulin , cholesterol , and adiponectin levels can be measured .
plasma lps and tnf- concentrations can be determined to examine the inflammatory status of db / db mice .
cecal content should be collected for bacterial 16s rrna gene sequence analysis after the mice are killed .
the link between alteration of microbial - community composition in the distal intestine and metabolic amelioration should be evaluated .
furthermore , to avoid the interaction between modulating gut microbiota and other mechanisms of berberine such as activation of ampk , some methods that do not change the gut microbiota should be carried out .
the diabetic mice can be treated with berberine through intravenous injection , which does not change the gastrointestinal flora markedly .
using germ - free db / db diabetic mice in the research can exclude the gut microbiota modulation effect of berberine .
we have sufficient reason to believe that modulating gut microbiota is an antidiabetic mechanism of berberine . our hypothesis might provide a novel elucidation for berberine s therapeutic effect in patients with diabetes mellitus . with the aid of well - designed prospective studies | summaryberberine , one of the main constituents of a chinese traditional herb used to treat bacterial diarrhea , has an effect of lowering glucose , which has been recently confirmed by many studies .
however , the mechanism of berberine s antidiabetic effect has not yet been well explained .
recent evidence suggests that the gut microbiota composition is associated with obesity and type 2 diabetes , which are closely associated with a low - grade inflammatory state .
the protective effect against diabetes of gut microbiota modulation with probiotics or antibiotics has been confirmed in recent observations .
berberine has significant antimicrobial activity against several microbes through inhibiting the assembly function of ftsz and halting the bacteria cell division .
because berberine acts topically in the gastrointestinal tract and it is poorly absorbed , berberine might modulate gut microbiota without systemic anti - infective activity .
our hypothesis is that gut microbiota modulation may be one mechanism of the antidiabetic effect of berberine .
our hypothesis may provide a novel explanation for berberine s therapeutic effect in patients with diabetes mellitus . |
retinitis pigmentosa ( rp ) is known to be caused by various genetic changes in the components of the phototransduction pathway .
some genetic changes may alter rod cells to be more susceptible to oxidative damage in the retina , which is exposed directly to the effects of light , leading to progressive retinal degeneration ( 1 , 2 ) .
in other genetic mutations , rod cells may die from other primary processes ; and then when the rod cells die , the oxygen content in the retina rises , causing oxidative stress and secondary degeneration of the remaining cone cells .
various anti - oxidants have been considered as possible candidates that might delay this progression .
there have been numerous reports on the effects of antioxidants in delaying the degeneration of photoreceptors in animal models of rp ( 3 ) .
anti - oxidant supplementation has been found to be effective in the intervention of atrophic age - related macular degeneration ( 4 ) .
human clinical trials for antioxidants such as vitamins a and e , lutein , and docosahexaenoic acid have been conducted for rp with some beneficial effects noted ( 5 - 8 ) .
additionally , continuous modifications of anti - oxidant dosages and regimens have been tried as possible candidates for nutritional supplementation in rp .
recently , patients consuming a diet rich in omega-3 fatty acids and receiving 15,000 iu vitamin a daily were found to have a slower progression in decline of visual acuity over 4 to 6 yr ( 9 ) , raising hope for a possible delay of progression in rp patients .
chlorogenic acid ( cga ) is a polyphenol found in various agricultural products such as coffee , beans , potatoes , and apples , and is formed by the esterification of caffeic and quinic acids .
the antibacterial , anti - inflammatory , anti - oxidant , and anti - carcinogenic activities of cga have been demonstrated in many studies ( 10 - 13 ) .
cga showed an anti - oxidant effect in vitro ( 14 ) , in cultured human endothelial cells ( 15 ) , and in rat models of ischemia / reperfusion injury to the liver ( 16 ) .
the beneficial effect of cga in coffee has been suggested in a human study monitoring the oxidation of proteins , dna membrane lipids , and other redox biomarkers ( 17 ) .
multifocal electroretinography ( merg ) has recently been evaluated as an objective electrophysiological method to evaluate changes in retinal function in rp patients ( 20 ) .
the response amplitude has been found to follow a similar exponential decay as the visual field and may provide reproducible responses , even if standard electroretinography ( erg ) is nearly extinguished . in order to evaluate the effect of cga supplementation in patients with rp
, we utilized merg to objectively evaluate changes in visual function , along with visual acuity , visual field , standard electroretinography ( erg ) results , and contrast sensitivity .
eighteen patients diagnosed with rp were enrolled in this prospective , non - comparative , single - arm study from october 2010 to january 2011 .
patients that were already taking other anti - oxidants , such as lutein and zeaxanthin , were kept on their regimens . the best corrected visual acuity ( measured using early treatment diabetic retinopathy study [ etdrs ] letters ) , merg ( retiscan system ; roland consult , brandenbrug , germany ) , visual field ( measured using humphrey field analyzer [ hfa ] ii [ carl zeiss meditech , inc . ,
pleasanton , ca , usa ] ) , erg , and contrast sensitivity ( vistech 6500 chart , vistech consultants , inc .
, dayton , oh , usa ) were measured at baseline and then measured again after 3 months of cga supplementation . rp patients with a residual visual field of less than 10 degrees in any direction on the visual field examination , a best corrected visual acuity of less than 50 etdrs letters , a history of cga supplementation within the prior 3 months , or abnormal liver enzyme ( aspartate and alanine aminotrasferase > 40
iu / l ) , creatinine ( > 1.4 mg / dl ) , or blood urea nitrogen levels ( > 26 mg / dl ) suggestive of liver or kidney dysfunction were excluded from the study .
the eye with the larger amplitude on ring 1 of merg was selected for analysis .
additionally , the total point score on hfa with target size iii was used for the visual field evaluation .
full - field erg followed the guidelines of the international society for clinical electrophysiology of vision ( iscev ) ( 21 ) ; rod - specific and standard bright flash erg were both recorded after a minimum of 20 min of dark adaptation , and the photopic 30 hz flicker and single flash cone response was recorded following 10 min of light adaptation .
the full - field rod , cone and combined response amplitude was also studied as a secondary outcome measure . the log10 ( contrast sensitivity ) at each spatial frequency ( 1.5 , 3 , 6 , 12 , and 18 cycles / degree ) was also used as a secondary outcome measure . any systemic adverse effects and subjective improvement of vision were also evaluated .
ibm spss statistics 21 for windows ( ibm , armonk , ny , usa ) was used for statistical analyses .
the wilcoxon signed - rank test was used for analysis of parameters before and after cga supplementation . a p value
the study protocol was approved by the institutional review board of seoul national university hospital ( irb no . 1008 - 131 - 329 ) , and the study conformed to the declaration of helsinki .
eighteen patients diagnosed with rp were enrolled in this prospective , non - comparative , single - arm study from october 2010 to january 2011 .
patients that were already taking other anti - oxidants , such as lutein and zeaxanthin , were kept on their regimens . the best corrected visual acuity ( measured using early treatment diabetic retinopathy study [ etdrs ] letters ) , merg ( retiscan system ; roland consult , brandenbrug , germany ) , visual field ( measured using humphrey field analyzer [ hfa ] ii [ carl zeiss meditech , inc . ,
pleasanton , ca , usa ] ) , erg , and contrast sensitivity ( vistech 6500 chart , vistech consultants , inc .
, dayton , oh , usa ) were measured at baseline and then measured again after 3 months of cga supplementation . rp patients with a residual visual field of less than 10 degrees in any direction on the visual field examination , a best corrected visual acuity of less than 50 etdrs letters , a history of cga supplementation within the prior 3 months , or abnormal liver enzyme ( aspartate and alanine aminotrasferase > 40
iu / l ) , creatinine ( > 1.4 mg / dl ) , or blood urea nitrogen levels ( > 26 mg / dl ) suggestive of liver or kidney dysfunction were excluded from the study .
the eye with the larger amplitude on ring 1 of merg was selected for analysis .
additionally , the total point score on hfa with target size iii was used for the visual field evaluation .
full - field erg followed the guidelines of the international society for clinical electrophysiology of vision ( iscev ) ( 21 ) ; rod - specific and standard bright flash erg were both recorded after a minimum of 20 min of dark adaptation , and the photopic 30 hz flicker and single flash cone response was recorded following 10 min of light adaptation .
the full - field rod , cone and combined response amplitude was also studied as a secondary outcome measure . the log10 ( contrast sensitivity ) at each spatial frequency ( 1.5 , 3 , 6 , 12 , and 18 cycles / degree ) was also used as a secondary outcome measure . any systemic adverse effects and subjective improvement of vision were also evaluated .
ibm spss statistics 21 for windows ( ibm , armonk , ny , usa ) was used for statistical analyses .
the wilcoxon signed - rank test was used for analysis of parameters before and after cga supplementation .
the study protocol was approved by the institutional review board of seoul national university hospital ( irb no . 1008 - 131 - 329 ) , and the study conformed to the declaration of helsinki .
the mean age of the patients included in the study was 31.7 9.9 yr . the mean visual acuity was 77.3 9.0 letters .
there was no significant change in best corrected visual acuity , total point score on hfa , rod , cone and combined response amplitude on standard erg , or contrast sensitivity ; however , the amplitude of ring 5 on merg was significantly higher after 3 months of cga supplementation ( 7.2 9.5 vs 8.3 10.8 nv / deg , mean standard deviation , p = 0.022 , wilcoxon signed - rank test ) ( fig .
five patients expressed a subjective improvement of visual function , while 12 patients said they experienced no change .
this study demonstrated that cga significantly increased the amplitude of merg , raising the possibility of a beneficial effect on the peripheral area at the margins of the retinal degeneration .
however , this study failed to show a significant improvement in visual outcome measurements after cga supplementation . considering the pathophysiology of photoreceptor cell damage in hereditary retinal degeneration such as rp , various anti - oxidants
however , there are difficulties in evaluating the effect on the degree of progression for slowly progressing diseases like rp , as conducting clinical trials for extended periods of time and controlling dietary intake is challenging .
therefore , the effect of anti - oxidants in rp patients is generally based on theoretical background , while the evidence from clinical trials is sparse .
vitamin a has been found to decrease the rate of decline in erg amplitude and marginally affect the rate of decline in the visual field area ( 22 ) .
docosahexaenoic acid , an omega-3 fatty acid , was found to have an additional benefit in patients naive to vitamin a supplementation in the initial 2 yr ( 8) .
lutein supplementation slowed the loss of the mid - peripheral visual field in nonsmoking adults with rp taking vitamin a ( 5 ) .
peripheral ring 5 amplitude on merg increased with cga intake in our study . in a previous study ( 20 ) , merg was suggested as an effective tool for the long - term follow - up of disease progression and as an objective outcome measure in studies evaluating treatment outcome in rp patients .
ring 5 amplitude was found to be strongly correlated with the scotopic erg mixed cone - rod response amplitude , but only mildly correlated with the visual field area .
other previous studies evaluating the effect of antioxidants in rp have not utilized merg as a visual function evaluation tool ( 5 - 9 ) .
this increase in amplitude with cga intake may indicate the possibility of a peripheral scotopic functional improvement effect , and raises the necessity of a long - term study verifying this effect of cga using other more gross measurement tools , such as visual field measurement .
changes in merg could be detected in korean rp patients , who had been reported to have more advanced peripheral visual field defects , while central vision was more preserved ( 23 ) ; these patients may be possible candidates to benefit most from cga supplementation to improve peripheral visual function .
previous studies that have noted the beneficial effect of anti - oxidants on the progression of rp have also identified improved visual function at the periphery of the retina , which is the site where degeneration occurs .
berson et al . ( 5 ) identified that lutein supplementation of 12 mg / d slowed the loss of the mid - peripheral visual field in nonsmoking adults with rp taking vitamin a. our finding of improvement in the most - peripheral ring 5 amplitude on merg is consistent with this hypothesis .
there has only been 1 study to date that has reported a benefit in the preservation of central visual function , which was a long - term study of 4 to 6 yr of omega-3 supplementation in rp patients taking vitamin a ( 9 ) .
parameters associated with central visual function such as total point score on the central visual field examination on hfa , contrast sensitivity , and photopic 30-hz flicker amplitude on erg need a longer follow - up time , and as expected , showed no significant change with short - term cga supplementation . in order to evaluate the possible benefits to central visual function , a long - term study with a supplementation period of several years should be conducted .
the follow - up period was short to evaluate the effects of nutrition supplementation , and the long - term benefit effect remains to be clarified .
in addition , this study enrolled a small number of subjects with no control group , raising the possibility of bias .
this study included a heterogeneous study population with various degrees of rp progression , various genetic types , altered proteins , and mutations .
another possible factor is that the diseases of patients included in this study were not very advanced , and the patients had considerably larger residual visual function .
a randomized , placebo - controlled , long - term clinical trial in a large scale must be conducted to confirm the possible benefit of cga supplementation in rp patients .
additionally , comparative studies among anti - oxidative agents may be necessary , and cga should be considered as an anti - oxidant that can be used in the management of rp . in conclusion , this study confirmed the objective effectiveness of cga in rp patients , and a clinical benefit of anti - oxidant supplementation in rp .
the increased activity in the peripheral retina may raise the possibility of delaying the progressive decline in visual function in hereditary retinal degeneration . | to evaluate the effect of chlorogenic acid supplementation in patients with retinitis pigmentosa , we evaluated objective change in visual function with multifocal electroretinography , along with visual acuity , visual field , standard electroretinography , and contrast sensitivity .
eighteen patients diagnosed with retinitis pigmentosa were enrolled in this prospective , non - comparative , single - arm study .
multifocal electroretinography , best - corrected visual acuity in early treatment diabetic retinopathy study letters , total point score on visual field examination with humphrey field analyzer ii , electroretinography , and contrast sensitivity were measured and repeated after 3 months supplementation with chlorogenic acid .
the amplitude of ring 5 was significantly higher on multifocal electroretinography after 3 months of chlorogenic acid supplementation ( 7.2 9.5 vs 8.3 10.8 nv / deg2 , mean standard deviation , p = 0.022 ) .
there were no significant changes in the best - corrected visual acuity , total point score on humphrey field analyzer , 30 hz flicker amplitude on standard electroretinography , or contrast sensitivity .
chlorogenic acid may have a beneficial effect on the peripheral area at the margins of retinal degeneration , and should be considered as an anti - oxidant for the management of retinitis pigmentosa . |
multiple sclerosis ( ms ) is an autoimmune disease with unknown etiology , primarily targeting the myelin sheaths of neuronal axons in the central nervous system .
the strongest and earliest identified genetic association with ms is within the major histocompatibility complex ( mhc ) , localizing to hla - dr2 ( drb1 * 1501)/dq6 ( dqb1 * 0602 ) .
subsequent gene mapping and genome - wide association studies ( gwas ) identified additional susceptibility loci outside of the mhc , including the il2ra and il7ra receptors . since 2007 , numerous additional risk variants have been identified outside of the mhc , primarily through large - scale replication , additional gwas and meta - analysis of gwas . a complementary identification strategy exploited targeted genotyping of susceptibility variants across several autoimmune diseases , constructing a customized immunochip ' to study 194 non - mhc loci having genome - wide significant association with at least one autoimmune disease .
this strategy resulted in 48 new susceptibility variants , bringing the most recent number of established ms risk variants outside of the mhc to > 100 .
a variety of biological pathways have been implicated in ms etiology based on gene enrichment analysis , including cell adhesion , leukocyte activation , apoptosis , janus kinase ( jak)-stat signaling , nf-b activation , and t - cell activation and proliferation .
further efforts may be enhanced by the identification of novel risk loci for follow - up functional analysis , examination of common functions and molecular interactions across high - probability candidate genes , and the detailed articulation of biological mechanisms that unify clinical observations in ms with the numerous genetic variants that are suggested to affect disease liability .
gwas noise reduction ( gwas - nr ) is an approach to extend the power of gwas studies to detect novel associations beyond those detected by traditional gwas . in a genomic context ,
noise ' represents random differences in the distribution of alleles between cases and controls that are not driven by the signal of interest : true linkage disequilibrium ( ld ) with a susceptibility or causative variant .
gwas - nr amplifies association signals that are locally replicated at nearby markers and across independent data subsets , attenuating the effect of random uncorrelated noise . in simulations comparing gwas - nr with single - marker joint analysis and fisher 's tests ,
the gwas - nr algorithm has demonstrated greater power to detect true positives in a variety of disease models .
the gwas - nr algorithm was applied here to genome - wide data generated by the international multiple sclerosis genetics consortium ( imsgc ) .
the most significant association region identified by gwas - nr was a 4.4 mb span covering the mhc on chromosome 6 ( 29 616 - 33 997 kb ) . using significance threshold of p<1.0e-04 , 191 significant ld blocks in this region
were identified , separated by an average of 9 kb and no more than 332 kb .
the most significant ld block ( p=2.6e-55 ) was 1 kb upstream of hla - dra , which encodes one of the hla class ii alpha chain paralogues . among 162 363 ld blocks outside of the mhc , 221 significant blocks ( p<1.0e-4 )
were identified , 215 of which had at least one other significant block in close proximity .
merging adjacent significant blocks separated by < 250 kb produced 84 distinct genomic clusters comprising the strongest gwas - nr association signals in ms outside of the mhc , corresponding to 220 candidate genes .
significant association clusters identified by gwas - nr that did not overlap regions within 1 mb of the 97 high - confidence loci based on the imsgc immunochip analysis were examined , where the 1 mb threshold was chosen to conservatively define novel findings . among the novel immune - related
ms , susceptibility candidates within or immediately adjacent to these non - overlapping regions are : map3k14 , which encodes nf-b inducing kinase ( nik ) , a central component of non - canonical nf-b activation ; rela , which encodes the nf-b p65 subunit ; ubash3b , which cooperates with inpp5d to negatively regulate nf-b signaling downstream of fc receptor activation ;
ncoa2 , a dose - dependent regulator of nf-b activity ;
tnfaip8 , a negative regulator of nf-b activation that maintains immune homeostasis ;
kat5 , which acts as a molecular bridge between rela activation and nf-b target genes ;
csf2rb , a mediator of cytokine - induced jak / stat signaling cascades ;
stim2 , which regulates autoimmune effector functions of th1 and t helper type 17 ( th17 ) cells ;
txk , a th1 cell - specific transcription factor ;
dpp4 ( cd26 ) , a t - cell activation marker implicated in autoimmune pathology and highly expressed on th17 cells ;
cadm1 , a cell adhesion molecule that promotes cell - mediated cytotoxicity ;
dap , a positive regulator of ifn- mediated cell - death and rgcc , a regulator of t - cell - mediated apoptosis .
significant gwas - nr association clusters more than 1 mb from immunochip loci also include three immune - related genes for which suggestive association with ms has been reported in prior studies .
these are : nfkbiz , which encodes ikb , a repressor of nf-b signaling activator upstream of il6 production and th17 differentiation ;
inpp5d , which encodes a negative regulator of nf-b signaling and il12 production in macrophages ; and tec , which encodes a protein tyrosine kinase required for lipopolysaccharide - induced expression of tumor necrosis factor ( tnf)- , il1 and il6 .
six genes identified by gwas - nr and having previously reported significance in ms are not covered within 1 mb of the 97 immunochip loci .
these include mertk , a receptor tyrosine kinase that negatively regulates lipopolysaccharide - induced nf-b activation ;
malt1 , which forms a trimeric complex with bcl10 and card11 to regulate nf-b signaling necessary for the generation of neuroinflammatory th17 cells ;
il12b , an activator of stat4 upstream of th1 differentiation ;
batf , which encodes basic leucine zipper transcription factor , which along with rorc ( rort ) is part of the molecular signature for all il-17 producing cells in vivo ,
sox8 , a transcription factor expressed in neural crest development , and znf746 , which transcriptionally represses the ppar- coactivator .
to evaluate the stringency of the a priori ld block significance threshold ( p<1.0e-04 ) , ld blocks were identified corresponding to 111 genes closest ( defined by genomic position ) to loci having previously reported significance in ms based on single - marker analyses .
fifty - nine of these genes ( 53.2% ) were included among ld blocks meeting this significance threshold , with four additional genes of previously reported significance captured by clusters joining significant ld blocks within 250 kb proximity .
an alternative ld block significance level of 1.0e-03 would increase the number of susceptibility candidates from 220 to 568 . based on 162 363 ld blocks outside of the mhc , 30 blocks satisfying a bonferroni - corrected significance level of p<3.1e-07 were identified .
known immune - related candidates in these ld blocks , based on immport classification ( https://immport.niaid.nih.gov ) and review of current literature , included clec16a , cd86 ( b7 - 2 ) , ptger4 , il2ra , fam213b , gfi1 , tnfrsf1a , eomes , stat3 , fcrl3 , rps6kb1 , gpr65 , tnfsf14 and cd58 .
although manba is not classified as immune - related , the most significant ld block associated with this gene is immediately adjacent to nfkb1 .
only 13 of the 111 genes with previously reported significance in ms were associated with ld blocks meeting the bonferroni threshold .
the 220 non - mhc candidate susceptibility genes identified by gwas - nr are listed in supplementary table 1 . among these candidate genes ,
57 were located > 1 mb from significant immunochip loci , with the remaining 163 candidates located within 1 mb of those loci .
the 220 genes in the gwas - nr candidate set were examined for functional enrichment in biological pathways .
the strongest functional enrichment reported by david for the candidate gene set was positive regulation of cell activation ( go term 0050867 , p=6.1e-15 ) with a number of highly synonymous pathways ( for example , positive regulation of lymphocyte activation , positive regulation of leukocyte activation ) reflecting the same subset of genes .
additional significant pathways included positive regulation of immune system process ( go term 0002684 , p=2.6e-13 ) , regulation of lymphocyte proliferation ( go term 00050670 , p=2.2e-09 ) , regulation of lymphocyte mediated immunity ( go term 0002706 , p=8.2e-09 ) and regulation of cytokine production ( go term 0001817 , p=1.7e-08 ) .
common biological functions of ms candidate gene set identified by gwas - nr , and specific genes responsible for pathway enrichment , are reported in supplementary table 2 . for comparative purposes , genes outside the mhc
the strongest functional enrichment reported by david for this gene set was also positive regulation of cell activation , but with lower significance ( p=2.3e-12 ) compared with the gwas - nr candidate set , resulting from the inclusion of fewer relevant genes in this pathway .
compared with the significance of pathway enrichment among gwas - nr candidates , uniformly less significant enrichment was observed among the 10 highest - ranked biological pathways using these genes defined by single - marker significance , and in 90% of the 20 highest - ranked pathways , even when matched for identical gene ontology terms . to identify potentially important candidates not captured by gwas - nr at the a priori significance level of 1.0e-04 , regions within 250 kb of the 97 immunochip loci were examined .
511 genes in these regions were not included in the candidate set identified by gwas - nr . as observed among the gwas - nr candidate set , the strongest functional enrichments reported by david for these additional genes were in domains related to cell - mediated immunity , particularly regulation of lymphocyte differentiation ( go term 00045619 , p=1.8e-04 ) and regulation of leukocyte activation ( go term 0002694 , p=3.1e-04 ) .
our results indicate that outside of the mhc region , the genetic background of ms is enriched with numerous variants that regulate the activation and proliferation of immune effector cells , extending genetic evidence that implicates ms as an autoimmune inflammatory condition .
gwas - nr prioritizes a combined 5.2 mb span of the non - mhc genome , including several novel regions of association .
the resulting candidate gene set includes central components of nf-b signaling ( map3k14 , rela ) , and provides novel or additional evidence for regulation of nf-b activation by membrane - bound receptors ( ubash3b , inpp5d , ncoa2 , tnfaip8 , kat5 and nfkbiz ) .
these results reinforce a previously suggestive role for nf-b activation in ms implicated by significant association loci adjacent to bcl10 and card11 ( ref .
12 ) and functional analysis of ms - associated variants proximal to nf-b1 and in an intron of tnfrsf1a .
additional novel candidates identified by gwas - nr include regulators of cd4 + th1 and th17 induction ( stim2 , dpp4 , txk ) , and apoptosis ( cadm1 , dap , rgcc ) , providing further support for involvement of these pathways in ms .
several immune - related candidates identified by gwas - nr within 1 mb of significant immunochip loci are not included among genes with previously reported significance in ms , including socs1 , tnfrsf14 , gfi1 , stat1 , stat5a , cd5 , klrb1 , amica1 , il12rb1 , tbkbp1 , cd80 and rel .
examination of ld block scores of genes with previously reported significance in ms suggests that the a priori ld block significance level ( p<1.0e-04 ) chosen for reporting purposes is conservative .
the identified blocks span ~0.2% of the non - mhc genome , and capture over 50% of the genes previously reported as proximal to at least one genome - wide significant single - nucleotide polymorphism ( snp ) in ms based on previous published single - marker analyses .
although the gwas - nr algorithm is well - suited to identifying and prioritizing candidates and regions for follow - up study , the filtering process results in p - values that are less likely to achieve genome - wide significance , compared with single - marker p - values that may benefit from contributions of noise .
moreover , gwas - nr can achieve no advantage over single - marker analysis when flanking markers provide little supplementary information , which may occur when a true locus is typed directly and a single - marker association method is used .
it is likely that additional genetic variation related to ms remains unidentified by any genome - wide association technique .
thus , the candidate susceptibility loci identified by gwas - nr are not exhaustive , and can be viewed as complementary to high - confidence loci identified by alternative methods .
gwas - nr reduces statistical noise by exploiting local correlation of association signals across multiple data sets , whereas biological pathway analysis exploits the concurrence of multiple candidate genes within various biological processes to identify functions that are highly represented among these candidate genes .
the grouping of candidates into local association clusters can also provide informative context , both because association signals at a given locus may reflect a causal variant at a different location in ld , and because local clusters of susceptibility loci may include functionally related genes .
applied jointly , we observe that the majority of association clusters identified by gwas - nr include one or more immune - related genes having molecular functions common to those in other clusters .
although it is not possible to exclude a role in ms for candidate susceptibility genes that are uncharacterized or have no common or known immunological function , numerous immune - related candidates identified and prioritized by gwas - nr have well - established roles in biologically relevant pathways such as tnf- signaling , nf-b transcription , cytokine production , jak / stat signaling , t - cell differentiation and cell adhesion .
several of these biological functions have been individually implicated in ms based on gene previous enrichment analyses .
we propose that in ms , these functions are not distinct from one another , but instead comprise a coordinated mechanism that may contribute to ms pathology .
based on previously reported molecular functions and interactions among the ms candidate genes identified by gwas - nr , the genes responsible for significant enrichment in common biological functions were found to interact in a tractable pathway regulating the nf-b - mediated induction and infiltration of pro - inflammatory th1/th17 t - cell lineages , and the maintenance of immune tolerance by suppressive t - regulatory cells .
figure 2 , constructed on the basis of reported interactions among the ms susceptibility candidate genes prioritized by gwas - nr , provides a graphic overview of this pathway .
we propose this mechanism as relevant to ms pathology based on numerous risk genes implicated by gwas - nr that act as key mediators with cooperative roles at each level of this pathway , comprising both novel candidates and previously reported ms risk genes .
the regulation of nf-b - mediated th1/th17 inflammation and treg tolerance involves a chain of interactions comprising receptor activation , signal transduction , nf-b transcription , cytokine production , jak / stat signaling , t - cell induction and adhesion - mediated cell motility , and can be well - described with specific reference to interactions between these candidate genes .
nf-b signaling is a critical regulator of the balance between inflammation and tolerance , as nf-b activation appears to be required both for the induction of pro - inflammatory th17 cells and immunosuppressive treg cells .
activation of tumor necrosis superfamily member tnfrsf14 by its ligand tnfsf14 , both candidates identified by gwas - nr , acts as a t - cell costimulatory signal that results in nf-b - mediated induction of pro - inflammatory genes .
induction of nf-b by tnf- also promotes the expression of cell adhesion molecules icam1 and vcam1 .
although nonspecific anti - tnf therapy does not result in remission of symptoms in ms , selective blocking of tnfrsf1a significantly suppresses the infiltration of inflammatory th1 and th17 cells , and significantly improves clinical outcomes in murine experimental autoimmune encelphalomyelitis , a primary animal model of ms pathology .
conversely , defective clearance of tnfrsf1a accumulation is associated with nf-b activation , excessive il1 secretion and chronic inflammation .
nf-b is a transcription factor composed of homo- or heterodimers of five subunits : rela , relb , rel , nfkb1 and nfkb2 .
canonical nf-b activation triggers the production of cytokines such as il1 , il6 and il12 , and is inhibited by rps6kb1 ( s6k1 ) .
cytokine response to nf-b signaling is dependent on the profile of subunit activation , and is subject to coordinated regulation by other signaling pathways .
optimal t - cell receptor activation of the canonical nf-b pathway requires costimulation by cd80 ( b7 - 1 ) or cd86 ( b7 - 2 ) , and is mediated by a trimeric complex of adapter proteins known as the card11-bcl10-malt1 ( cbm ) complex .
the non - canonical nf-b pathway involves activation of map3k14 ( nik ' ) by members of the tnf family , such as by cd40 activation of traf3 .
nik also coordinates signaling by t - cell receptor and il6 to activate stat3 , contributing to the induction of th17 cells .
cd4 + t - cells differentiate into effector cells or t - regulatory cells depending on lineage - specific signals determined by the pathogen and resulting microenvironment that is encountered .
cytokine production activates the jak signal transducers and activators of transcription ( stat ) pathway .
uninfected dendritic cells produce tgf , which activates stat5 induction of foxp3 , the lineage - specific transcription factor for regulatory t cells .
th1 differentiation is dependent on il12 and ifn- activation of stat4 and stat1 , respectively , resulting in induction of the transcription factor tbx21 .
the th17 differentiation program involves il6 activation of stat3 , which is enhanced by socs1 , and induces the lineage - specific transcription factor rorc .
as il6 is one of the most strongly induced nf-b - dependent cytokines , nf-b has a particularly important role in the induction of inflammatory th17 cells .
rel and rela directly induce rorc , and nf-b also regulates the production of il6 and il23 .
expression of il2ra and the presence of il2 are critical for the generation of treg cells .
il2 has a central role in the maintenance of peripheral tolerance by eliminating self - reactive t cells , and knockout of il2ra ( cd25 ) results in autoimmune disease in mice .
il7 is essential for t - cell survival , and the high - affinity receptor il7r is also expressed on treg cells .
however , the presence of il7 impairs the ability of treg cells to suppress proliferation of autoreactive t cells in response to t - cell receptor activation by autoantigens , promoting their uncontrolled expansion il7-rich environments .
the expression of cell adhesion molecules icam1 and vcam1 on vascular endothelia is promoted by nf-b activation and inhibited by s1pr5 .
knockdown of s1pr5 abolishes quiescence of the blood brain barrier and increases the expression of icam1 and vcam1 in an nf-b - dependent manner , creating a permissive environment for the infiltration of inflammatory t cells into the cns .
amica1 also contributes to the binding of leukocytes expressing 41 integrin ( vla-4 ) on their plasma membranes to endothelial adhesion molecules .
the regulation of nf-b - mediated th1/th17 inflammation and treg tolerance implicated by the gwas - nr candidate gene set is consistent with clinical observations in ms .
blockade of il6r signaling reduces this production , elevates il10 release by activated cd4 + t - cells and restores the ability of hydrocortisone to inhibit t - cell proliferation .
a positive association between th17 and treg cell percentages is found in ms patients with remission , but not during relapse , while prevalence of th17 cells in csf is significantly elevated during relapse .
elevated expression of ifn- is observed during both early phase and relapse , and stat3 expression is persistently higher in cd4 + t - cells in patients who subsequently convert to clinically defined ms than in patients who do not and controls .
this cytokine profile is consistent with infiltration of pro - inflammatory th1/th17 lineages during exacerbations , with immunosuppression by treg cells during remission . at the transcriptional level ,
analysis of the t - cell transcriptome identifies aberrant regulation of gene expression by nf-b as a biomarker of relapse in ms based on 43 differentially expressed genes .
upregulation of nuclear staining for both nf-b and jnk was found on a large proportion of oligodendrocytes at the edge of active lesions and on microglia / macrophages throughout actively demyelenating plaques .
the data set used for analysis included 9772 individuals with ms and 17 376 healthy controls from 15 countries ; the majority of which were recruited by the imsgc and the wellcome trust case control consortium .
all individuals in this study gave informed consent with approval from the relevant local ethical committees or institutional review boards .
details of the ascertainment scheme and quality control of samples were described previously . to maximize the capability of gwas - nr in amplifying association signals that are locally replicated across loci in independent data subsets , the samples were divided into seven population specific stratum : australia ( 793 ms , 2160 control ) , central europe ( 2242 ms , 2046 control ) , mediterranean ( 950 ms , 1317 control ) , finland ( 581 ms , 2165 control ) , scandinavia ( 1970 ms , 2049 control ) , united kingdom ( 1854 ms , 5175 control ) and united states ( 1382 ms , 2464 control ) .
therefore , in addition to the snp quality control that was done previously , resulting in 465 434 autosomal snps , quality control was done within each of these assigned strata .
this included removing snps with low call rate ( < 95% ) , with differential missingness between individuals with ms and healthy controls ( p<1.0e-03 ) , and which were out of hardy - weinberg equilibrium ( hwe ) ( p<1.0e-05 ) .
this resulted in 447 419 snps which passed quality control in all seven strata and were subsequently analyzed .
eigenstrat was run separately within each stratum using 43k independent snps ( r<0.1 ) with minor allele frequency 0.1 across the genome .
logistic regression analysis was run within each stratum using plink and adjusting for the first five principal components from eigenstrat to account for residual population stratification . after sample size adjustment ,
the genomic inflation factor in each stratum was < 1.05 , indicating minimal evidence for residual population stratification .
a joint p - value across all strata was obtained using an inverse - variance meta - analysis of the seven strata , under a fixed effects model .
the p - values from each of the strata and from the joint analysis were then input into the gwas - nr algorithm . the gwas - nr algorithm ( version 2 )
noise reduction is achieved by applying a linear filter within a sliding window to identify genomic regions demonstrating correlated profiles of association across multiple subsets ; capturing association signals from surrounding markers in ld and suppressing signals that are not replicated in multiple data sets or across flanking markers .
the algorithm produces a weighted p - value for each snp , for use in prioritizing genomic regions and associated genes for follow - up study .
the gwas - nr and joint analysis methods were compared for sensitivity and specificity in identifying target loci defined , for comparative purposes , as all genotyped loci within 250 kb of 97 independent ms association signals previously identified by the imsgc using a custom immunochip ' genotyping array with 29 300 subjects with ms and 50 794 controls .
area under the receiver operating characteristic curve was 0.635 for joint analysis , and 0.719 for gwas - nr . at each level of specificity ,
gwas - nr demonstrated higher sensitivity than joint analysis in identifying these target loci . to evaluate the results of gwas - nr from multiple perspectives ,
significant loci were examined across individual genes , ld blocks , and association clusters defined by multiple significant ld blocks in close proximity .
this context was viewed as potentially informative as strong association signals at a given marker may reflect a causal variant at a different location in ld with that marker , and because physical clusters of susceptibility loci in the same region of association may include functionally related genes .
ld blocks were defined using plink and according to the method proposed by gabriel et al .
single snps not in an ld block with surrounding markers and between two ld blocks were treated as single - snp blocks .
any snp located between two snps within the same ld block was assigned the same block i d .
the significance of these ld blocks was computed using the truncated product method of zaykin .
a combined score for the markers in each ld block was calculated , based on the product of gwas - nr p - values that were below a threshold of 0.05 .
a monte carlo algorithm was used to test the significance of the combined score . to generate a reduced set of high - priority association candidates , an a priori significance level of p<1.0e-04
was chosen for reporting purposes . to identify distinct genomic regions comprising the strongest gwas - nr association signals ,
association clusters were defined by joining adjacent significant ld blocks ( p<1.0e-04 ) separated by < 250 kb . for clusters comprising more than one ld block ,
the lowest p - value among these blocks was identified for purposes of sorting and discussion .
the candidate gene set was defined to include the gene nearest ( defined by genomic position ) to each snp within each cluster , as well as the gene nearest to each snp within 25 kb of cluster boundaries .
immune - related genes and marginally significant ld blocks ( p<1.1e-04 ) immediately adjacent to significant association clusters were also examined as potential candidates .
candidate genes were classified as demonstrating previously reported significance in ms if included among 111 genes reported by the imsgc as proximal to a snp demonstrating genome - wide significance ( p<5.0e-08 ) or strong association ( p<5.0e-07 ) based on gwas and immunochip genotyping analysis .
although this classification is indicative of candidate genes corresponding to statistically significant and previously reported association loci , it does not represent an exhaustive list of genes that may otherwise be implicated in ms , and the associated variants may not be established as relevant to ms based on functional evidence . to investigate functional relationships among genes in the candidate set , each candidate was manually annotated and cross - referenced , based on a review of current literature , with attention to molecular function and directly interacting proteins .
candidate genes were classified as immune - related based on reported involvement in immune function , or inclusion in the comprehensive list of immune - related genes maintained by the immunology database and analysis portal ( immport https://immport.niaid.nih.gov ) .
supplementary functional annotations and pathway enrichment scores were generated using david ( the database for annotation , visualization and integrated discovery ) version 6.7 .
the gwas - nr was applied to ms data , with the objective of prioritizing genomic regions and associated genes for biological pathway analysis , targeted sequencing and follow - up functional analysis .
gwas - nr prioritizes a combined 5.2 mb span comprising ~0.2% of the non - mhc genome .
the resulting candidate set of 220 ms susceptibility genes is highly enriched with genes involved in the positive regulation of cell - mediated immunity .
novel candidates include key regulators of nf-b signaling and regulation of cd4+th1 and th17 lineages . based on previously reported molecular functions and interactions , a large subset of the ms candidate genes
were found to interact in a tractable pathway regulating the nf-b - mediated induction and infiltration of pro - inflammatory th1/th17 t - cell lineages , and the maintenance of immune tolerance by suppressive t - regulatory cells .
this mechanism provides a biological context that potentially links clinical observations in ms to the underlying genetic landscape that may confer susceptibility , and may help to inform efforts to develop targeted therapies .
| to identify genes and biologically relevant pathways associated with risk to develop multiple sclerosis ( ms ) , the genome - wide association studies noise reduction method ( gwas - nr ) was applied to ms genotyping data .
regions of association were defined based on the significance of linkage disequilibrium blocks .
candidate genes were cross - referenced based on a review of current literature , with attention to molecular function and directly interacting proteins .
supplementary annotations and pathway enrichment scores were generated using the database for annotation , visualization and integrated discovery .
the candidate set of 220 ms susceptibility genes prioritized by gwas - nr was highly enriched with genes involved in biological pathways related to positive regulation of cell , lymphocyte and leukocyte activation ( p=6.1e-15 , 1.2e-14 and 5.0e-14 , respectively ) .
novel gene candidates include key regulators of nf-b signaling and cd4 + t helper type 1 ( th1 ) and t helper type 17 ( th17 ) lineages .
a large subset of ms candidate genes prioritized by gwas - nr were found to interact in a tractable pathway regulating the nf-b - mediated induction and infiltration of pro - inflammatory th1/th17 t - cell lineages , and maintenance of immune tolerance by t - regulatory cells .
this mechanism provides a biological context that potentially links clinical observations in ms to the underlying genetic landscape that may confer susceptibility . |
many studies have shown that lyric / aeg-1 has a pivotal role in the development and progression of tumourigenesis ( brown and ruoslahti , 2004 ; chen et al . ; emdad et al . , 2009 ; hu et al . , 2009 ; jiang et al . , 2012 ;
, 2011 ; song et al . , 2009 ; wang et al . , 2011 ; xia et al . ; yoo et al . , 2009 ; yu et al . ,
differential lyric / aeg-1 expression and altered localisation has been demonstrated in a variety of cancers , with increased expression frequently linked to a poor prognosis ( chen et al . ; jiang et al .
, 2012 ; lee et al . , 2009 ; li et al . , 2008 ;
liao et al . , 2011 ; song et al . , 2009 ; thirkettle et al .
, 2011 ; xia et al . ; yoo et al . , 2009 ; yu et al . , 2009 ) .
previous studies have shown that lyric / aeg-1 increases chemo - resistance and cell survival in response to hypoxia and glucose starvation , possibly by initiating protective autophagy ( bhutia et al . ;
lyric / aeg-1 is regulated by h - ras and pi3k and there is mounting evidence for its role in regulating ubiquitination ( ash et al . , 2008 ; emdad et al . , 2006 ;
ubiquitin modifications are known to result in a broad range of responses , dependent upon the number of ubiquitin molecules and their linkage ( denuc and marfany , 2010 ; ikeda and dikic , 2008 ) .
the addition of k48-linked ubiquitin moieties is associated with proteasomal dependent degradation of the substrate protein , whereas k63-linked ubiquitin moieties are associated with endocytosis and dna repair { aguilar and wendland , 2003 # 48}. signalling activation and protein trafficking are more closely associated with monoubiqutination .
ubiquitin monomers can also elicit a response analogous to phosphorylation ( ikeda and dikic , 2008 ) that regulates signalling or sub - cellular localisation of proteins such as pten , p53 and foxo3a ( salmena and pandolfi , 2007 ) .
topors is a unique e3 ligase which can ligate both ubiquitin and small ubiquitin - like modifier ( sumo ) to substrate proteins ( denuc and marfany , 2010 ) .
it contains an n - terminal ring - domain which ligates ubiquitin to its target proteins including dna topoisomerase i , p53 and nkx3.1 ( guan et al . , 2008 ;
depending on cellular context , topors has been documented as a proto - oncogene or tumour suppressor ( guan et al .
lyric / aeg-1 contains an usually large number of lysine residues ( > 12% ) with the majority clustered within extended nuclear localisation signal regions ( exnls ) .
these regions have previously been defined as exnls-1 ( aa78130 ) , exnls-2 ( aa415486 ) and exnls-3 ( aa546582 ) and shown to act as nuclear and nucleolar localisation signals ( thirkettle et al . , 2009a ) .
however , only exnls-2 , which contains 18 lysine residues , was shown to be modified by mono - ubiquitin ( thirkettle et al . ,
2009a ) causing almost all of lyric / aeg-1 to run at a higher mw of 105 kda .
here we identify the specific sites of lyric / aeg-1 ubiquitination by topors in the exnls-2 region .
mutagenesis of these residues leads to a highly significant reduction in lyric / aeg-1 ubiquitination which results in altered sub - cellular distribution and impairs the interaction between p65 , importin- and lyric / aeg-1 .
gfp - tagged lyric / aeg-1 constructs were created using pqbi25-fc3 plasmid ( q - biogene ) as described ( thirkettle et al . , 2009a ) .
site directed mutagenesis was performed using quickchange ii site - directed mutagenesis kit ( stratagene ) following the manufacturer 's instructions to create gfp - k486r - lyric / aeg-1 alone , gfp - k491r - lyric / aeg-1 alone or a gfp - k486r / k491r - lyric / aeg-1 double mutant .
stefan weger ( the free university of berlin , germany ) ( weger et al . , 2005 ) .
cos-7 cells purchased from the cancer research uk cell bank were routinely cultured in dmem ( gibco ) supplemented with 10% fetal bovine serum ( labtech ) . for transfection cells were grown to 40% confluency .
dna ( 1 g/6 wells of a 24 well plate or 6 g / per 14 cm dish ) was transfected using fugene 6 ( roche ) following the manufacturer 's protocol .
cells were grown for a further 48 h prior to confocal microscopy or western analysis .
all western blotting procedures were performed as described ( whitaker et al . , 2007 ) including the addition of 0.5% ( w / v ) sodium deoxycholate .
membranes were incubated with primary antibody ; anti - gfp ( 1:5000 ) , anti - actin ( 1:5000 ) , anti - p65 ( 1:1000 ) , anti - importin- ( 1:5000 ) ( all from abcam ) , anti - ubiquitin ( 1:1000 , santa cruz biotechnology ) and anti - topors ( 1:1000 , sigma ) .
protein bands were detected with ecl - plus ( ge healthcare ) and where they exceeded the dynamic range of film diaminobenzidine ( vector laboratories ) was used .
immunoprecipitation was performed at 4 c using the method previously described ( thirkettle et al . , 2009a ) .
for densitometry all available western blots ( n > 3 ) were scanned using a typhoon scanner ( ge healthcare ) and analysed using imagequant tl ( ge healthcare ) . to overcome any variation in transfection / translation efficiency
the amount of ubiquitination was normalised using gfp - lyric / aeg-1 values ( ubiquitination / gfp - lyric / aeg-1 ) .
proteins were separated using pre - cast 8% gels ( pierce ) and stained using biosafe coomassie ( molecular probes ) .
protein bands were excised and incubated overnight with destain ( 1:1 ratio of acetonitrile and 10 mm triethylammonium bicarbonate ) .
bands were washed in destain , dehydrated and reduced using 10 mm dtt , alkylated with 50 mm iodoacetamide then washed and dehydrated once more .
proteins were digested at 37 c overnight using porcine trypsin ( promega ) ( 10 ng/l ) .
digestion was stopped by flash freezing before the addition of 5% formic acid ( fluka , uk ) and sonication for 15 min , and repeated prior to sample dehydration .
digested samples were resuspended in 8 l 1% formic acid for 20 min prior to analysis using a nano - reverse phase lc- ms using a qtof 6510 mass spectrometer with chip cube source interface and 1200 series hplc running masshunter b.01.03 ( agilent , usa ) .
samples were loaded onto a chip containing integrated 40 nl enrichment and 150 mm analytical columns and electrospray needle using 3 l / min 0.1% formic acid .
nl / min using a linear gradient from 10 to 70% elution buffer ( 80% acetonitrile/20% 0.1% formic acid ( v / v ) ) for 10 min .
after 1 min the gradient was increased to 100% elution buffer and 100% buffer was continued for a further 6 min .
ms data was recorded in the 2902500 m / z range at 6 spectra per second .
ms / ms data was acquired in the 573000 m / z range at 4 spectra per second .
data was searched against uniprot ( kb15.5j ) and the known lyric / aeg-1 constructs using mascot daemon version 2.2.2 ( matrix science , uk ) using a peptide tolerance of 10 ppm and a fragment tolerance of 0.05 da in addition to statistical scoring , the validity of the data was confirmed by manual assessment of the data using scaffold software 2.1 ( proteome science , usa ) .
data were acquired using the agilent 's lc - chip cube- 6510 qtof data - dependent mode with the settings described above with the exception of collecting profile data .
data was acquired randomly as determined by list randomizer ( http://www.random.org/lists/ ) . using trapper version 4.2.0 ( natalie tasman , institute for systems biology , usa ) files were converted to mzxml format and imported into progenesis lc - ms version 2.5.3478.16299 ( nonlinear dynamics , uk ) for lc - ms run alignment and ms peak extraction for label - free quantitation after ion peak identification by mascot .
peaks of interest were analysed and validated using the statistical software r ( version 2.10.1 ) ( team , 2010 ) . to normalize for variability in protein input
the data was analysed using a mixed effects model , where the two fixed effects were gfp - lyric / aeg-1 constructs and ubiquitin . to reflect the pairing of measurements within these fixed effects ( induced by the fact that each replicate provides two measurements ) we also had a single mixed effects term indicating which replicate ion abundance mass spectrometric measurement it came from ( analogous to the paired t - test that would have been carried out for a single fixed effect ) .
analysed data was the log 2 median of the unmodified ubiquitin normalized abundances obtained from progenesis lc - ms label - free quantitation software .
cells were fixed stained and mounted as previously described ( thirkettle et al . , 2009a ) .
a yeast two - hybrid assay was performed by dualsystems biotech ag , zurich , switzerland using plexa - dir - lyric / aeg-1 aa73 - 582 as bait and a human placental cdna library as described ( thirkettle et al . , 2009b ) .
previously , we have demonstrated that cytoplasmic lyric / aeg-1 is modified within the exnls-2 region by mono - ubiquitin , leading to a 105 kda mw band and no increase in lyric / aeg-1 degradation ( thirkettle et al . , 2009a ) .
using immunoprecipitation of gfp - tagged wtlyric / aeg-1 and exnls constructs ( shown in figure 1a ) we have previously demonstrated that deletion of the entire exnls-2 region ( aa415486 ) , and not exnls-1 ( aa78130 ) or exnls-3 ( aa546582 ) , leads to an almost complete loss of lyric / aeg-1 mono - ubiquitination ( thirkettle et al . , 2009a ) .
we identified two potential specific mono - ubiquitination sites by mass spectrometry at lysine residues k486 and k491 .
k486 lies within the exnls-2 region while k491 lies 5 residues upstream of exnls-2 ( figure 1b , top panel ) . to determine if k486 and k491 are the primary sites of ubiquitin modification single and double point mutations were made to ablate mono - ubiquitination without disrupting the protein charge or the potential to act as an nls ( k486r , k491r and k486/491r ) .
gfp - tagged wild - type and mutant constructs and ubiquitin were over - expressed in mammalian cells and immunoprecipitated using the gfp - tag .
mutation of either k486 or k491 resulted in a significant reduction in lyric / aeg-1 mono - ubiquitination . when both k486 and k491 were mutated mono - ubiquitination was almost entirely ablated ( figure 1b ) . to validate these findings we employed a mass spectrometry quantification method .
ubiquitination was normalised to the amount of gfp - tagged construct using the number of defined gfp peptides per construct as a control for gfp - lyric / aeg-1 input ( figure 1c ) . due to the lysine - rich nature of lyric / aeg-1
the standard mass spectrometry methods were unsuitable and ubiquitination of each construct was quantified using a label - free mass spectrometry and a mixed effects model . using this method the most consistent and robust ubiquitin and gfp peptides that could be detected
were defined and measured for each construct ( shown in supplementary figures s2 and s3 ) . using mass spectrometry
we confirmed that deletion of exnls-2 resulted in a significant loss of ubiquitination compared to wtlyric / aeg-1 ( p = 0.0012 ) ( figures 1a and 1c ) . when k486 and k491 were modified a highly significant loss of ubiquitination
was seen ; k486r mutant ( p = 0.0028 ) , k491r mutant ( p = 0.0004 ) and the k486/491r double point mutant ( p = 0.0009 ) ( figures 1b and 1c ) .
there was also a small , but not significant , loss of ubiquitination when exnls1/3 was deleted ( p = 0.43 ) .
we have previously demonstrated that deletion of the entire exnls-2 region had no effect on lyric / aeg-1 function or stability and that treatment with mg132 failed to promote lyric / aeg-1 poly - ubiquitination , both of which are consistent with mono - ubiquitination ( thirkettle et al . , 2009a ) .
to establish if ubiquitination of lyric / aeg-1 had any effect on its function we examined the interaction between lyric / aeg-1 and p65 , a known interacting protein proposed to shuttle into the nucleus with lyric / aeg-1 ( emdad et al .
wtlyric / aeg-1 interacts with p65 but this interaction was lost with the non - ubiquitinated k486r / k491r mutant ( figure 2a ) .
mass spectrometry data identified a number of peptides corresponding to importin- , a nuclear import chaperone , suggesting that it may interact with lyric / aeg-1 ( supplementary figure s4 ) ( harel and forbes , 2004 ) .
this interaction was confirmed by immunoprecipitating gfp - lyric / aeg-1 or the gfp - lyric / aeg-1 k486/491r mutant from cos-7 cells overexpressing the constructs .
importin- was shown to interact with the wtlyric / aeg-1 but only at very low levels in the k486/491r mutant suggesting the ubiquitination of lyric / aeg-1 might regulate its interaction with importin-. using confocal microscopy we examined the localisation of the gfp - tagged lyric / aeg-1 constructs .
previously we have reported that wtlyric / aeg-1 localises throughout the cytoplasm , nucleolus and to a lesser extent the nucleoplasm , as shown by co - localisation with fibrillarin ( figure 3 ) ( thirkettle et al . , 2009a ) .
all of the lyric / aeg-1 ubiquitin mutants are expressed at lower levels than wtlyric / aeg-1 despite being transfected with identical amounts of dna .
all of these mutants exhibit loss of the wild - type diffuse cytoplasmic and nucleoplasmic localisation . k491r and
all nuclear lyric / aeg-1 in the k486r , k491r and k486r / k491r double mutants resides within the nucleolus and nuclear bodies and is lost from the nucleoplasm .
to identify potential e3 ligases for the ubiquitination of lyric / aeg-1 we used a yeast two - hybrid assay with c - terminal lyric / aeg-1 , ( aa 73582 ) , as bait ( thirkettle et al .
the screen identified only one e3 ligase , the n - terminal fragment of topors ( aa 45250 ) , containing the ring finger domain , as an lyric / aeg-1 interacting partner ( figure 4a ) ( rajendra et al . , 2004 ) .
the interaction between wtlyric / aeg-1 was supported using immunoprecipitation of gfp - tagged wtlyric / aeg-1 from cos-7 cells co - transfected with wtgfp - lyric / aeg-1 and topors ( figure 4b ) . to determine if lyric / aeg-1 is a potential substrate for the topors ubiquitin e3 ligase activity we immunoprecipitated wtlyric / aeg-1 from cos-7 cells transfected with an increasing concentration of topors ( figure 4c ) .
western blot analysis showed that the ubiquitination of lyric / aeg-1 increased with increasing topors expression .
we have shown in previous studies using mg132 that ubiquitination of lyric / aeg-1 does not regulate its degradation by the proteasome ( thirkettle et al . , 2009a ) .
mono - biquitination is known to regulate the localisation and function of a number of proteins including pten , p53 and foxo3a ( denuc and marfany , 2010 ; salmena and pandolfi , 2007 ) .
the large number of functions attributed to lyric / aeg-1 suggests it plays a pivotal role in tumourigenesis ( ash et al . , 2008 ;
bhutia et al . ; brown and ruoslahti , 2004 ; emdad et al . , 2009 ; hu et al .
, 2009 ; li et al . , 2012 ; yoo et al . ) .
previously we have shown that the exnls-2 region of lyric / aeg-1 is modified by mono - ubiquitin , rather than poly - ubiquitin and that this modification does not alter lyric protein stability ( thirkettle et al . , 2009a ) .
we also saw modification by sumo-1 , but at much lower levels than mono - ubiquitin suggesting that mono - ubiquitin may play a more important role in post - translational modification of lyric / aeg-1 .
deletion of exnls-2 , but not exnls-1 or exnls-3 , results in an abrogation of lyric / aeg-1 modification by mono - ubiquitin ( figure 1a ) .
we have previously reported that lyric / aeg-1 localisation and expression is up - regulated in prostate tumourigenesis supported by reports of overexpression multiple other cancers ( brown and ruoslahti , 2004 ; chen et al . ;
, 2011 , 2009 , 2008 ; liao et al . , 2011 ; song et al . ;
; yoo et al . , 2009 ; yu et al . , 2009 ) .
we and others have previously shown that lyric / aeg-1 can move between the cytoplasm and nucleus ( figure 3 ) ( emdad et al . , 2006 ;
sarkar et al . , 2008 ; thirkettle et al . , 2009a ) .
we have demonstrated that shuttling into the nucleus and interaction with proteins known to shuttle into the nucleus is attenuated by mono - ubiquitination of two key lysine regions at k486 and k491 ( figure 3 ) .
mutating k486 and k491 of lyric / aeg-1 resulted in a dramatic effect on its localisation to peri- and sub - nuclear compartments and complete loss of cytoplasmic and nucleoplasmic lyric / aeg-1 ( figure 3 ) .
this suggests the retention of non - ubiquitinated lyric / aeg-1 within inactive sub - cellular compartments where it is unable to bind with known interacting protein p65 and the nuclear import protein importin- ( figure 2 and supplementary figure s4 ) ( emdad et al . , 2006 ;
we have shown that mutation of k486 and k491 results in a dramatic loss of lyric / aeg-1 mono - ubiquitination ( figure 1b and c ) .
this suggests that k486 and k491 are the major sites of mono - ubiquitination and that multiple other neighbouring lysine residues do not act co - operatively to compensate for the loss of a single lysine at either k486 or k491 .
although k486 lies with the reported exnls-2 region , k491 lies just outside the exnls-2 region previously identified as the mono - ubiquitinated region of lyric - aeg-1 .
this is consistent either with co - operativity between the k486 and k491 mono - ubiquitination sites or binding of the e3 ligase needed to modify k491 to a site within exnls-2 .
lyric / aeg-1 has previously been demonstrated to interact with brca2 and cdkn1a - interacting protein and indirectly alter its stability ( ash et al . ,
we identified topors , an oncogene and e3 ligase known to ubiquitinate proteins including p53 , topoisomerase i and ib kinase epsilon ( renner et al . ) , as an e3 ligase that potentially interacts with lyric / aeg-1 .
topors was shown to interact with lyric / aeg-1 by yeast two - hybrid assay and co - immunoprecipitation figure 4a and b. in addition , increasing amounts of topors alters lyric / aeg-1 ubiquitination in a dose - dependent manner ( figure 4c ) .
lyric / aeg-1 has been implicated in tumour development , progression and treatment resistance ( bhutia et al . ;
chen et al . ; hu et al . , 2009 ; jiang et al .
, 2012 ; lee et al . , 2009 ; li et al . , 2012 , 2008 ;
thirkettle et al . , 2009a ; wang et al . , 2011 ; xia et al .
understanding the mechanism of lyric / aeg-1 modification by ubiquitin and its effects on lyric / aeg-1 sub - cellular trafficking provides new information on the how lyric / aeg-1 function is regulated in cells and offers potential new avenues for therapeutic intervention . | the pivotal role of lyric / aeg-1 in malignant transformation , tumourigenesis and chemo - resistance has previously been demonstrated in different cell types and sub - cellular compartments .
the localisation of lyric / aeg-1 appears crucial to its function and is regulated by three lysine - rich nuclear localisation signal regions , one of which was previously demonstrated to be modified by ubiquitin . here
we show that mutation of lyric / aeg-1 at k486 and k491 results in a loss of ubiquitination .
a k486/491r double mutant that is incapable of ubiquitination shows reduced binding to the nfb subunit p65 or importin- resulting in a distinctive peri - nuclear localisation of lyric / aeg-1 .
we also provide evidence to suggest that topors , an e3 ligase that also regulates p53 modification may be responsible for lyric / aeg-1 ubiquitin modification .
overall we demonstrate that specific sites of lyric / aeg-1 ubiquitination are essential for regulating lyric / aeg-1 localisation and functionally interacting proteins . |
the main incentive for all efforts and developments in human societies is to improve and maintain human health ( 1 ) .
the health of the general population depends on improving the health of pregnant women and infants ( 2 ) .
every year there are 132 million births in the world ( 3 ) , providing high - quality and suitable services to maternal satisfaction and improving their health status plays a leading role in health systems ( 4 ) .
access to high - quality services in the area of pregnancy health is being considered as a right of people , and governments have a commitment to provide it ( 5 ) .
there are many definitions of quality of pregnancy health services , but all of them have a point in common : that good quality of services need educated staff providing services with respect in an appropriate place for applicants ( 6 ) .
quality maternal care is referred care that is appropriate , satisfactory , affordable , and easily accessible and enables one to make healthy lifestyle choices ( 7 ) .
quality of care can be measured in two dimensions : first , the way of providing care technically ; second , experiences of care users that are of great importance in evaluating services ( 8) . in fact , customer expectations are beliefs in the assessment of health provisions that are employed as a point of reference and standards to exactly make sense of service quality ( 9 ) .
depending on what redshaw has stated , allowing for women to express their perspectives at different stages of receiving care , a variety of sites and providers have built an accurate picture of received care ( 10 ) .
negative experience of pregnant women when receiving care may have an adverse impact on them ( 11 ) .
the studies have shown that poor quality of care leads to the occurrence of disease , more disabilities , higher cost ( 12 ) , and less use of care by women ( 13 , 14 ) .
generally , clients being satisfied continue to use and encourage others to use services ( 15 ) .
the world health organization ( who ) presented the concept of responsiveness , so that it could corroborate a patient s expectations in the standard and combining form .
responsiveness as a special concept in the health system deals with how to provide services and only nonclinical aspects of care .
this concept , based on the donabedian quality framework , shows the ability of health systems in fulfilling people s expectations in connection with nonclinical aspects of them .
taking responsiveness for patient wants along with health improvement and equitable financing is of main goals of who ( 1 ) .
hence , at the international level , health systems make attempts to better their own responsiveness for patients and people ( 17 ) .
responsiveness makes up eight dimensions , including dignity , autonomy , confidentiality , communication , prompt attention , provision social - needs , basic amenities , choice of provider ( 1 ) .
the who , based on experience of patients in 79 european countries , has used this model to the assessment of satisfaction with health systems ( 18 ) .
wisniewski illustrated that the expectations of patients are higher than their perceptions , there having been space for the betterment of patient satisfaction and providing desirable healthcare ( 19 ) .
good quality of care requires that healthcare workers obtain the necessary skills and give serious considerations to the needs of pregnant women .
healthcare organizations require essential equipment and furthermore , referral centers should be efficient when they provide healthcare for the treatment of pregnancy and labor complications .
these issues are not feasible except by supervision and assessment of related units as well as attention to service measurement and improvement ( 8 , 20 ) . with regard to customer
s contribution in healthcare and the advent of total quality management as an approach to quality improvement , health organizations and policymakers have to recognize what health customers think so that they can improve quality of maternal child health services . because there is no study on provided maternal health services based on the responsiveness model in iran , the aim of the present study was to investigate quality of maternal child health services based on the responsiveness model of who in selected tehran hospitals in 2015 .
this is a cross - sectional study conducted on women visiting hospitals in tehran in 2015 .
thus , two hospitals affiliated with each of tehran university of medical sciences , iran university of medical sciences , and shahid beheshti university of medical sciences and one hospital belonging to bagiyat allah university were selected randomly . sampling in each hospital
the inclusion criteria consisted of having interest in participating in the study , being a resident in tehran , having the ability to write and read , and being between 37 and 41 weeks of pregnancy .
after obtaining written informed consent from women , the questionnaires were filled by themselves . a questionnaire containing demographic and obstetric information and a researcher - made questionnaire based on the responsiveness model of who were used to collect data . the questionnaire included 37 items on dignity ( eight items ) , autonomy ( five questions ) , confidentiality ( one question ) , communication ( four questions ) , prompt attention ( four questions ) , provision social - needs ( five questions ) , basic amenities ( seven questions ) , and choice of provider ( three questions ) .
the format of questionnaire s questions was a 5-point likert scale ( where 1=completely disagree and 5 = completely agree ) .
content validity of questionnaire was examined and confirmed by 10 specialists and face validity confirmed by 30 pregnant women after applying their point of view .
reliability of the questionnaire was checked and confirmed by internal consistency ( cronbach s alpha ) based on 30 samples of interest for each element and entire questionnaire ( total alpha=0.94 ) and test - retest reliability ( a comparison of the results of the questionnaire in the same condition conducted on 30 pregnant women after 10 days from initial tests passed ) was conducted and intra - class correlation was 0.96 . to study the perspective of pregnant women on satisfaction with the care , pregnant women were asked to score on a scale from 0 to 20 for every hospital in regards to satisfaction with maternal care during the period of pregnancy .
, armonk , ny , usa ) . to describe the data , measures of central tendency and dispersion were used . to examine the relationship between demographic variables and dimensions of the questionnaire , after ensuring that they are normally distributed , kolmogorov
smirnov test , anova , t - test , and pearson s correlation ( for quantitative data ) and spearman correlation ( for ordinal data ) were used .
this is a cross - sectional study conducted on women visiting hospitals in tehran in 2015 .
thus , two hospitals affiliated with each of tehran university of medical sciences , iran university of medical sciences , and shahid beheshti university of medical sciences and one hospital belonging to bagiyat allah university were selected randomly . sampling in each hospital
the inclusion criteria consisted of having interest in participating in the study , being a resident in tehran , having the ability to write and read , and being between 37 and 41 weeks of pregnancy .
after obtaining written informed consent from women , the questionnaires were filled by themselves . a questionnaire containing demographic and obstetric information and a researcher - made questionnaire based on the responsiveness model of who were used to collect data .
the questionnaire included 37 items on dignity ( eight items ) , autonomy ( five questions ) , confidentiality ( one question ) , communication ( four questions ) , prompt attention ( four questions ) , provision social - needs ( five questions ) , basic amenities ( seven questions ) , and choice of provider ( three questions ) .
the format of questionnaire s questions was a 5-point likert scale ( where 1=completely disagree and 5 = completely agree ) .
content validity of questionnaire was examined and confirmed by 10 specialists and face validity confirmed by 30 pregnant women after applying their point of view .
reliability of the questionnaire was checked and confirmed by internal consistency ( cronbach s alpha ) based on 30 samples of interest for each element and entire questionnaire ( total alpha=0.94 ) and test - retest reliability ( a comparison of the results of the questionnaire in the same condition conducted on 30 pregnant women after 10 days from initial tests passed ) was conducted and intra - class correlation was 0.96 . to study the perspective of pregnant women on satisfaction with the care , pregnant women were asked to score on a scale from 0 to 20 for every hospital in regards to satisfaction with maternal care during the period of pregnancy .
the obtained data were analyzed by ibm spss statistics version 20 ( ibm corp . , armonk , ny , usa ) . to describe the data , measures of central tendency and dispersion were used . to examine the relationship between demographic variables and dimensions of the questionnaire , after ensuring that they are normally distributed , kolmogorov
smirnov test , anova , t - test , and pearson s correlation ( for quantitative data ) and spearman correlation ( for ordinal data ) were used .
the mean age of pregnant women was 28.74.9 and 96.9% of them were covered by insurance .
the mean score given to the hospital was 17.042.4 from a score of 20 . according to the statements of 91.5% of pregnant women ,
there were no significant relationships among age , education , employment , planned pregnancy , rank of pregnancy , and dimension of responsiveness ( p>0.05 ) .
findings related to the determination of the quality of pregnancy care suggested that scores of total responsiveness of service recipients were 69.46 14.65 in the range of 0 to 100 ( table 2 ) .
the obtained scores of different dimensions showed that , in the range of 0 to 100 , 73.02 were about basic amenities ( the most score ) , 72.93 about dignity , 70.91 about communication , 70.76 about confidentiality , 66.30 about provision social - needs , 65.96 about choice of provider , 65.92 about autonomy , and 52.65 about prompt attention , which are representing the average level of service quality . if we consider the tendency to return the same hospital as a sign of maternal satisfaction from pregnancy care , it is possible to express that whereas satisfaction from services is high , but service quality is at the middle level .
findings obtained utilizing one - way anova suggested a significant relationship between insurance type and dimensions of choice of provider ( p=0.03 ) and communication ( p=0.03 ) ( table 3 ) . the associations between participating in preparation classes of labor and each of the responsiveness dimensions have shown significant relationships between participating in preparation class of labor and dignity ( p<0.001 ) , autonomy ( p=0.01 ) , provision social needs ( p=0.01 ) , and overall responsiveness ( p=0.03 ) ( table 4 ) .
those participating in the classes gave high scores for all dimensions except for basic amenities .
finally , the pearson correlation test for determining correlation between scores given to hospitals ( in the range of 0 to 20 ) and dimensions of responsiveness showed no significant relationship between scores and dimensions of responsiveness ( table 5 ) .
the purpose of this study is to assess the performance of responsiveness of obstetric services provided for pregnant women in tehran .
findings suggested that the mean of total responsiveness scores was 69.64 from 100 showing average level of responsiveness . the best and poorest performances in terms of responsiveness were dimensions of basic amenities and prompt attention , respectively , which indicate a positive perspective of pregnant women on quality of settings providing services . on the other hand , negative perspective of them
implies dimensions of prompt attention , failure in urgently providing vital services and long waiting time . in a study by ameriuon et al . in military hospitals of tehran in 2014 , the mean and standard deviation of total responsiveness scores were 977.5 from 160 , representing upper middle mean of responsiveness score , which has concordance with the results of this study ( 21 ) . in a study by gharaee and associations on government hospitals , private and charity hospitals of hamedan and yazd provinces , which is conducted in 2013 ,
all dimensions of autonomy , basic amenities , confidentiality of information and having the right to select were assessed as average level and participants gave high scores to dimensions of dignity , communication , prompt attention and provision social - needs in both cities ( 22 ) .
responsiveness to the hospital patient needs in poland recommended that total responsiveness score was 73% and scores of other dimensions were respectively , from much to less , 96% for provision social - needs , 92% for dignity , 85% for communication , 76% for autonomy , 70% for prompt attention , 64% for confidentiality , and 41% for choice of provider .
this study illustrated that outpatient needs had been satisfied in dimensions of provision social - needs , dignity and communication ( 23 ) .
responsiveness of health system for patients suffering from heart failure in tehran examined by karami et al . showed that total responsiveness score is 84% . in this study 66.5% of outpatients and
the highest scores had given by outpatients and inpatients to dimensions of confidentiality and dignity ( 24 ) .
health system responsiveness for delivery care in southern thailand , by liabsuetrakul in thailand , showed that there has been a little difference between dimensions of responsiveness and the lowest score was related to the choice of provider . in this study ,
type of hospital had significant relationship with all of responsiveness dimensions and insurance type had significant association with dimensions of dignity , choice of provider , prompt attention .
the relationships between the rank of pregnancy and provision social - needs , choice of provider , prompt attention and confidentiality were significant ( 25 ) . in the study of patient experiences and responsiveness of health system in south africa conducted by peltzer , the lowest scores that outpatients and inpatients dedicated to aforementioned dimensions were related to waiting time and choice of provider respectively .
the percentages of people having given high or very high score were lower in general health centers .
8.16% of patients admitted to public centers had given scores that were bad or very bad , while only 2.3% of patients referred to private hospitals gave scores that were bad or very bad ( 26 ) . according to bramesfeld , a study about assessing mental health system utilizing responsiveness model in germany , the best performances obtained in terms of confidentiality and then in dignity dimensions .
outpatients in comparison with inpatients had given the lower scores to responsiveness dimensions ( 27 ) . in assessing of health system in region of seventeen in tehran by rashidian ,
the best dimensions from the inpatient views were respect and dignity and confidentiality while from outpatient views were dignity and autonomy .
the lowest score had been given to basic amenities and autonomy dimensions ( 28 ) . in a study by mohammadi for assessing responsiveness of health system to outpatients ,
there were relationships between age and dignity , autonomy , choice of provider , and total responsiveness .
there were also significant associations between level of education and dignity , autonomy , communication and total responsiveness ( 29 ) .
njeru et al . in a study about assessment of responsiveness tool of who in malindi city located in kenya , suggested that in the dimension of choice of provider , majority of participants believed that there is no choice to select care unit or providers ( 30 ) .
a study by ugurluoglu and colleagues in 2006 about responsiveness of health systems from the perspective of managers in turkey hospitals showed that the highest score ( 7.27 ) , on a scale of 010 ( 0=poorest ; 10=the best ) , belongs to the dimension of confidentiality .
the highest mean scores were dedicated to the dimensions of adherence to dignity and confidentiality during treatment and diagnosis , and the lowest scores to having choice of provider over selecting physicians and healthcare providers ( 31 ) . except for dignity in this study and most studies , which is ranked first or second ,
it appears that differences between the present study and research mentioned above is due to the difference in the research population .
participants in this study were only pregnant women , while in other studies of men , women and patients with chronic diseases , etc .
the mean score given to hospitals was 17.042.4 from 20 and 91.5% of women who stated that they have a tendency to visit the same hospitals in the case of becoming pregnant once again .
this matter implies to the positive attitude of women and their satisfaction with services provided .
sawyer and colleagues in a study in 2013 entitled parents experiences and satisfaction with care during the birth of their very preterm baby , 80% of participants were satisfied ( 32 ) . a study in nepal by paudel showed that the satisfaction score in basic amenities was 86% , service provider skills 85% , politeness of staff 83% , waiting time 80% , and participation in decision making 77% , cleanliness 70% , receiving information 69% , and confidentiality 67% , respectively ( 33 ) .
melese and colleagues in a study in ethiopia presented that the percentage of women being completely satisfied with labor care is between 4.2% and 21% and the lowest scores were in relation to satisfaction with pain management ( 82% dissatisfaction ) ( 34 ) .
bleich et al . conducted a study entitled how does satisfaction with the health - care system relate to patient experience in 21 european countries .
they suggested that , in all countries excluding five of them , 50% of respondents were relatively perfectly satisfied with their own health systems .
all dimensions of responsiveness had a significant and positive relationship with satisfaction ( p<0.01 ) , which is consistent with findings of this study ( 18 ) .
depending on existing evidence , utilizing data obtained from customers to assess and assure quality of healthcare , then the active involvement of customers plays an important role in making policies and improving service quality . wholly , the mean score of service quality in the present investigation illustrated that nonclinical dimensions have been disregarded , and it has potential to be better without making a huge investment in technology , human resources , and new regulations . | introductionthe world health organization ( who ) responsiveness model showing the ability of health systems in fulfilling people s expectations in connection with nonclinical aspects is an appropriate pattern to assess healthcare . the purpose of this study was to determine the status of pregnancy care provisions based on the responsiveness model.methodsthis was a cross - sectional study conducted by randomly sampling 130 women visiting selected hospitals in tehran in 2015 . a researcher - made questionnaire based on the responsiveness model of who was used to collect data .
we determined the face validity and content validity of the questionnaire , and its reliability was confirmed by cronbach s alpha coefficient ( 0.94 ) and test - retest analysis ( 0.96 ) .
the obtained data were analyzed by spss version 20 descriptive statistics , t - test , one - way anova , pearson product - moment correlation coefficient , and spearman correlation.resultstotal responsiveness from the perspective of service recipients was 69.4614.65 from 100 .
the obtained scores showed that , in the range of 0 to 100 , 73.02 were about basic amenities ( the most score ) , 72.93 about dignity , 70.91 about communication , 70.76 about confidentiality , 66.30 about provision social needs , 65.96 about choice of provider , 65.92 about autonomy , and 52.65 about prompt attention ( the lowest score ) , which are representing the average level of service quality .
there were significant relationships between participating in preparation class of labor and dignity ( p<0.001 ) , autonomy ( p=0.01 ) , provision social needs ( p=0.01 ) , and overall responsiveness ( p=0.03 ) .
it was obtained that there is a significant linear relationship between scores given to hospitals and dimensions of responsiveness ( p=0.05 ) .
findings indicated a significant relationship between insurance type and dimensions of choice of provider ( p=0.03 ) and communication ( p=0.03).conclusionthe mean score of service quality in the present investigation illustrated that nonclinical dimensions have been disregarded and it has potential to be better . so some grand plans are needed . |
obtaining accurate and repeatable measurements of anterior segment parameters is a mandatory step in achieving the best outcomes in refractive anterior segment surgery .
the corneal power ( k ) value is entered into any intraocular lens ( iol ) power formula [ 14 ] , while corneal astigmatism measurements are needed when planning toric iols implantation .
anterior chamber depth ( acd ) is required by the haigis , holladay 2 , and shammas formulae , which can be used to assess the risk for angle closure [ 8 , 9 ] and investigate changes in the anterior eye segment during accommodation .
corneal power is important to the evaluation of corneal ectasia and the diagnosis of keratoconus .
finally , the white to white ( wtw ) distance can be used to estimate phakic intraocular lens ( iol ) size .
anterior segment measurements can now be obtained by a number of instruments , including manual and automated keratometers , corneal topographers , scheimpflug cameras , and optical coherence tomographers .
the iolmaster ( carl zeiss meditec , germany ) and sirius ( cso , italy ) are two validated and widely used instruments [ 1316 ] . to our knowledge ,
therefore , the current study was performed to evaluate and compare k , axis of the corneal astigmatism , acd , and wtw measurements of the sirius and iolmaster devices .
this prospective study adhered to the tenets of the declaration of helsinki and was approved by the research review board at wenzhou medical university . after learning about the study procedure and purpose , all subjects provided written informed consent .
ninety eyes of 90 patients ( 40 men and 50 women ) with a mean age of 65.46 9.78 years ( range , 38 to 84 years ) were included in this study .
all subjects received a full ophthalmologic examination and all eyes involved in the study had clinically significant cataracts .
the exclusion criteria included active inflammation , previous refractive surgery , corneal scarring , a history of contact lens wear within the previous 4 weeks , and severe systemic diseases that made patients intolerant to the operation .
moreover , those patients who could not successfully fixate their eyes or had severe blepharophimosis were also excluded .
the iolmaster measures the radius of anterior corneal curvature and the respective axial orientation using the data from six light reflections oriented in an approximately 2.3 mm - diameter hexagonal pattern .
the corneal refractive index used to calculate the keratometric power and keratometric astigmatism for this study was 1.3375 .
the device measures the acd ( i.e. , distance from the corneal epithelium to the anterior lens surface ) through a 0.7 mm - wide lateral slit illumination at 30. the sirius combines a single - scheimpflug rotating camera with placido disk topography to measure and image the anterior eye segment . within a single scan
, it can simultaneously acquire more than 30,000 points on the corneal anterior and posterior surfaces and 25 radial sections of the cornea and anterior chamber .
the system acquires the radius curvature measurements in the flat and steep meridians on a 3.0 mm - diameter field of the central cornea .
each patient was guided to a seat in front of the equipment and placed their chin on a chinrest and their forehead against the forehead strap .
the patients were instructed to fixate on an internal fixation target within each device and permitted to blink completely just before each measurement to spread an optically smooth tear film over the cornea and keep the eye open during image acquisition .
with sirius , scans had to show the ok signal , meaning that placido and scheimpflug acquisition was above the required quality specification for coverage and centration .
with iolmaster , the optimum measurement setting ( green traffic light ) had been reached . to avoid the effects of diurnal variation in corneal shape and thickness
after the first device 's measurements were taken , the patients were asked to rest with their eyes before undergoing an examination by the other one 3 minutes later .
all measurements were performed by the same experienced examiner between 10 a.m. and 5 p.m. to eliminate operator - induced error and diurnal variations .
statistical analyses were performed using spss software ( version 17 ; spss inc . , chicago , il , usa ) .
the normality of all data distributions was confirmed using the kolmogorov - smirnov test ( p > 0.05 ) .
comparisons between the sirius and iolmaster measurements were conducted using paired t - tests to assess the mean differences in the anterior segment parameters and bland - altman plots to assess the degree of agreement between the two methods . in this analysis
, bias was defined as a significant difference in the means of the two methods ; 95% limits of agreement ( loa ) were calculated as the mean difference 1.96 sd .
table 1 shows the measured parameters : mean acd , flattest keratometry ( kf ) , steepest keratometry ( ks ) , mean keratometry ( km ) , astigmatism axis , and wtw .
table 2 shows the mean difference , sd , p values , and 95% loa .
the acd , kf , ks and km taken with the sirius were statistically significantly higher than that taken with the iolmaster ( p < 0.05 ) ; however , the sirius significantly underestimated the wtw values compared with the iolmaster biometer ( p < 0.05 ) .
figures 1 , 2 , 3 , 4 , 5 , and 6 show bland - altman plots for the anterior parameter comparisons between the sirius and iolmaster .
except for the astigmatism axis and wtw value , the bland - altman plots showed that the mean differences between the two devices were not significantly different for other comparisons of anterior parameters , which implied good agreement .
this investigation showed that , with the exception of astigmatism axis and wtw , the anterior segment measurements taken by the sirius are similar to those obtained by the iolmaster , which means that they show good agreement and may be used interchangeably in patients with cataracts .
achieving similar measurements could be useful in several clinical applications , including intraocular lens calculation .
accurate and precise determination of the anterior ocular segment is fundamental to many clinical and research applications in ophthalmology .
the sirius device is both noncontact and easy to use and showed good repeatability of the anterior segment measurements in healthy eyes as well as those after refractive surgery or with keratoconus . as far as we are concerned , this is the first prospectively designed comparative study of the differences and agreement of the measurement of the anterior segment using both devices in patients with cataracts . in the current study , the corneal power measurements ( ks , km ) obtained by the sirius and iolmaster showed a high level of agreement . although such good agreement suggests that their measurements could be used interchangeably , we always recommend optimizing the iol constants when shifting from one instrument to another .
the k value obtained by the sirius device was slightly higher than that produced by the iolmaster , but the difference in averages was too small to be clinically relevant .
these findings are consistent with previous studies of the iolmaster [ 17 , 22 ] .
however , for corneal astigmatism axis , the range of the 95% loa was too broad to be accepted in clinical practice .
compared the corneal powers obtained using four different instruments in 20 healthy volunteers and found that the values obtained by the galilei ( ziemer , port , switzerland ) , which uses dual scheimpflug cameras and a placido disk , were highly comparable with those obtained by the iolmaster .
the mean central corneal powers difference between them was only 0.12 diopter ( d ) .
this finding implied that the technique combining a scheimpflug camera and a placido disk could obtain valid and accurate corneal power in clinical application .
in addition , symes et al . found that the scheimpflug system was comparable to the iolmaster device and might have better accuracy in eyes with higher delta k values ( mean 2.13 diopters ) and a greater degree of preoperative astigmatism .
the values obtained from the sirius device in our study were a bit lower than those of de la parra - colin et al .
that study compared the anterior segment biometry parameters obtained from the sirius and a pentacam ( oculus , germany ) in unoperated eyes of healthy subjects , while we recruited patients with cataracts .
moreover , the age range was also different , as that in the previous study was younger ( mean age , 24.6 1.64 years ) .
because a cross - sectional sampling showed that corneal curvature tended to increase with age , the discrepancy would be due to the differences in study populations . with regard to acd measurements , the mean values were 3.13 0.41 mm versus 3.04 0.40 mm by the sirius and iolmaster , respectively .
it was a little bit higher when the acd was measured with the sirius but the difference was not statistically significant .
we attributed this divergence to alignment differences , as the iolmaster measures acd along the visual axis whereas the sirius measures acd along the optical axis , which usually represents the deepest central acd .
the other variable , instrument - specific corrective factors , may be responsible for those systematic differences . although no studies have directly compared the acd measurement by the sirius and iolmaster , indirect study results did provide some indications .
compared acd measurements taken with the pentacam , iolmaster , and orbscan in 42 volunteers . although the average iolmaster acd measurement was 0.11 mm smaller than that of the pentacam , the observed mean error was too small to create any noticeable difference in refractive outcome in cataract surgery in the clinical setting .
interestingly , de la parra - colin et al . evaluated the repeatability and comparability of acd measured by the sirius and pentacam and found that both devices showed adequate agreement in unoperated healthy eyes .
these findings implied that the sirius combined with scheimpflug / placido was comparable with iolmaster in acd measurements .
although recent studies have shown that the wtw can not accurately predict the real sulcus - to - sulcus distance , it remains an important biometric parameter for phakic iol diameter calculation . because patients are not comfortable due to direct contact measurements by ultrasound biomicroscopy , most surgeons rely on noncontact devices such as the iolmaster .
compared manual and automated methods to measure the wtw and found that automated devices provide more precise and reliable results and that the iolmaster has the highest reliability for measuring corneal diameter . in our study
, we found that this measurement was 11.42 0.46 mm using the sirius , lower than the average value obtained by the iolmaster ( 11.8 10.42 mm ) .
the sirius could be expected to read as much as 0.37 mm above to 1.15 mm below the iolmaster for wtw measurement .
this disparity was statistically significant and may have clinical implications ( e.g. , relying on sirius measurements may lead to an incorrect sizing of phakic iols ) .
thus , these devices should not be considered interchangeable for wtw assessments in clinical practice . although we do not yet know the exact reason for the differences in the values obtained by these two imaging devices , we speculate that fundamental methods for acquiring and analyzing images were responsible to the disparity .
this study has some limitations . from a practical point of view , when the sirius is used to calculate iols , the use of other devices to obtain the axial length is also required . as such , having the ability to measure the axial length would expand the clinical application of the sirius .
studies have shown that the scheimpflug photography feature provided precise and valid measurements for iol calculation .
enrolled 43 consecutive patients who were scheduled to undergo phacoemulsification to evaluate the accuracy of the galilei dual scheimpflug analyzer and a placido disk corneal topography system ( optikon 2000 spa ; keratron , italy ) for iol calculation , which found that corneal power measurements provided by the scheimpflug camera and placido disk corneal topographer obtained accurate iol power calculation .
the same team also confirmed that corneal power measurements provided by the sirius were successfully entered into third - generation iol power calculation formulas in eyes with cataracts .
different constants must be used for adjusting the postoperative iol position . on the other hand
, some studies suggested that the iols could influence measurement accuracy [ 34 , 35 ] .
since both devices were not used in the patients after cataract surgery in the same prospective study , we could not determine which one was better for predicting the iol power calculation in patients with cataracts , which requires further investigation .
in summary , with the exception of astigmatism axis and wtw measurements , the sirius scheimpflug - placido topographer and iolmaster partial coherence interferometer showed good agreement in anterior segment measurements , indicating that they may be used interchangeably in most clinical applications . | purpose . to assess
the consistency of anterior segment measurements obtained using a sirius scheimpflug / placido photography - based topography system ( cso , italy ) and iolmaster partial coherence interferometry ( carl zeiss meditec , germany ) in eyes with cataracts . methods .
a total of 90 eyes of 90 patients were included in this prospective study . the anterior chamber depth ( acd ) , keratometry ( k ) , corneal astigmatism axis , and white to white ( wtw ) values
were randomly measured three times with sirius and iolmaster .
concordance between them was assessed by calculating 95% limits of agreement ( loa ) .
results .
the acd and k taken with the sirius were statistically significantly higher than that taken with the iolmaster ; however , the sirius significantly underestimated the wtw values compared with the iolmaster .
good agreement was found for km and acd measurements , with 95% loa of 0.20 to 0.54 mm and 0.16 to 0.34 mm , respectively .
poor agreement was observed for astigmatism axis and wtw measurements , as the 95% loa was 23.96 to 23.36 and 1.15 to 0.37 mm , respectively . conclusion . with the exception of astigmatism axis and wtw , anterior segment measurements taken by sirius and iolmaster devices showed good agreement and may be used interchangeably in patients with cataracts . |
these are the malignant neoplasms mainly affecting lymph nodes , spleen and other nonhematopoietic tissues .
they are divided into hodgkin 's and non - hodgkin 's subtypes and are of b - cell or t - cell origin .
the majority of hodgkin 's lymphomas presents as a nodal disease involving mainly cervical and axillary nodes . among the 40% non - hodgkin 's lymphomas presenting extranodally , gastrointestinal tract , waldeyer 's ring , spleen , salivary gland are the most common sites of occurrence and are rarely seen in the oral cavity .
it may arise from preexisting low - grade lymphomas or as a de novo neoplasms . in the paraoral and primarily oral lymphomas , diffuse large b - cell lymphomas ( dlbcl ) are most common and is show a male predominance .
the most common sites of involvement intraorally are palate , buccal mucosa and tongue with the prevalence of only 35% .
a 79-year - old male patient reported to the department of oral pathology and microbiology with a chief complaint of pain and swelling on the upper left buccal mucosa since 1-month .
extraorally , facial asymmetry was noticed with diffuse swelling on the left sided cheek region , approximately 3 cm 2 cm in size , irregular in shape , vertically extending from inferior border of zygomatic arch up to 12 cm above corner of mouth and was extending from ala of nose up to tragus of the ear mediolaterally .
on intraoral examination , the swelling was diffuse , soft and roughly oval in shape [ figure 1 ] .
the swelling was situated in maxillary buccal vestibule extending anteroposteriorly from 26 to 28 region and mediolaterally from buccal mucosa of left side obliterating the vestibule , crossing the alveolar ridge and extending onto palatal mucosa .
the swelling was approximately 4 cm 2 cm in size and was of smooth surface texture .
clinical image shows diffuse , roughly oval shape swelling situated in maxillary buccal vestibule radiologically , on waters view haziness was noticed in the left maxillary sinus region as compared to the right side , extending from superior border up to inferior border of sinus vertically and extending from medial to the lateral border of sinus mediolaterally [ figure 2 ] .
radiographic images shows haziness in left maxillary sinus region an incisional biopsy was advised from buccal mucosa of left side under local anesthesia after routine blood investigations , which were within the normal limits . human immunodeficiency virus ( hiv )
on histopathological examination , the hematoxylin and eosin stained tissue section showed diffuse , monotonous proliferation of round cells , in the form of sheets within the loose fibrillar connective tissue stroma [ figure 3 ] .
these cells were having large nuclei with a small amount of cytoplasm resembling lymphoid cells .
it also revealed the presence of cells with large nuclei showing prominent nucleoli arranged at the periphery , exhibiting condensation and vesiculation of chromatin with cellular and nuclear pleomorphism .
these cells resembled centroblasts [ figure 4 ] and immunoblastic cells [ figure 5 ] .
the adjacent minor salivary gland tissue with mucous acini showed infiltration of these cells , disturbing the normal architecture .
histopathological image shows diffuse monotonous proliferation of round cells in the form of sheets within a loose fibrillar stroma ( h&e stain , 100 ) histopathological image shows round centroblastic cells with large nuclei ( h&e stain , 400 ) histopathological image shows round immunoblastic cells ( h&e stain , 400 ) overall histopathological features suggest the diagnosis of atypical lymphoproliferative lesion . for confirmation
on immunohistochemical analysis , cd45 ( leukocyte common antigen ) [ figure 6 ] positivity distinguished malignant lymphomas from other non - lymphoid neoplasms , strong positivity for cd20 ( b - cell marker ) [ figure 7 ] and weak expression of cd3 ( t - cell ) marker suggests the b - cell origin of the lesion and ki-67 ( proliferation marker ) positivity indicates the proliferative potential of the lesion .
immunohistochemical image shows strong positivity for cd45 ( ihc stain , 100 ) immunohistochemical image shows strong positivity for cd20 ( ihc stain , 100 )
in head and neck region , lymphomas are the second most common neoplasms . among 40% extranodal non - hodgkin 's lymphomas majority of cases
b - cell lymphomas are classified as precursor b - cell neoplasm and peripheral b - cell neoplasm . according to revised european - american lymphoma classification of non - hodgkin 's lymphomas , dlbcl is a peripheral b - cell neoplasm . world health organization in 2008
further categorized aggressive b - cell lymphomas as :
dlbcl not otherwise specifieddlbcl associated with chronic inflammationlarge b - cell lymphoma arising in human herpes virus ( hhv)-8 associated castelman 's diseaseb - cell lymphoma unclassifiable andburkitt 's lymphoma .
dlbcl not otherwise specified dlbcl associated with chronic inflammation large b - cell lymphoma arising in human herpes virus ( hhv)-8 associated castelman 's disease b - cell lymphoma unclassifiable and burkitt 's lymphoma . the kiel classification subdivided large b - cell lymphomas by pure morphology into centroblastic and immunoblastic lymphomas .
the working formulation , on the other hand , subdivided large b - cell lymphomas according to their biological behavior into an intermediate - grade ( large cleaved and large noncleaved ) and a high - grade ( immunoblastic ) category . among dlbcls ,
dlbcl not otherwise specified is the most common subtype and it accounts for up to 2030% cases seen in elderly , above 70 years of age .
it is characterized by large neoplastic b lymphocytes diffusely proliferating , with nuclei larger than or equal to normal macrophage nuclei in size , or more than twice the size of normal lymphocyte and are seen arising de novo .
various studies have shown that dlbcls shows an average age of presentation that is above 5055 years with a male predominance .
the present case was also reported in elderly ( 79-year - old ) male patient .
gastrointestinal tract , thyroid , bone , skin and lungs are the most frequent extranodal sites and in orofacial region waldeyer 's ring , salivary gland , tonsils , nasopharynx , base of the tongue are frequently involved .
intraorally it shows maxillary predominance ( 77% ) and the majority of cases are reported on the palate .
this is in accordance with the present case which was also reported in maxillary vestibular region and extended towards the palate .
histopathologically , the pattern of invasion in lymph nodes or extranodal sites is diffuse , with frequent peripheral tissue involvement .
the plasmablastic form is another morphologic variant but is more commonly associated with epstein - barr virus or anaplastic lymphoma kinase positive b - cell lymphomas . in the present case , there was a presence of diffuse involvement of mucosa and infiltration of adjacent minor salivary glands , main cell types seen in this case were centroblastic and immunoblastic variants showing cells with larger nuclei and prominent nucleoli , chromatin condensation and pleomorphism .
differential diagnosis includes reactive non - neoplastic processes in which aggregates of lymphocytes are evident but they did not show atypical features . in case of burkitt 's lymphoma the cells are of homogeneous size and shape giving starry sky appearance due to presence of macrophages which were absent in this case . in plasmablastic lymphomas
classic hodgkin 's lymphoma is predominantly the disease of lymph nodes and rarely affects oral cavity showing reed
other malignancies such as liposarcoma , round cell type show sheets of poorly differentiated round cells with finely vacuolated or granular cytoplasm and cells may appear epitheloid or pericytoid type .
carcinomas of poorly differentiated type show atypical round cells with eosinophilic cytoplasm , but centroblastic and immunoblastic cells are not seen .
thus , after excluding all these possibilities and the presence of centroblastic and immunoblastic cells a diagnosis of dlbcl was suggested .
the beginning ihc panel includes cd20 , cd3 , cd45 and if alteration of b - cell areas are seen then antibodies against cd5 , cd10 , cd23 , cd43 , bcl2 , bcl6 protein , etc . ,
previous studies have done an immunohistochemical analysis of leukocyte common antigen ( lca ) , cd20 , cd5 to confirm the diagnosis . here , in the present case ki-67 ( proliferative marker ) , cd45 ( common leukocyte antigen ) , cd20 ( b - cell marker ) analysis were done , which showed strong positivity .
it can be best treated by surgical excision with adjuvant radiotherapy if the lesion is localized ; chemotherapy is the treatment of choice in case of diffuse and aggressive lesions .
no significant difference in prognosis has been found between the three major groups : centroblastic , immunoblastic and anaplastic . in diffuse cases and hiv patients ,
the prognosis is worse than that in follicular , nodal and hiv - negative cases .
the diagnosis of these lesions is challenging due to their resemblance to inflammatory and benign lesions , chances of misdiagnosis are more and the treatment is prolonged . hence , the clinicians must consider it as a possible differential diagnosis since the lesions are aggressive and efforts should be taken to diagnose these lesions as early as possible as they demonstrate fatal outcomes .
| lymphomas are the group of neoplasms originating from lymphoreticular system mainly from lymph nodes , among them up to 40% of non - hodgkin 's lymphomas present extra nodally . in oral cavity , lymphomas are least common and account for 35% of all malignancies , presenting mainly in older age groups with male predominance . according to revised european - american lymphoma classification , among b - cell and t - cell subtypes of non - hodgkin 's lymphomas , diffuse large b - cell lymphoma ( dlbcl ) is the most common , characterized by diffuse proliferation of large neoplastic b lymphoid cells . here we present a case report of dlbcl affecting oral cavity involving left buccal vestibule and extending onto the palate , along with its clinical , histopathologic and immunohistochemical features . |
the foundation of all known life , deoxyribonucleic acid ( dna ) , performs the fundamental task of storing an organism s genetic information . in recent times , however , it has attracted attention due to its possible applications in biotechnology and nano - electronics . of particular interest to scientists
is the nature of charge transport in dna molecules , a topic surrounded by significant controversy .
the idea that a dna molecule could be treated like a semi - conducting nano - wire originated with eley and spivey , who performed experiments on the dc conductivity of dna molecules in the dry state .
more recently , there has been substantial interest , both experimental and theoretical , in the study of charge transport in this important biomolecule [ 4 - 14 ] .
this study investigates the electric field dependence of the conductivity of nano - scale ( ~100 nm ) samples of poly(dg)poly(dc ) and poly(da)poly(dt ) ( uniformly sequenced ) dna at fixed temperatures in an attempt to better understand the precise nature of the charge transport mechanism .
experiments performed by yoo et al . measured the electric field dependence of the current through samples of poly(dg)poly(dc ) and poly(da)poly(dt ) dna .
they used molecules with lengths ranging from 0.52.9 m , which were then folded to a maximum length of 100 nm and suspended between au / ti electrodes spaced 20 nm apart .
their experiment measured the current voltage characteristics of single- and double - stranded herring dna at three different values of relative humidity .
they demonstrate that at high relative humidity , the conductivity of their dna samples is similar to that of distilled water and accredit this to the hydrolysis of a thin film of adsorbed water .
the work of kutnjak et al . on calf - thymus li - dna displays a non - linear current voltage relationship similar to that described in ,
although the base pair sequence is not uniform in this case and the samples are macroscopic .
the voltage dependence of the current in their samples was studied at 75% relative humidity .
also measured the temperature dependence of the conductivity of their samples at 75% relative humidity and dried in a vacuum .
their results indicate a decrease of nearly one order of magnitude in the conductivity in a vacuum while the activation energy increased by approximately 0.15 ev , based on the arrhenius model they used to explain their curves .
they attribute this relatively large change in conductivity to an electric field - induced increase in the number of charge carriers .
lastly , an attempt was made to explain this behaviour with a variable range hopping model . using a power law , kutnjak et al . found the model fit their data well for temperatures below 220 k. although there is significant controversy over the manner of charge transport in dna , with some experiments
indicate that dna acts more like an insulator or that the charge migration is strongly assisted by an adsorbed layer of water molecules , there is evidence that dna generally behaves like a semiconductor .
performed experiments on dna molecules covalently bound to coaxial single - walled carbon nanotubes attached to au / ti electrodes spaced approximately 27 nm apart and found a non - linear current voltage relationship as well .
they were able to measure a gate voltage bias effect on this relationship and determined that their samples of avian influenza genes behaved as p - type semi - conductors .
this observation is in agreement with yoo et al . who showed their samples of poly(dg)poly(dc ) dna were also p - type semi - conductors .
conversely , it has been shown that poly(da)poly(dt ) dna behaves like an n - type semiconductor .
the lower oxidation potential of gc base pairs leads to the localization of holes , which allows the formation and hopping conduction of polarons .
we consider that charge carriers are localized on a base pair and interact with phonons generated by motions in the double helix .
this interaction leads to the formation of a localized polaron , whose states are distributed randomly in energy and space coordinates .
polarons move throughout the dna molecule by random hops in this hopping space .
the recent work of singh develops a theoretical approach to the phonon - assisted hopping conduction mechanism of polarons in dna , based on the general molecular crystal model employed by triberis and friedman . by treating dna as a quasi - one - dimensional molecular crystal ,
a 2/3 power law for the temperature dependence for the conductivity is developed and is found to agree well with the experimental results of kutnjak et al . .
in this study , we extend the work of singh to develop a model to explain the non - linear current voltage relationship for poly(dg)poly(dc ) and poly(da)poly(dt ) dna molecules , using a 2/3 power law for temperature .
a good agreement is found between the model and data obtained from yoo et al . .
the 2/3 power law is used to obtain the fitting parameter td , which contains information about the density of states in the dna , from the temperature - dependent conductance .
this value is , in turn , used to find the characteristic current i0 and temperature - dependent polaron hopping distance a from the i v curves of poly(da)poly(dt ) at 143 , 161 , and 223 k. we find that our analysis yields a linear hopping distance in temperature .
a sugar ( deoxyribose ) and phosphate ion combine with one of four amino acids .
these are guanine ( g ) , cytosine ( c ) , adenine ( a ) , and thymine ( t ) .
nucleotides form pairs in such a way that those consisting of guanine only bond with a nucleotide containing cytosine . similarly
these nucleotide pairs are then stacked to produce a double - helix structure , with deoxyribose forming the backbone of the molecule and the amino acids forming base pairs . due to the stacked nature of the nucleotides , there is an overlap of the molecular orbitals of the base pairs in the direction parallel to the long axis of the dna .
this is thought to facilitate charge transfer along the base pair sequence of the molecule by allowing a charge carrier to exist in a delocalized state .
it is believed that there are two primary modes of long - distance charge migration .
one is a coherent tunnelling process whereby a charge carrier is transferred from a donor to acceptor without exchanging energy with the dna molecule itself .
the effect of this method is thought to be minor over sufficiently long distances at the temperatures commonly encountered in experiment [ 1,5 - 9 ] .
the second is an incoherent multi - step process where a charge carrier on a base pair is localized and interacts with phonons in the molecule , which are generated by thermal and twisting motions of the double - helix , forming a phonon .
we have used the following model conductance expression which is based on quasi - one - dimensional polaron hopping in dna .
the conductance depends on the electric field and temperature and is written as the basis for this expression can be found in in terms of electrical conductivity .
equation 1 is a modified form of this in that the temperature dependence has been simplified and the electric field dependence has been changed to a hyperbolic function .
, who demonstrate a relationship between electric current and electric field for a given temperature with a function proportional to sinh(v ) . here , is a constant and has units of v. similarly , td is a constant and has units of temperature .
they are defined as here , q is the elementary charge , a is the localization length , n0 is the number density of hopping sites , dd is the density of states , and d is the spacing between the electrodes in the experiment ( d = 20 nm in the case of ) .
note that when the electric field is weak in the dna , the conductance expression reduces to the 2/3 power law was obtained using the polaron variable range hopping mechanism in dna .
it is considered that the charge carriers are localized at the bases in dna . the h - bonds and pi
the electron orbitals of bases overlap quite well with each other along the long axis of the dna .
the charge carriers are localized on bases due the disorder in the system and strong thermal fluctuations in these structures .
the interaction between localized carriers and phonons create polarons which are responsible for charge conduction in dna due to the variable range hopping mechanism .
yoo et al . have found that the parameter appearing in eq . 1 is independent of temperature
2 , we get the parameters g0, , and td can be obtained from experiments .
data were numerically calculated by yoo et al . for zero voltage from their i
v curves plot of electric current as a function of voltage for two samples of poly(da)poly(dt ) dna at 143 , 161 , and 223 k. fits to the data are represented by solid curves
experiments performed by yoo et al . measured the current voltage characteristics of poly(dg)poly(dc ) and poly(da)poly(dt ) dna as well as the temperature dependence of their conductances at zero voltage .
they found that as temperature decreased , the behaviour of the current in poly(da)poly(dt ) dna became increasingly non - linear , and temperature independent at temperatures below 50 k . in this discussion , we compare our model , based on that discussed in and the earlier work of singh , to the current voltage relationship for poly(da)poly(dt ) dna at 143 , 161 , and 223 k. there is good agreement between our model and the data obtained from yoo et al .
4to calculate the temperature - dependent conductance , which is compared to experimental data to determine the value of td for poly(da)poly(dt ) at temperatures > 100 k. figure 1 displays the conductance versus inverse temperature ( crosses ) and the solid curve representing eq .
4 , which is used to fit the data . the fitting parameters g0 and td were found to have the values of 78.05 and 3.030 10 k , respectively .
it is difficult to estimate the conductivity of the sample in , due to the absence of the precise dimensions of the samples used . assuming the samples have lengths on the order of 10 m and cross - sectional areas of ~10 m
, we find the quantity 0 = g0l / a to have a value on the order of 10 ( cm ) .
the value of td agrees within an order of magnitude of the values of 6.7 10 and 1.07 10 k presented by singh .
however , those values correspond to non - uniformly sequenced samples of dna and , as such , only serve as a guideline .
we use the td obtained in fig . 1 to determine the fitting parameters for the i v curves presented in fig .
2 . by letting i0,t = i0 exp[(td / t ) ] , we are able to determine i0 using the 2/3 power law for the polaron hopping model developed by singh . with this expression
, we find i0 to be 3.65 10 a at 143 k , 6.87 10 a at 161 k , and 2.36 10 a at 223 k. a decreasing trend in temperature for the scaling factor 0 is predicted by singh , which corresponds to our i0 .
the parameter takes the value of 0.68 v for poly(da)poly(dt ) at the temperatures in question .
, who offer little explanation for this choice . nonetheless , there is good agreement between the model and experiment . from eq . 5
the corresponding values of a for each of our fits at 143 , 161 , and 223 k are , respectively , 3.35 , 3.77 , and 5.23 .
further study will yield a better understanding of charge transport in dna molecules and the relationship between temperature and polaron hopping distance .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . | we investigate the current voltage relationship and the temperature - dependent conductance of nano - scale samples of poly(da)poly(dt ) dna molecules .
a polaron hopping model has been used to calculate the i v characteristic of nano - scale samples of dna .
this model agrees with the data for current versus voltage at temperatures greater than 100 k. the quantities g0 , i0 , and t1d are determined empirically , and the conductivity is estimated for samples of poly(da)poly(dt ) . |
chronic obstructive pulmonary disease ( copd ) is associated with a two- to threefold increased risk of cardiovascular events.1,2 in fact , autopsy studies revealed cardiovascular disease ( cvd ) as the most frequent , but undetected and potentially modifiable , cause of death in patients with copd.3 the underlying mechanisms for the relationship between copd and cvd exceed the effects of traditional risk factors and remain largely unknown .
a long latency period between disease initiation and clinical manifestation allows the identification of subjects at risk at a subclinical , potentially reversible stage of cvd . for this purpose ,
several markers of subclinical atherosclerosis have been identified as predictors of cardiovascular risk.46 intima - media thickness of the brachial artery ( b - imt)7 is a novel and easily accessible parameter for the quantification of subclinical atherosclerosis .
recent studies validating markers of subclinical atherosclerosis against coronary angiography revealed b - imt as an independent predictor of cardiovascular events.8 in a 12-year follow - up study , b - imt clearly outperformed the predictive power of flow - mediated dilation ( fmd ) , the gold standard of noninvasive cardiovascular risk prediction via endothelial function.9 b - imt has not been studied in copd so far . consequently , this study aimed to assess b - imt in patients with copd compared to healthy controls and to investigate potential associations between b - imt and clinical as well as laboratory markers of copd ( ie , airflow limitation and systemic inflammation ) . in this context , the present analysis specifically aimed to assess the relationship between asymmetric dimethylarginine ( adma ) and copd .
adma is an endogenous inhibitor of nitric oxide synthase , providing a potential link between chronic inflammatory disorders and subclinical atherosclerosis.10,11 it was hypothesized that :
b - imt is significantly increased in patients with copd compared to smoking and nonsmoking controls.b - imt is significantly correlated with markers of systemic inflammation as well as adma levels in patients with copd .
b - imt is significantly increased in patients with copd compared to smoking and nonsmoking controls .
b - imt is significantly correlated with markers of systemic inflammation as well as adma levels in patients with copd .
for this observational study , a sample of patients with copd ( n=60 ) previously recruited via the outpatient clinic database of the otto wagner hospital , vienna , was analyzed .
inclusion criteria comprised evidence of airflow limitation on spirometry according to current guidelines,12 a smoking history of at least 20 pack - years and age > 40 years .
subjects were excluded if they were on long - term oxygen therapy , had evidence of an acute exacerbation of copd within the previous 4 months ( requiring antibiotics , oral steroids , and/or hospitalization ) , or suffered from any other relevant lung disease .
other exclusion criteria comprised overt cardiovascular disorders ( ie , coronary artery disease , chronic heart failure , cardiomyopathy , arterial hypertension , atrial fibrillation , and peripheral arterial disease ) , vasoactive medication , malignant diseases , diabetes mellitus , chronic renal failure , or chronic inflammatory conditions other than copd .
the control groups consisted of lifelong nonsmokers ( n=20 ) as well as apparently healthy smokers ( n=20 ) .
exclusion criteria were the same as in the study group plus the absence of obstructive lung disease confirmed by clinical evaluation and spirometry .
control subjects were recruited from the general population during a public awareness campaign on spirometry at the otto wagner hospital . the study was planned and conducted in accordance with the declaration of helsinki .
data handling and reporting followed the strobe - statement ( strengthening the reporting of observational studies in epidemiology).13 ultrasound studies of the brachial artery were conducted as previously reported.8 subjects were instructed to refrain from food , caffeine , tobacco , and vasoactive drugs for 46 h prior to the study visit .
measurements were performed at the antecubital fossa of the right arm after 10 min of rest with subjects placed in a supine position .
investigators blinded to clinical and spirometric results used high - resolution ultrasound ( 13 mhz ) for depiction of the intimal and medial layer of the brachial artery in a longitudinal plane . using a stereotactic device ,
the transducer position was fixed and images were stored at an adequate gain for subsequent offline analysis .
b - imt was assessed from the leading edge between lumen and intima to the leading edge between media and adventitia .
a cutoff value 0.345 mm was regarded as elevated b - imt14 and was therefore used as a discriminator for cardiovascular risk using the framingham risk score.15 lumen diameter was measured as the distance between the leading edge of the near and far intima lumen interface .
finally , the cross - sectional intima - media area ( b - ima ) was calculated following a previously described equation.16 lung function testing was performed according to the current guidelines12 for the verification of copd diagnosis , the classification of the gold stage , and the quantification of the distinct parameters of lung function ( forced expiratory volume in 1 s [ fev1 ] , % predicted ; forced vital capacity [ fvc ] , % predicted ; fev1/fvc ratio ) . a physical examination was conducted in every participant for height , weight , heart rate , and arterial blood pressure measurement .
venous blood samples were collected and analyzed for inflammatory biomarkers ( neutrophil count , interleukin [ il]-6 ) and levels of n - type pro - bnp ( pro b - type natriuretic peptide ) .
circulating adma concentrations were measured via reversed - phase high - performance liquid chromatography following a previously described method.17 the 10-year risk for cardiac events was calculated by means of the framingham risk score15 including the independent cardiovascular risk factors : age , sex , smoking history , arterial blood pressure , cholesterol ( total and high - density lipoprotein [ hdl]-cholesterol ) , and diabetes .
variables of interest are expressed as counts ( % ) for categorical variables , as mean standard deviation or median and interquartile range for interval variables depending on the respective data distribution .
between - group comparisons were performed using independent samples t - test or mann whitney u - test depending on the respective data distribution .
univariate correlations were performed by means of pearson s correlation coefficient or kendall s tau depending on the respective data distribution .
a multiple linear regression model was used to identify the strongest predictors of b - imt . through forward selection ,
the sequence of considered covariates was chosen by means of the highest increase in r. variables remained in the model if they altered the coefficient by at least 10% .
analyses were performed using the statistical package for social sciences ( spss [ version 15 for windows ] ; spss inc . ,
artwork was created using the graphpad software , version 5 for windows ( graphpad inc .
for this observational study , a sample of patients with copd ( n=60 ) previously recruited via the outpatient clinic database of the otto wagner hospital , vienna , was analyzed .
inclusion criteria comprised evidence of airflow limitation on spirometry according to current guidelines,12 a smoking history of at least 20 pack - years and age > 40 years .
subjects were excluded if they were on long - term oxygen therapy , had evidence of an acute exacerbation of copd within the previous 4 months ( requiring antibiotics , oral steroids , and/or hospitalization ) , or suffered from any other relevant lung disease .
other exclusion criteria comprised overt cardiovascular disorders ( ie , coronary artery disease , chronic heart failure , cardiomyopathy , arterial hypertension , atrial fibrillation , and peripheral arterial disease ) , vasoactive medication , malignant diseases , diabetes mellitus , chronic renal failure , or chronic inflammatory conditions other than copd .
the control groups consisted of lifelong nonsmokers ( n=20 ) as well as apparently healthy smokers ( n=20 ) .
exclusion criteria were the same as in the study group plus the absence of obstructive lung disease confirmed by clinical evaluation and spirometry .
control subjects were recruited from the general population during a public awareness campaign on spirometry at the otto wagner hospital . the study was planned and conducted in accordance with the declaration of helsinki .
data handling and reporting followed the strobe - statement ( strengthening the reporting of observational studies in epidemiology).13
ultrasound studies of the brachial artery were conducted as previously reported.8 subjects were instructed to refrain from food , caffeine , tobacco , and vasoactive drugs for 46 h prior to the study visit .
measurements were performed at the antecubital fossa of the right arm after 10 min of rest with subjects placed in a supine position .
investigators blinded to clinical and spirometric results used high - resolution ultrasound ( 13 mhz ) for depiction of the intimal and medial layer of the brachial artery in a longitudinal plane . using a stereotactic device ,
the transducer position was fixed and images were stored at an adequate gain for subsequent offline analysis .
b - imt was assessed from the leading edge between lumen and intima to the leading edge between media and adventitia .
a cutoff value 0.345 mm was regarded as elevated b - imt14 and was therefore used as a discriminator for cardiovascular risk using the framingham risk score.15 lumen diameter was measured as the distance between the leading edge of the near and far intima lumen interface .
finally , the cross - sectional intima - media area ( b - ima ) was calculated following a previously described equation.16 lung function testing was performed according to the current guidelines12 for the verification of copd diagnosis , the classification of the gold stage , and the quantification of the distinct parameters of lung function ( forced expiratory volume in 1 s [ fev1 ] , % predicted ; forced vital capacity [ fvc ] , % predicted ; fev1/fvc ratio ) .
a physical examination was conducted in every participant for height , weight , heart rate , and arterial blood pressure measurement .
venous blood samples were collected and analyzed for inflammatory biomarkers ( neutrophil count , interleukin [ il]-6 ) and levels of n - type pro - bnp ( pro b - type natriuretic peptide ) .
circulating adma concentrations were measured via reversed - phase high - performance liquid chromatography following a previously described method.17 the 10-year risk for cardiac events was calculated by means of the framingham risk score15 including the independent cardiovascular risk factors : age , sex , smoking history , arterial blood pressure , cholesterol ( total and high - density lipoprotein [ hdl]-cholesterol ) , and diabetes .
ultrasound studies of the brachial artery were conducted as previously reported.8 subjects were instructed to refrain from food , caffeine , tobacco , and vasoactive drugs for 46 h prior to the study visit .
measurements were performed at the antecubital fossa of the right arm after 10 min of rest with subjects placed in a supine position .
investigators blinded to clinical and spirometric results used high - resolution ultrasound ( 13 mhz ) for depiction of the intimal and medial layer of the brachial artery in a longitudinal plane . using a stereotactic device ,
the transducer position was fixed and images were stored at an adequate gain for subsequent offline analysis .
b - imt was assessed from the leading edge between lumen and intima to the leading edge between media and adventitia .
a cutoff value 0.345 mm was regarded as elevated b - imt14 and was therefore used as a discriminator for cardiovascular risk using the framingham risk score.15 lumen diameter was measured as the distance between the leading edge of the near and far intima lumen interface .
finally , the cross - sectional intima - media area ( b - ima ) was calculated following a previously described equation.16
lung function testing was performed according to the current guidelines12 for the verification of copd diagnosis , the classification of the gold stage , and the quantification of the distinct parameters of lung function ( forced expiratory volume in 1 s [ fev1 ] , % predicted ; forced vital capacity [ fvc ] , % predicted ; fev1/fvc ratio ) .
a physical examination was conducted in every participant for height , weight , heart rate , and arterial blood pressure measurement .
venous blood samples were collected and analyzed for inflammatory biomarkers ( neutrophil count , interleukin [ il]-6 ) and levels of n - type pro - bnp ( pro b - type natriuretic peptide ) .
circulating adma concentrations were measured via reversed - phase high - performance liquid chromatography following a previously described method.17 the 10-year risk for cardiac events was calculated by means of the framingham risk score15 including the independent cardiovascular risk factors : age , sex , smoking history , arterial blood pressure , cholesterol ( total and high - density lipoprotein [ hdl]-cholesterol ) , and diabetes .
variables of interest are expressed as counts ( % ) for categorical variables , as mean standard deviation or median and interquartile range for interval variables depending on the respective data distribution .
between - group comparisons were performed using independent samples t - test or mann whitney u - test depending on the respective data distribution .
univariate correlations were performed by means of pearson s correlation coefficient or kendall s tau depending on the respective data distribution .
a multiple linear regression model was used to identify the strongest predictors of b - imt . through forward selection ,
the sequence of considered covariates was chosen by means of the highest increase in r. variables remained in the model if they altered the coefficient by at least 10% .
analyses were performed using the statistical package for social sciences ( spss [ version 15 for windows ] ; spss inc . ,
artwork was created using the graphpad software , version 5 for windows ( graphpad inc . , san diego , ca , usa , 2005 ) .
sixty patients with stable copd , free from overt cardiovascular disorders , were available for analysis .
the sample of copd patients comprised 32% gold stage ii , 32% gold stage iii , and 36% gold stage iv .
the three groups were matched for age , sex , body mass index , blood pressure , and lipid levels .
there were significant differences in heart rate , smoking history , and lung function between patients with copd and smoking , as well as nonsmoking controls ( p<0.05 ) .
results of b - imt measurements for the different study groups are depicted in figure 1 . in patients with copd ,
significantly higher b - imt ( 0.370.08 mm vs 0.320.06 mm ; p=0.007 ; vs 0.330.06 mm ; p=0.033 ) as well as b - ima ( 4.71.4 mm vs 3.91.2 mm ; p=0.027
; vs 3.91.3 mm ; p=0.022 ) was observed compared to smoking and nonsmoking controls . within the copd group ,
male participants showed significantly higher b - imt levels ( 0.410.06 mm ) than females ( 0.330.07 mm ) ( p<0.001 ) .
prediction of cardiovascular risk through the framingham risk score revealed a twofold increased 10-year risk for cardiovascular events in copd patients with elevated b - imt ( 127.6 ) compared to those with b - imt levels below the recommended cutoff ( 5.91.8 ) ( p=0.002 ) ( figure 2 ) .
univariate correlations between b - imt and variables of interest are listed in table 2 .
white blood cell count showed normal levels of neutrophils in the study sample without significant differences between copd patients , smokers , and nonsmokers .
there was a significant positive correlation between il-6 plasma levels and b - imt in patients with copd ( figure 3 ) .
there were no significant differences of circulating adma levels between the three study groups . in patients with copd ,
b - imt , however , was positively correlated with adma levels ( figure 4 ) , an observation that was not observed in controls . in a multivariable linear regression model ( table 3 ) with b - imt as the dependent variable , this study included age , male sex , systolic blood pressure , exacerbation rate , fev1/fvc ratio , ln il-6 , ln adma , and ln pro - bnp as independent variables . through forward selection , male sex , and adma remained independent predictors of b - imt in the model .
sixty patients with stable copd , free from overt cardiovascular disorders , were available for analysis .
the sample of copd patients comprised 32% gold stage ii , 32% gold stage iii , and 36% gold stage iv .
the three groups were matched for age , sex , body mass index , blood pressure , and lipid levels .
there were significant differences in heart rate , smoking history , and lung function between patients with copd and smoking , as well as nonsmoking controls ( p<0.05 ) .
results of b - imt measurements for the different study groups are depicted in figure 1 . in patients with copd , significantly higher b - imt ( 0.370.08 mm vs 0.320.06 mm ; p=0.007 ; vs 0.330.06 mm ; p=0.033 ) as well as b - ima ( 4.71.4 mm vs 3.91.2 mm ; p=0.027 ; vs 3.91.3 mm ; p=0.022 ) was observed compared to smoking and nonsmoking controls . within the copd group ,
male participants showed significantly higher b - imt levels ( 0.410.06 mm ) than females ( 0.330.07 mm ) ( p<0.001 ) .
prediction of cardiovascular risk through the framingham risk score revealed a twofold increased 10-year risk for cardiovascular events in copd patients with elevated b - imt ( 127.6 ) compared to those with b - imt levels below the recommended cutoff ( 5.91.8 ) ( p=0.002 ) ( figure 2 ) .
univariate correlations between b - imt and variables of interest are listed in table 2 .
white blood cell count showed normal levels of neutrophils in the study sample without significant differences between copd patients , smokers , and nonsmokers .
there was a significant positive correlation between il-6 plasma levels and b - imt in patients with copd ( figure 3 ) .
there were no significant differences of circulating adma levels between the three study groups . in patients with copd ,
b - imt , however , was positively correlated with adma levels ( figure 4 ) , an observation that was not observed in controls . in a multivariable linear regression model ( table 3 ) with b - imt as the dependent variable , this study included age , male sex , systolic blood pressure , exacerbation rate , fev1/fvc ratio , ln il-6 , ln adma , and ln pro - bnp as independent variables . through forward selection , male sex , and adma remained independent predictors of b - imt in the model .
in the present study , significantly higher b - imt was observed in patients with copd compared to smoking and nonsmoking controls .
patients with elevated b - imt had a twofold higher risk for future cardiovascular events calculated by means of the framingham risk score .
b - imt was correlated with airflow limitation , systemic inflammation , and circulating levels of adma .
the data support further evidence for an increased cardiovascular risk in patients with stable copd , independent from traditional cardiovascular risk factors .
although adma plasma levels were not different between the groups , the correlation with b - imt in copd patients suggests an interaction with circulating adma levels as a determinant of subclinical atherosclerosis in copd .
this is the first study to investigate b - imt in patients with copd free from overt cardiovascular disorders .
these findings support a previous study by iwamoto et al , who assessed intima - media thickness of the carotid artery as a predictor of cvd risk in smokers with airflow limitation.18 the authors similarly observed a thickening of the arterial wall in copd compared to healthy smoking and nonsmoking controls .
direct comparison of brachial versus carotid artery intima - media thickness in patients with coronary artery disease showed comparable diagnostic accuracy of both markers to detect diffuse coronary arteriosclerotic lesions.19 using the brachial artery , however , enables study of both structural and functional properties of systemic vasculature . using brachial artery measurements of fmd ,
impaired endothelial function has been previously demonstrated in copd compared to matched controls.2022 in a direct comparison , however , b - imt appears to be a better and independent predictor of coronary artery disease when compared with fmd , in both cross - sectional23 and prospective studies.9 indeed , stratification of the copd sample via a previously suggested cutoff14 for b - imt of 0.345 mm revealed a twofold higher risk of cardiac events in the framingham risk score for copd patients with elevated b - imt compared to those below the recommended cutoff .
the available literature suggests several potential mechanisms contributing to elevated subclinical atherosclerosis in copd . in this study ,
this observation is consistent with findings from the epidemiological gutenberg heart study.24 in the absence of traditional cardiovascular risk factors , these findings suggest potential vasoprotective effects of estrogen , a hypothesis that is supported by other studies.2527 tobacco smoke remains the major risk factor for both copd and cvd .
patients with copd , however , are at an increased risk of cvd , independent of smoking.21,28,29 indeed , analyses of the framingham study identified airflow obstruction as an independent predictor of cardiac events , with an effect size stronger than traditional cardiovascular risk factors.30,31 in line with these findings , the univariate analysis in this study showed an inverse correlation between b - imt and fev1/fvc ratio in patients with copd . both patients with airflow obstruction and those with cvd exhibit systemic inflammation.28,32,33 significantly higher levels of circulating il-6 were observed in patients with copd compared to controls .
these observations are consistent with previous reports suggesting a link between systemic inflammation and subclinical atherosclerosis.18,34 systemic inflammation may drive the development of cvd via impairments of the nitric oxide pathway , a hypothesis that is supported by earlier findings of the relationship between circulating inflammatory markers and endothelial function in copd.21 adma is an endogenous inhibitor of nitric oxide synthase , and implicated in the pathogenesis of cvd.35 indeed , a meta - analysis10 of 22 original articles recently confirmed the relationship between adma and b - imt .
furthermore , circulating adma levels predict clinical endpoints , such as myocardial infarction or stroke.36,37 the lung has been identified as one of the most important sources of adma formation;38 however , the role of adma in copd remains unknown yet .
recent studies provide conflicting results , demonstrating either higher39 or lower40 circulating adma concentrations in patients with copd compared with controls .
lower concentrations of adma were also found in smokers without copd compared to nonsmokers.41,42 in the present report , there were no statistically significant differences between copd patients and controls with respect to blood adma levels ; however , a relationship of the latter with b - imt exists exclusively in copd .
thus , these findings may suggest a potential role for adma in the development of subclinical atherosclerosis in copd , rather than a marker of lung function impairment as a consequence of smoking .
the relationship between adma and impaired b - imt in copd in this report is novel and may extend earlier findings , providing a further potential interaction between structural , functional , and molecular properties of the systemic vasculature .
a number of potential limitations in the current study need to be acknowledged including but not limited to the small sample size and a highly selected patient population , free from overt cvds . however , the present study aimed to investigate pathobiological associations by stringent exclusion of potentially confounding comorbid conditions .
nevertheless , it has to be admitted that the exclusion of coronary artery disease was based on patient surveys and medical history alone , as coronary angiography was not performed in this asymptomatic population .
finally , due to the observational nature of the study further data are needed to characterize the role of adma within the observed associations between intima - media thickness and cardiovascular risk in patients with copd .
in conclusion , the present study reveals b - imt as a promising and easily accessible tool for stratification of future cardiovascular risk in patients with copd . confirming previous observations , a significant association was found between subclinical atherosclerosis and systemic inflammation .
additionally , adma was characterized as a potential cofactor in the pathogenesis of copd related cardiovascular comorbidity .
these findings help in understanding the complex mechanisms linking copd to the associated excess cardiovascular risk . | backgroundchronic obstructive pulmonary disease ( copd ) is associated with an increased cardiovascular risk .
however , the mechanisms for this association are yet unclear .
the aim of this study was to investigate the relationship between brachial intima - media thickness ( b - imt ) , an independent predictor of cardiovascular risk , systemic inflammation , and asymmetric dimethylarginine ( adma ) , an endogenous inhibitor of nitric oxide synthase , in patients with copd and respective controls.methodsthe study sample consisted of 60 patients with stable copd , free from overt cardiovascular disorders , as well as 20 smoking and 20 nonsmoking controls .
ultrasound assessment of b - imt , spirometry , venous blood sampling for quantification of inflammatory markers and adma levels were carried out , and individual cardiovascular risk was calculated via the framingham risk score.resultspatients with copd showed significantly higher b - imt compared to smoking ( p=0.007 ) and nonsmoking controls ( p=0.033 ) .
copd patients with elevated b - imt had a twofold increased calculated 10-year risk for cardiovascular events compared to those below the recommended cutoff ( p=0.002 ) .
b - imt was significantly associated with systemic inflammation ( interleukin-6 [ il-6 ] ; r=0.365 , p=0.006 ) and adma ( r=0.331 , p=0.013 ) in copd .
multivariate linear regression revealed male sex and adma as independent predictors of b - imt in this study sample.conclusionb-imt is significantly increased in patients with copd and is associated with systemic inflammation and adma levels . |
since the development of chlorpromazine in the 1950s antipsychotic drugs have been the primary treatment choice for schizophrenia .
the common pharmacological antagonism of dopamine ( da ) d2 receptors by all antipsychotics and direct link with clinical improvement led to the theory of excess dopaminergic neurotransmission precipitating psychotic states [ 24 ] .
later , advances in animal , postmortem , and neuroimaging studies led to refinements of the dopamine hypothesis and a regional specificity of abnormal da signalling was proposed . negative symptoms of schizophrenia ( such as anhedonia , flat or blunted affect , alogia , and avolition ) as well as cognitive impairments ( including deficits in executive functions , attention , and working memory ) were postulated to be caused by deficiencies in da transmission at d1 receptors in mesocortical projections to the prefrontal cortex ( pfc ) .
this dysregulation in cortical da pathways , through a reciprocal relationship with subcortical da projections , was hypothesised to cause a hyperdopaminergic state at d2 receptors in mesolimbic da projections , resulting in positive symptoms of the disorder ( such as hallucinations and delusions ) [ 58 ] .
psychotomimetic effects of indirect da agonists , such as amphetamines , in healthy individuals [ 9 , 10 ] as well as more recent neuroimaging findings - linking increased da synthesis at presynaptic striatal d2 receptors to positive symptoms [ 11 , 12 ] and da deficiencies in pfc areas to cognitive deficits [ 1315 ] have lent further support to the dopamine hypothesis .
in addition , associations between specific candidate genes and dopaminergic dysfunction in schizophrenia have been identified [ 1618 ] . whilst first - generation antipsychotics ( fgas )
are characterised by their principal blockade of d2 receptors , second - generation atypical compounds comprise a more heterogeneous pharmacological profile involving actions on multiple neurotransmitter systems [ 19 , 20 ] . despite widespread anticipation of better tolerability of these newer agents ( particularly in regards to extrapyramidal side effects associated with fgas ) , metabolic complications such as weight gain , impaired glucose tolerance , and dyslipidaemia are commonly occurring side effects .
further , it is estimated that one third of patients do not respond adequately to antipsychotic medication [ 2224 ] , with only clozapine showing better efficacy than fgas in treatment - resistant schizophrenia [ 25 , 26 ] .
while positive symptoms are generally reasonably well controlled by antipsychotic treatment , negative and cognitive symptom clusters commonly fail to respond in a large proportion of patients [ 2729 ] , though their severity is associated with longer - term clinical outcomes [ 3032 ] .
these factors underline the urgent need for novel compounds with improved tolerability and efficacy , particularly for negative and cognitive symptoms .
research has identified other neurotransmitter systems in addition to dopamine in the pathology of schizophrenia [ 3335 ] .
most prominently , work on the role of glutamate the primary excitatory neurotransmitter in the central nervous system forms the basis of efforts into developing the first nondopaminergic compounds in sixty years of pharmacotherapeutic treatment of schizophrenia [ 3642 ] .
two main types of receptors underlie glutamatergic neurotransmission : the ligand - gated ionotropic receptor family , divided into -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ( ampa ) , n - methyl - d - aspartate ( nmda ) and kainate receptors , and the g - protein coupled metabotropic receptor ( mglur ) family comprising groups i to iii with a total of eight identified subtypes . while ionotropic receptors , in particular ampa and nmda , mediate fast excitatory transmission at the glutamate synapse , ligand binding at metabotropic receptors leads to conformational changes directly or indirectly affecting neurotransmission via second messenger pathways . a number of shortcomings of the historically prevailing dopamine hypothesis
most prominently the suboptimal or lacking clinical response of negative and cognitive symptoms to d2 antagonism and findings of structural brain changes associated with the schizophrenia inspired the integration of glutamate into a theoretical framework of the disorder [ 4547 ] .
the observation that administration of noncompetitive nmda receptor ( nmdar ) antagonists like phencyclidine ( pcp ) and ketamine could mimic symptoms of schizophrenia in healthy individuals gave rise to the hypothesis of impaired nmdar functioning contributing to its pathophysiology [ 4850 ] .
importantly , it was found that pcp and ketamine immediately and reliably induced symptom patterns identical to the cognitive impairments and negative symptoms of schizophrenia not observed under amphetamine challenge . in recent years , several lines of evidence have suggested that reduced glutamatergic excitation of subcortical gamma - amino - butyric acid ( gaba ) interneurons , through an initial hypofunction of nmdar , results in disinhibition of glutamate ( as well as dopamine and acetylcholine ) neurotransmission to the cortex [ 49 , 5254 ] .
in addition , increased glutamate signalling mainly at ampa receptors has been found to be a possible downstream effect of nmdar blockade [ 38 , 55 ] .
hence , refinements of the glutamate hypothesis postulate that behavioural and cognitive symptoms of schizophrenia appear to be caused by a dysregulation of glutamatergic neurotransmission , characterised by nmdar hypofunction and subsequent excess glutamatergic activity . evidence for glutamatergic abnormalities in schizophrenia comprises findings from a range of animal models and human methodologies . in rodents , higher cortical glutamate levels after nmdar antagonist injections into thalamic structures
have been linked to cortical neurotoxic changes reminiscent of grey matter volume reductions in patients with schizophrenia [ 49 , 56 , 57 ] . in addition ,
persistent , long - term cognitive impairments similar to cognitive dysfunction in schizophrenia have been observed in rodents as a result of nmdar antagonist - induced neurotoxicity in prefrontal cortical areas . despite lacking evidence for abnormal cerebrospinal fluid glutamate levels in patients with schizophrenia [ 48 , 59 ] , recent advances in neuroimaging
employed single photon emission tomography ( spet ) in unmedicated patients with schizophrenia and demonstrated a reduction in nmda receptor binding compared to healthy controls in hippocampal areas .
a number of proton magnetic resonance spectroscopy ( h - mrs ) studies have found increased levels of prefrontal glutamatergic neurotransmission in unmedicated patients during early disease stages , which appeared to be normalised in chronic patients [ 41 , 6163 ] , reflecting a possible influence of antipsychotic treatment and/or disease progression on glutamate signalling .
structural abnormalities of nmda receptors in schizophrenia as well as reduced levels of the endogenous neurotransmitter d - serine have further been documented [ 64 , 65 ] . as either glycine or d - serine
is required as a coagonist with glutamate for channel opening at the nmdar glycine site , d - serine levels appear to reflect glutamatergic neurotransmission .
finally , a number of possible candidate risk genes for schizophrenia involved in glutamate signalling have been proposed , including polymorphisms of several nmdar subunits and allelic variations of mglu receptors [ 6770 ] .
several unifying approaches have been made in recent years to integrate the evidence for glutamatergic abnormalities and dopamine in the pathogenesis and current treatment of schizophrenia [ 34 , 42 , 71 , 72 ] .
a current , widely supported theory states that dopaminergic imbalances in striatal and cortical areas are preceded and modulated by nmdar hypofunction in the pfc . in turn
, the arising dopaminergic dysregulation might further disrupt glutamatergic signalling at nmda receptors [ 39 , 73 , 74 ] .
dopaminergic dysregulation might thus be the result of primary impairments of glutamate neurotransmission and a subsequent reinforcing factor in maintaining these impairments .
in fact , lower glutamate levels in hippocampal areas of individuals in the prodromal states of schizophrenia , but not in healthy controls , have been found to be linked to increased dopaminergic neurotransmission .
several compounds approved for other indications , however , have been identified as additionally affecting glutamate neurotransmission .
lamotrigine , an anticonvulsant , inhibits glutamate release by acting on presynaptic sodium channels and has been experimentally used as an adjunct treatment in schizophrenia .
although a cochrane review concluded with a lack of robust evidence for its efficacy , a more recent meta - analysis suggests moderate effects for use in patients with suboptimal response to clozapine .
another anticonvulsant , topiramate , exhibits antagonistic actions at ampa and kainate receptors and has been used to augment standard antipsychotic treatment , though its efficacy is modest [ 82 , 83 ] .
there is similarly some evidence for the tetracycline antibiotic minocycline , which exhibits neuroprotective actions by depressing glutamate - induced excitotoxicity [ 8487 ] . nevertheless , current data is underwhelming , and further large - scale randomised controlled trials into all these agents are required .
the development of novel drugs with antipsychotic properties through direct binding on the glutamate site of the nmda receptor has proven challenging . its wide distribution in the central nervous system associated with decreased tolerability and
the potentially neurotoxic effects of receptor overactivation have stimulated the search for compounds more indirectly affecting glutamatergic signalling [ 36 , 88 ] .
attempts to develop nmda receptor enhancing treatments have most notably focused on full agonists of the glycine modulatory site of the nmdar glycine and d - serine as well as sarcosine , a glycine transporter type 1 ( glyt1 ) inhibitor .
discouragingly , the cognitive and negative symptoms in schizophrenia trial ( consist ) found no beneficial effect of using glycine as an add - on treatment in chronic schizophrenia . a recent meta - analysis by tsai and lin found a consistently good tolerability of both glycine and d - serine as adjunct treatments but failed to demonstrate more convincing evidence for their efficacy than moderate effect sizes for negative symptoms and small effect sizes for positive and cognitive symptoms .
interestingly , no additional benefits could be observed for patients receiving clozapine treatment , possibly due to clozapine 's action as a partial nmdar agonist and the generally later disease stage in this group of patients [ 91 , 92 ] . whilst evidence to date for adjunctive
d - serine has been weak , there is some data to indicate this might be due to inadequate dosing .
trials of indirectly increasing d - serine by inhibiting the metabolising enzyme d - amino acid oxidase ( daao ) have not yet advanced to clinical phases [ 94 , 95 ] .
an alternative approach to enhancing glycine levels is the inhibition of glycine reuptake via type 1 glycine transporters ( glyt1 ) .
sarcosine , the endogenous glyt1 inhibitor , has demonstrated efficacy in alleviating positive , negative , and general symptoms of schizophrenia as an adjunct treatment to nonclozapine antipsychotics , both in the acute phase and in chronically ill patients [ 98 , 99 ] .
lane et al . investigated sarcosine as monotherapy for schizophrenia and found some efficacy , with 2 g / day producing stronger symptom reduction than 1 g / day , but lacked placebo as well as active control groups .
while no published results are available for the selective glyt1 inhibitor org25935 developed by merck ( phase ii completed ) , roche 's compound rg1678/ro4917838 has entered phase iii testing after moderately beneficial results in reducing positive and negative symptoms as an adjunct treatment were reported [ 101 , 102 ] .
a multitude of potential new antipsychotic drugs target the less widely distributed metabotropic glutamate receptors ( mglurs ) as agonists or positive allosteric modulators ( pams ) .
selective agonists at mglu2/3 receptors are thought to exert their antipsychotic actions by limiting glutamate release presynaptically and have been shown to attenuate the effects of nmdar antagonists in humans [ 38 , 103 ] .
animal models have suggested reduced pcp - induced dopamine signalling in striatal areas and increased dopaminergic neurotransmission in the pfc after administration of mglu2/3 agonists [ 56 , 104 , 105 ] . as a result , compounds of this type have been predicted to alleviate negative , cognitive , and positive symptoms .
monotherapy with pomaglumetad methionil ( ly2140023 , an oral prodrug of ly404039 ) developed by eli lilly demonstrated superior efficacy to placebo and equal results to olanzapine in reducing positive and negative symptoms of schizophrenia in a phase ii trial while being well - tolerated .
these findings could not be replicated in a follow - up trial as neither pomaglumetad nor olanzapine showed greater efficacy than placebo while the incidence of a number of adverse events limited the drug 's tolerability . nevertheless , the compound has recently entered phase iii for long - term testing and head - to - head comparisons with atypical antipsychotics ( http://www.clinicaltrials.gov/ ) .
in contrast to mglu2/3 agonists , selective agonists at mglu5 receptors which are located mainly postsynaptically and are thought to modulate nmdar function by increasing nmdar - mediated current have not yet reached clinical stages . while the potential of alleviating cognitive symptoms in schizophrenia via mglu5r agonism has stimulated interest in this target , full agonistic action at orthosteric sites of mglurs ( that is , primary sites of endogenous ligand binding ) has generally been associated with receptor downregulation and an increased risk of neurotoxic effects [ 109111 ] .
consequently , a number of compounds targeting the allosteric site of mglurs have been developed , some of which have reached clinical testing phases . as binding to an allosteric site only modulates the receptor in the presence of an endogenous ligand therefore causing transient and activity - dependent changes
several generations of selective positive allosteric modulators at mglu5 receptors ( mglu5r pams ) have shown potential in animal models for their antipsychotic properties [ 114 , 115 ] .
most notably , potential reductions in all symptom areas of schizophrenia have been suggested for the compound adx-47273 currently in preclinical development by addex [ 116 , 117 ] and transition into clinical testing is awaited .
although an mglu2/3 receptor pam compound ( azd8529 ) , developed by astrazeneca , has recently completed a phase ii trial , no reports have been published to date and it is unclear if further development of this drug can be expected .
encouraging results from preclinical studies , however , have suggested antipsychotic potential for selective positive allosteric modulators at the mglu2 receptor . as growing evidence from animal studies has emphasised the critical role of the mglu2 receptor in mediating the clinical effects of this group of compounds [ 118 , 119 ] , potent selectivity for this subtype might yield increased efficacy .
notably , strong potential for antipsychotic action has been demonstrated for the two major prototypes , ly487379 and biphenylindanone a ( bina ) , resembling results of the aforementioned orthosteric mglu2/3 agonists [ 120122 ] .
in addition to targeting nmdar and metabotropic glutamate receptors , several research efforts have focused on pams for the ionotropic ampa receptor , so - called ampakines .
based on animal models of improved memory , attention and learning through enhanced glutamatergic signalling [ 123 , 124 ] , the ampa pam prototype cx516 , developed by shire and servier and cortex , showed preliminary evidence in clinical trials for enhancing cognitive function as an adjuvant treatment in schizophrenia . in line with the failure to reproduce these results in a larger trial , phase
ii trials of the related compound farampator ( org24448 ) developed by cortex were terminated and results have not been published .
while it remains unclear whether research into these agents for the treatment of schizophrenia will be continued , a number of nonglutamatergic targets indirectly modulating the glutamate system have shown promise .
in particular , potential antipsychotic properties have been preliminary demonstrated for the nonhallucinogenic cannabinoid cannabidiol ( cbd ) [ 128130 ] as well as the selective muscarinic m1/m4 receptor agonist xanomeline in development by lilly [ 131 , 132 ] .
further preclinical work is required , however , to evaluate their suitability for use as antipsychotic treatment .
although supported by a large body of evidence and dominating decades of research into aetiology and treatment , the dopamine hypothesis of schizophrenia insufficiently accounts for the complexities of the disorder .
both generations of dopaminergic antipsychotics leave a substantial proportion of patients suboptimally treated , particularly regarding negative and cognitive symptoms , as well as having significant side effects that limit patient concordance with treatment .
the clinical and financial costs of psychosis are enormous and the need for novel antipsychotic compounds is acute .
better understanding of the neuropathology of schizophrenia has highlighted the involvement of alternative neurotransmitter systems and , in particular , the potential of the glutamate synapse for providing new pharmacological targets has arisen .
abnormal glutamate neurotransmission has been linked to the prodromal phase of schizophrenia and early psychotic episodes and with the frequently treatment refractory processes of negative and cognitive symptoms .
consequently , compounds aiming at restoring glutamatergic dysregulation could provide relief for symptoms not optimally treated by current antipsychotic drugs , intervene in a potentially critical disease stage and have downstream effects on dopaminergic neurotransmission . a variety of glutamatergic agents in development are currently undergoing preclinical and clinical testing .
a number of compounds licensed for medical conditions , several nmdar enhancing drugs as well as different mglu agonists and pams have accumulated preclinical and , in part , early clinical evidence for use in antipsychotic treatment .
most notably , a glyt1 inhibitor and an agent modulating glutamate signalling through selective agonistic action at mglu2/3 receptors have shown promising antipsychotic efficacy as well as favourable side effect profiles and have recently been entered into phase iii trials . while these encouraging advances , together with the emergence of new potential agents , instil confidence in a timely identification of novel antipsychotic drugs , moderate or inconclusive results of several compounds emphasise the need for further large - scale , high - quality research efforts . in particular , efficacy of promising candidate drugs , their role in antipsychotic monotherapy or adjunct treatment as well as their long - term safety and tolerability require confirmation and clarification . | the dopamine hypothesis of schizophrenia remains the primary theoretical framework for the pharmacological treatment of the disorder . despite various lines of evidence of dopaminergic abnormalities and reasonable efficacy of current antipsychotic medication , a significant proportion of patients show suboptimal treatment responses , poor tolerability , and a subsequent lack of treatment concordance . in recent decades ,
intriguing evidence for the critical involvement of other neurotransmitter systems in the pathophysiology of schizophrenia has emerged , most notably of dysfunctions within the glutamate pathways .
consequently , the glutamate synapse has arisen as a promising target for urgently needed novel antipsychotic compounds particularly in regards to debilitating negative and cognitive symptoms poorly controlled by currently available drugs . in this paper ,
recent findings integrating glutamatergic and dopaminergic abnormalities in schizophrenia and their implications for novel pharmacological targets are discussed .
an overview of compounds in various stages of development is given : drugs enhancing nmda receptor function as well as metabotropic glutamate receptor ( mglur ) agonist and positive allosteric modulators ( pams ) are emphasised .
together with other agents more indirectly affecting glutamatergic neurotransmission , their potential future role in the pharmacotherapy of schizophrenia is critically evaluated . |
to cope with an ever - increasing number of published scientific articles that are spread over a likewise growing number of scientific journals , new effective and easy - to - use tools are required to support scientists to stay up - to - date with the new findings in their specific area of research as well as to give a liable overview when entering a new scientific field ( 13 ) . the ncbi pubmed - medline service ( )
provides a comprehensive resource of freely available scientific abstracts in the area of biomedical research .
therefore , it is well - suited to develop and test new approaches in text mining methodologies .
normal keyword searching is the most frequently applied way to query the medline database ( 4 ) .
useful abstracts are often buried in hundreds of less relevant papers and it is often not easy to evaluate if a retrieved abstract matches well with the desired topic . even for very specific subjects ,
it is not always clear how to narrow the search to focus on the most relevant papers . assuming a scientist is interested in the role of heparin as a potential drug in cancer treatment and queries the pubmed server with the terms heparin and cancer , this search currently returns more than 2000 references , which encompass many biological events of heparin like inflammation , blood coagulation and cancer .
several services exist where information provider like pubmed , biomail , jade , pubcrawler , ovid and sciencedirect notify their users periodically of new publications , report literature or other sources of information on subjects in which the users have specified their interest .
xplormed ( 6 ) ( ) is a web tool that aids medline searching by summarizing the subjects contained in the results of a pubmed keyword search .
liu and altman describe a program for incremental updates of a bibliography using the pubmed related articles function .
this approach is able to select a very narrowly defined set of articles from the literature ( 7 ) .
the aim of the pubfinder service ( ) is to support scientists to retrieve a higher rate of relevant papers for the subject they are interested in and thus reduce the necessary time to sort out many less relevant articles .
basic requirements for the broad acceptance of such an approach are : ( i ) the search must be easy to set up , ( ii ) minimal user interaction should be required , ( iii ) no special knowledge concerning the query - syntax should be necessary and ( iv ) the whole procedure should be highly automized . to be able to optimize search queries , the service should provide tools to evaluate , why a certain abstract has been highly ranked . additionally , the service should provide a convenient way to improve search queries based on previous runs .
the statistical approach is used to scan abstracts taken from pubmed - medline and to select and rank just those abstracts discussing a certain topic : first , a reference dictionary was generated containing the frequencies of the 100 000 most common words contained in all abstracts available through pubmed since 1990 . in a second step , discriminating words are derived from a set of user defined abstracts discussing the topic of interest the training set that appear at unexpectedly high or low frequencies compared with the reference dictionary . the frequency of occurrence of each word contained in the training set is compared with the frequency in the reference directory .
the first 100 words exhibiting the highest difference in occurrence between both data sets are assigned as discriminating words for a specific topic . in a third step
all selected abstracts are scanned for all discriminating words and scored for their likelihood of discussing the given topic .
the user has the possibility to display the automatically derived list of discriminating words and their frequencies in the training set ( see table 1 ) . in a fourth step after having scanned all defined volumes of abstracts a hit list of references in descending order of their likelihood score is presented .
a direct link to the corresponding pubmed page , information about authors , title and bibliographic data is given for each entry .
the above outlined algorithm was written in perl and is processed on a pc - linux cluster that currently consists of 11 standard pcs , each equipped with two processors ( 1.67 mhz ) , resulting in 22 cluster nodes . depending on the number of free processors a complete year of pubmed abstracts , which include roughly half a million abstracts , can be scanned in 13 min .
all administrative tasks to run the service are handled automatically or at least in a semi - automatic way , so that only little human supervision is required . to be able to efficiently scan all pubmed abstracts they were downloaded and stored in a local database ( starting from 1990 ) , skipping abstracts whose total length does not exceed 100 characters .
this daily update process takes < 5 min . the overall storage space required per stored pubmed year averages 650 mb , including the abstracts ' text , author information and additional data provided by pubmed .
a user registration is required to manage the access to queries and results at any time .
all scan results are stored and the short topic description is used to recall former queries at any time .
an online help and a collection of faqs ( ) provide a quick overview on how to use the pubfinder web service .
the user has to provide a list of abstracts ( indicated by pubmed - ids , see figure 1 ) , which describe well the scientific topic to be looked for .
however , the rule the more , the better is of course true and the number of highly relevant articles will increase with increasing homogeneity of the provided set of abstracts . a very small number of selected abstracts or the choice of a thematically inconsistent set of abstracts normally results in low scores .
the most practical way to include pubmed - ids is to scan for references using the text - mining tools provided by pubmed and copy - paste the ids of references , which are regarded as relevant into the input sheet of the new - scan window . after having selected a sufficient number of abstracts a meaningful title has to be provided , which will be subsequently used to identify and manage different queries .
having submitted the search , the status of the scan process can be pursued by a progress meter , which indicates the percentage of already performed search time of the overall estimated computing time .
depending on the free resources on the pc - cluster and the number of volumes of pubmed abstracts to be scanned , a search may take several minutes up to 1 or 2 h. the current load of the pc - server is always displayed .
the user will be informed by email as soon as a search has been finished . as result a hit list of references in descending order of their likelihood score
the number of retrieved relevant papers depends on the intensity a certain scientific question is investigated and thus the number of available publications . if a search reveals only references having low scores , this finding is a clear indication that the selected set of publications is too inhomogeneous and does not mimic a scientific topic well .
pubfinder offers the option to restart a search by indicating a certain number of references retrieved in a previous run .
the rescan option has been shown as an efficient way to improve the relevance of the number of retrieved papers .
a suitable procedure is as follows : in a first run 1020 abstracts are selected , which are known to cover a certain scientific subject well . from the retrieved entries
our experience shows , that this procedure works very well as long as consistent sets of references are selected .
confronted with an ever - increasing number of published scientific articles , new advanced , automatic and easy to use tools are required to improve the rate of retrieved publications that are relevant .
the algorithms and procedures implemented in pubfinder are capable of facilitating the task of staying up - to - date with current scientific knowledge for specific subject in biomedicine .
the required selection of a representative set of references that are central for a scientific topic should be easily manageable for scientists .
normally they are well aware of the key publications in their specific field and the required pubmed - ids can be easily retrieved and copied to pubfinder . in case researchers want to gain an overview in a new field , they can use the information extraction procedures provided by pubmed and thus select a preliminary set of relevant papers . the pubfinder option to restart searches allows beginning with a crude selection of papers to indicate more relevant articles from the retrieved references and thus optimize the set of relevant discriminating words consecutively .
a rescan needs only modest user interaction just an indication of relevant papers as the rest of the procedure runs completely in an automatic mode . additionally , to support the optimization process
, pubfinder displays a complete list of retrieved discriminating words and their ranking , which may help to evaluate how to optimize a query . with
the related articles function pubmed offers a similar service , which also uses a purely statistical approach .
it is able to select a very narrowly defined set of articles from the literature ( 9 ) .
the pubmed related articles function calculates the similarity of a given article with all other documents in pubmed mainly evaluating on the occurrence of terms in two articles and the rate of all abstracts , which contain the certain term . to allow an interactive response time
, closely related documents are pre - computed for each document in pubmed so that the service has only to recall this list . as described above , pubfinder uses discriminating words , derived from a user definable set of articles relevant for a specific topic , which are subsequently used to score all other abstracts .
as can be expected from the different kind of scoring functions applied , both approaches produce varying but complementary results . a detailed analysis and comparison of both approaches
it has turned out to be a very useful procedure , to define a set of relevant articles to start a pubfinder run by using the related articles function .
the immediate response time of the ncbi service is definitively one of its strong points .
however , this gain in speed has to be paid by an inflexibility , which does not allow influencing the criteria to retrieve similar articles .
in contrast , the user interoperability of pubfinder is one of its strong points allowing users to subsequently optimize the search queries according to their specific needs .
this method may also assist curators in acquiring pertinent literature in building biomedical databases dealing with specific topics ( 7 ) .
the list of discriminating words generated by pubfinder may also help to support the process of deriving a catalogue of meaningful objects describing a domain of scientific interest as required to set an ontology ( 10 ) .
therefore , the initial derivation of an optimal set of discriminating words , which demands scanning several volumes of pubmed abstracts , may require a considerable amount of computing time .
however , to keep up - to - date would not take up much time as only a relatively small amount of newly published abstracts would have to be reviewed .
after having provided a short description and selecting the starting year of the query , the user has to enter a set of pubmed ids , which represent the scientific topic to be scanned for . in this example we chose a set of 28 pubmed abstracts that deal with literature mining .
the scanned articles are listed in descending order according to their likelihood of discussing the user 's given topic .
the score is calculated as stated in ( 8) and represents the logarithm of the likelihood of belonging to the topic or not .
the 20 words that most discriminate abstracts of the example training set ( literature mining , see figure 1 ) from other non - related abstracts | since it is becoming increasingly laborious to manually extract useful information embedded in the ever - growing volumes of literature , automated intelligent text analysis tools are becoming more and more essential to assist in this task .
pubfinder ( ) is a publicly available web tool designed to improve the retrieval rate of scientific abstracts relevant for a specific scientific topic .
only the selection of a representative set of abstracts is required , which are central for a scientific topic .
no special knowledge concerning the query - syntax is necessary .
based on the selected abstracts , a list of discriminating words is automatically calculated , which is subsequently used for scoring all defined pubmed abstracts for their probability of belonging to the defined scientific topic .
this results in a hit - list of references in the descending order of their likelihood score .
the algorithms and procedures implemented in pubfinder facilitate the perpetual task for every scientist of staying up - to - date with current publications dealing with a specific subject in biomedicine . |
the fraction of no in exhaled air ( feno ) has been shown to be related the well to degree of eosinophil inflammation in the airways .
several studies have reported good correlations between feno levels and sputum eosinophils , as well as degree of bronchial hyperreactivity ( bhr ) .
accordingly , feno has been shown to be increased in asthma and also to discriminate subjects with asthmatic disease from those without [ 5 , 6 ] .
several studies suggest a moderate association between asthma control and concentration of no in exhaled air [ 7 , 8 ] . the mechanism behind the increased feno levels is believed to be related to an upregulation of inducible no - synthase on bronchial epithelial cells , which in turn is caused by a local activation of inflammatory cytokines in the airways .
this phenomenon is especially evident in allergic airways diseases which a recent study reporting decreased feno after three months of inhaled corticosteroid ( ics ) therapy in atopic asthmatics gives indirect evidence of . in clinical practise
, it appears that feno measurements can be of value both to diagnose asthma , and to monitor effect of asthma therapy such as ics longitudinally . in a study conducted on 47 consecutive cases referred for suspicion of asthma , feno and sputum eosinophilia proved to be the two best methods to discriminate between presence of disease or not
. however , there is still debate about the proper place for feno in the clinic and what levels of no that represent ongoing pathology in the airways in epidemiological studies , where the study population differ substantially from that in hospital - based studies , feno may be an important tool to discriminate subjects with airway inflammation supplementing respiratory questionnaires to improve validity .
we have previously shown that feno is related to atopy , height , and age in a random population study .
this indirectly indicates that feno is related to eosinophilic airway inflammation in a random population , but this has never been examined further . in that study
we also found a number of subjects with high feno without asthma or respiratory symptoms questioning the relation to eosinophilic airway inflammation .
a number of substances measured in exhaled breath condensate ( ebc ) have also been suggested as noninvasive markers of airway inflammation .
here , we have focused on measurement of markers for oxidative stress in ebc , such as hydrogen peroxide and malondialdehyde , the latter a marker of lipid peroxidation , and nitrotyrosine . for these substances ,
nitrotyrosine is regarded as marker for peroxynitrite formation ( onoo ) , a very short - lived and potent free radical , that previously has been shown to be increased in subjects with asthma although measured with a different method based on specific enzyme immunoassay ( eia ) . in the present study
, we investigated whether high feno levels found in a subgroup of subjects from a large population - based cohort could be used as a signal for inflammatory airways disease .
we also asked whether high feno found in this context was related to inflammatory cells and biomarkers in bronchoalveolar lavage and exhaled breath condensate ( ebc ) .
the study was performed within the followup in gothenburg of the european community respiratory health survey ( http://www.ecrhs.org/ ) , which design have been described elsewhere . shortly , during 19911993 , a random sample of 3600 subjects aged 20 to 44 years were asked to complete a short respiratory questionnaire by mail . among the responders a small random sample and all subjects affirming certain respiratory symptoms ( enriched sample )
were clinically investigated ( n = 682 ) . at followup , 1998 to 2002 , ( ecrhs ii ) the small random sample and the enriched sample were asked to participate in a clinical investigation . in gothenburg , the followup was attended by 548 subjects .
all completed a detailed questionnaire on details of respiratory symptoms , performed standard lung function test including spirometry and methacholine challenge , and serum ige levels ( elisa ) were recorded .
atopy was defined as presence of specific ige antibodies to any of the following allergen ( house dust mite , cat , timothy grass , and a local allergen ) as previously described .
feno was measured with aerocrine equipment and single breath maneuver , using an exhalation flow of 50 ml / s in accordance with ats criteria .
feno was measured in 295 subjects from the random sample and in 75 subjects from the enriched sample .
the feno levels were remeasured the morning before the subjects underwent bronchoscopy and used for all calculations .
subjective symptoms of allergic rhinitis or asthma were recorded , and any concomitant medication was registered . for or
the present study active smokers , ex - smokers < 10 years , and subjects using peroral steroids were excluded .
individuals with levels of feno higher than the 85th percentile ( > 30.3 ppb ) were identified and asked to participate in the study ( n = 30 ) .
28 of these subjects and 31 randomly selected subjects with normal feno ( feno 2575 percentile corresponding to 917 ppb ) were participating in an initial examination including spirometry , collection of exhaled breath condensate , and blood sampling .
19 of these subjects ( 9 with high feno ) were also willing to undergo bronchoscopy and were included in the present study .
exhaled breath condensate ( ebc ) was collected using the ecoscreen breath condenser ( jaeger ; wrzburg , germany ) . in short , after rinsing the mouth with purified water , the subjects performed tidal breathing through a mouthpiece attached to a two - way nonrebreathing valve . a saliva trap was connected in order to avoid contamination from saliva , and a nose clip prevented nose breathing during sampling .
the exhaled air passed through a cooling system consisting of a lamellar tube and an attached sample container . after collection , the ebc volume was determined gravimetrically , and the sample was subsequently centrifuged at 1,200 rpm for 2 min .
ph measurements were performed by deaeration / decarbonation of ebc through argon bubbling for five minutes , followed by ph determination using a minielectrode attached to a ph - meter ( ph/330 gmbh wtw , weilheim , germany ) .
all bronchoscopies were done by the same investigator ( gr ) after standard premedication and topical anaesthesia had been given .
all subjects received nebulized 2.5 mg salbutamol 30 minutes before the procedure , as well as continuous oxygen nasally during the bronchoscopy .
no complications were observed . bronchial wash ( bw ) was first sampled by infusion of 20 ml sterile pyrogen - free phosphate buffered saline ( pbs ) solution into the middle lobe bronchus .
bronchoalveolar lavage ( bal ) was then performed by infusion of 3 50 ml pbs into the same location with the bronchoscope in a wedged position .
the fluid was aspirated after each 50 ml aliquot , pooled in a sterile siliconized container , and immediately transported on ice to the laboratory .
cellular components were sedimented by centrifugation at 4c , 500 g for 10 minutes .
runcorn , uk ) were made from 100 l aliquots of the resuspended cell pellet .
slides were immediately fixed in 96% alcohol and stained with may grunwald giemsa for later identification of cell types on a morphological basis .
percentages of polymorphonuclear granulocytes , eosinophil granulocytes , lymphocytes , and macrophages were calculated in both bw and bal samples by counting 400 cells using a standard light microscope .
eosinophil cationic protein in bal was measured with a commercial pharmacia cap system feia ( pharmacia diagnostics , uppsala , sweden ) following the instructions by the manufacturer .
the study had been approved by the ethical committee of the university of gteborg ( 00 - 230 ) , and all subjects gave their consent to participate after written and oral information .
clinical characteristics and numerical data are expressed as medians and interquartile range ( iqr ) .
cross - sectional analysis of differences between the patient groups were performed with mann - whitney u - test .
thirty subjects agreed to participate in the bronchoscopy study . of these , nine subjects had high levels of feno ( > 85th percentile , median 67 ppb , iqr 33 ppb ) , and 21 were controls with median levels of feno ( 50th percentile , median 17 ppb , iqr 7 ppb ) .
the feno levels reassessed the morning before bronchoscopy correlated well with the previous screening feno ( = 0.95 , p < .0001 ) .
there were no significant differences in age , gender , lung function , serum ige levels , or number of ex - smokers > 10 years between the two groups , see table 1 .
however , the high - feno subjects were significantly more sensitive in methacholine challenge and had significantly more asthma and rhinitis symptoms compared to the normal feno group ( p < .01 ) .
all subjects with high feno had symptoms of allergic airways disease in the form of either asthma and/or rhinitis , and 6 had both . only one control subject had symptoms of both asthma and rhinitis . three subjects with high
feno used inhaled corticosteroids ( median 102 ppb , iqr 50 ppb ) , and one with normal feno ( 11 ppb ) used ics .
feno was significantly increased ( 55 ppb versus 22 ppb , p < .02 , figure 2 ) .
this was also true for subjects with rhinitis symptoms ( 47 ppb versus 21 ppb , p < .01 ) .
a multivariate analysis of the clinical factors and symptoms stated in table 1 using feno as the dependent variable revealed only asthma symptoms to be a significant factor ( p < .05 ) . as
only nine out of thirty subjects with high feno were willing to undergo bronchoscopy , there might be a risk of selection bias , that is , subjects included may have more / less severe disease than those not participating .
there were , however , no difference in lungfunction ( fev1 = 3.79 versus 3.80 l ) or presence of doctors ' diagnosed asthma ( 47% versus 53% ) between participants and nonparticipants although the prevalence of atopy was slightly lower among participants 40% versus 60% among nonparticipants .
subjects with high feno levels had significantly higher median percentage eosinophils both in bw ( 9.0 versus 0.7 , p < .001 ) and in bal compared to the controls ( 2.1 versus 0.6 , p < .006 , table 2 ) .
bal fluid ecp levels were also significantly higher in the high feno group ( mean = 5.3 mg / l versus 2.0
mg / l , median 2.2 mg / l ( iqr 5.1 ) versus 2.0 mg / l ( iqr 0.1 ) , p < .005 ) . in the group with high feno , levels of feno
were significantly associated with percentages of eosinophils in bal ( = 0.78 , p < .02 , figure 1 ) , and ecp in bal ( = 0.65 , p < .05 ) . in all subjects ,
feno was associated with percentage of eosinophils in bal ( = 0.6 , p < .002 , figure 1 ) , and ecp in bal ( = 0.65 , p < .05 ) , but not with age , gender , fev1% predicted , or degree of methacholine sensitivity .
median ebc condensate weight 2.5 g ( iqr 0.7 ) versus 2.7 g ( iqr 0.9 ) and ebc ph did not differ between the two groups . nor were any differences in ebc concentrations of na , nh4 , h2o2 , mda , or 3-nitrotyrosine found ( table 3 ) . in the group with high feno , percentage of bw neutrophils correlated positively with ebc h2o2 concentration ( = 0.86 , p < .05 , figure 3 ) .
in the present study , we found that nine subjects with high feno levels identified in a population study cohort had increased eosinophil airway inflammation .
these subjects also had clinical symptoms of allergic airways disease , either asthma and/or rhinitis , and increased bhr .
interestingly , the majority of the nine subjects were undiagnosed for having asthma , and only three of them had known asthmatic disease and treatment with ics .
the results are of importance for the applicability and interpretation of feno in epidemiological studies .
previous studies , comparing feno with inflammatory markers in bal , have been performed in clinical studies among patients with asthma and healthy controls , which may represent a biased selection , and the results , hence , are not fully transferable to the general population .
the fact that feno also in the general population is strongly associated to eosnophilic inflammation potentates its role as a useful marker for eosinophilic airway inflammation in epidemiological studies .
we have recently presented a large population study , where we found feno to be associated with current symptoms of asthma , especially symptoms within the last month , and that feno are increased in subjects with atopic disease , irrespective of respiratory symptom . in another population - based study , henriksen et al .
have found that suspected asthmatics with both ahr and atopy had the highest levels feno . in several previous clinical studies ,
the association between feno and markers for inflammation in bal has been examined . in a recent study in asthmatic patients using allergen installation during bronchoscopy as a model for local provocation , erpenbeck et al . showed that segmental no correlated well with signs of eosinophil inflammation in the airway .
clinical studies in children with asthma have shown that feno relate to airway eosinophilia in bronchial biopsies , presence of bal eosinophilia , and sputum eosinophilia .
this relationship was also described in a study of adult patients with either asthma or eosinophilic bronchitis , where increased levels of feno correlated well with eosinophils in both sputum , bronchial wash , and bal .
previous results are , however , not unambiguous ; in a recent study by lemiere , feno was associated with mucosal eosinophils from biopsies , but not with sputum eosinophils , in subjects with moderate and severe asthma . in an earlier study by lim et al .
, no correlation between feno and mucosal eosinophils in bronchial biopsies from adult patients with asthma was found .
even though most studies find an association between feno and the percentage of eosinophils in sputum and bal , the explanatory value of the correlations are low in the study by berry et al . , as low as 26% , indicating that factors other than eosinophils are determining feno levels .
a significant correlation of bw neutrophils with concentration of h2o2 in ebc was seen in the group with high feno which could be a signal of ongoing oxidative stress , a phenomenon described in asthma as well as other inflammatory airways diseases .
however , other biomarkers of oxidative stress in ebc were not elevated in the group with high feno .
there are many possible explanations for this , where the high variability seen in the ebc analytical methods and sampling procedure might be the most likely cause .
the results were similar if we corrected the concentrations of the different biomarkers in ebc for differences in dilution of water vapour by the means of the concentrations of sodium and potassium suggested by effros et al . .
our results thus add further support to the hypothesis that feno reflect ongoing allergic airway inflammation and have a potential as a noninvasive diagnostic tool for detecting eosinophilic airway inflammation in epidemiological studies .
g. c. riise carried out the bronchoscopy and bal in all subjects , and drafted the paper .
a. c. olin coordinated the study , examined all subjects clinically , and was responsible for ebc sampling .
| background .
measurement of fraction of exhaled nitric oxide ( feno ) is a promising tool to increase validity in epidemiological studies of asthma .
the association between airway inflammation and feno has , however , only been examined in clinical settings and may be biased by selection of patients with asthma .
methods . in a population study with feno registrations on 370 individuals
, we identified nine subjects out of thirty subjects with high levels of feno ( > 85th percentile , 30.3 ppb ) , irrespective of presence of respiratory symptoms , and 21 control subjects with feno at median levels ( 11.116.4 ppb ) willing to undergo bronchoscopy and bronchoalveolar lavage ( bal ) , all nonsmokers .
feno was measured in accordance with ats criteria , and the examination also included spirometry , methacholine challenge test , and sampling of exhaled breath condensate ( ebc ) .
results .
subjects with high feno levels had significantly higher median the percentage of eosinophils in bal than controls ( 2.1 versus 0.6 , p < .006 ) , and there was a significant association between feno and the percentage of eosinophils in bal ( =0.6 , p < .002 ) and ecp in bal ( =0.65 , p < .05 ) examining the whole group , but no association with gender , fev1 , or degree of metacholine sensitivity or any of the biomarkers in ebc . all subjects with high feno had respiratory symptoms , but only three had diagnosed asthma .
there were a significant association between hydrogen peroxide in ebc and the percentage of neutrophils in bronchial wash .
conclusion .
high feno levels signal asthmatic or allergic respiratory disease in a population - based study .
feno levels are associated with degree of eosinophil airway inflammation as measured by the percentage of eosinophils and ecp in bal . |
pulmonary alveolar microlithiasis ( pam ) is a very rare diffuse and chronic lung disease , characterized by the presence or deposition of small spherical calcium phosphate bodies containing concentric calcareous lamellas in pulmonary alveoli .
although the etiology still remains not clear , pam is considered to have an autosomal recessive pattern caused by mutations of the solute carrier family 34 ( sodium phosphate ) , member 2 gene ( the slc34a2 gene ) , which encodes a sodium phosphate cotransporter .
a 55-year - old man presented to our department with moderate to severe shortness of breath and dry cough that had worsened over the previous few years .
initially he became short of breath during moderate exertion and later he had shortness of breath even at rest .
he denied having any history of recent respiratory tract infection and old chronic infections like tuberculosis .
he did not have history of any chronic drug use or any chronic medical problems apart from the shortness of the breath .
vitals were within normal range , he had coarse crackles mainly at the basal and middle zone of the left lung .
a full blood count revealed an elevated erythrocyte count of 6.05 10 ml and hematocrit 55% .
arterial blood gas analysis values in room air were : ph : 7.43 , pao2 : 51 mmhg , paco2 : 29 mmhg , hco3 : 16 mmol l and o2 saturation 87% .
pulmonary function tests revealed typical features of a restrictive defect with a reduced total lung capacity of 56% and vital capacity of 46% , and a forced expiratory volume in 1 s / forced vital capacity ( fev1 /fvc):100 .
echocardiography showed severe pulmonary hypertension , 82 mmhg , a dilated right heart and moderate tricuspid regurgitation .
moreover , skin tests for tuberculosis and studies for acid - fast bacilli were negative and also there was negative urinalysis .
additionally , the inflammatory markers like esr , crp and even ferritin were within normal range . the plain chest x - ray films revealed a diffuse symmetric dense , micronodular ( sand - storm ) pattern mainly on the left side [ figure 1 ] .
a high - resolution ct ( hrct ) scan was obtained in the supine position at maximal inspiration at 10 mm intervals .
all scans were photographed with window / level settings of 770/10 , 1730/2810 and 1910/2500 hounsfield units .
the examination showed diffuse symmetric lung abnormalities characterized by ground - glass attenuation and septal thickening with calcified nodules , resulting in a crazy - paving pattern .
the patient had a transbronchial lung biopsy and histology revealed round , concentrically laminated microliths in the alveoli , which were periodic acid - schiff ( pas ) positive in keeping with the diagnosis of pam .
he was treated with alendronate 70 mg once weekly , steroid initially as prednisolone 30 mg / day reduced over a month to 10 mg / day , with further reduction over coming months until we stopped it completely and the total duration for steroid use was 1 year , he is still on alendronate .
furthermore , with the current treatment his pft ( fev1 and vc ) improved by 10% after a year of treatment .
illustrates the plain chest x - ray films which revealed a diffuse symmetric dense , micronodular ( sand - storm ) pattern mainly on the left side the hrct scan of the chest showed diffuse symmetric lung abnormalities characterized by ground - glass attenuation and septal thickening with calcified nodules , resulting in a crazy - paving pattern .
the main feature of pam is widespread laminated calcipherites in the alveolar spaces in the absence of any known disorder of calcium metabolism .
patients may remain asymptomatic for many years and do usually become symptomatic between the third and fourth decades .
the clinical presentation usually illustrate a restrictive pattern of lung disease as seen in our patient . in the early course of the disease pneumothorax may be observed .
adult patients commonly show progressive deterioration of the pulmonary function and death usually occurs in mid - life because of respiratory failure associated with cor pulmonale .
the chest radiograph usually shows small innumerable , widespread , and calcified nodules , which tend to be more at the base .
hrct scan has assisted in characterization of the imaging of the disease and can be beneficial in diagnosis .
ground - glass opacities due to volume averaging of tiny microliths are a common and major finding in the literature .
although the imaging can help in diagnosing the disease but the lung biopsy is the best for definitive diagnosis .
recommend that ( 18 ) f - sodium fluoride pet / ct is a superior modality in characterization and assessment of the extent of disease in pam compared to all other non - invasive imaging modalities thus ( 18 ) f - sodium fluoride pet / ct should be the investigation of choice in pam .
there is no known effective treatment for pam , with the exception of lung transplantation . to our knowledge ,
observational studies have found therapeutic bal to be ineffective , except for symptomatic improvement in one case , without improvement in conventional pulmonary radiography and hrct . | pulmonary alveolar microlithiasis ( pam ) is a very rare diffuse chronic lung disease characterized by deposition of small spherules of calcium phosphate within the alveolar cavity .
the disease is usually seen from birth up to 40 years of age and is usually diagnosed incidentally during radiography of the chest for other reasons .
most of patients are asymptomatic or having very mild symptoms and the majority of patients either have normal or restrictive pulmonary function test .
clinically , the course of the disease is different ; it remains static in few patients or it may progress to pulmonary fibrosis , respiratory failure and cor pulmonale in others . in this case report , we present a 55-year - old man who presented with moderate shortness of breath which has progressed from mild symptoms with in the previous years .
his chest high - resolution ct scan showed diffusely scattered , ill - defined little shadowy micronodules which involve the left lung ; lingula and left lower lobe in particular .
a lung biopsy confirmed the diagnosis of pam .
he was followed up for 1 year with treatment by steroid and alendronate , and no progression was noticed in fact improvement in pulmonary function test noticed .
this is the first case report of pam in kurdistan . |
post - fusion lumbar flatback deformity with global sagittal imbalance causes gait disturbances , chronic low back and refered leg pain , and reduced quality of life .
the incidence of patients requiring lumbar corrective osteotomy for post - fusion lumbar flatback deformity with sagittal imbalance is increasing as the numbers of spine fusion surgeries and overall average life expectancy increases12 ) . in patients with sagittal imbalance ,
adequate correction to restore an ideal lumbar lordosis relative to pelvic incidence ( pi ) in the sagittal plane is very important during corrective osteotomy3 ) . according to schwab et al.15 ) ,
corrective osteotomy for adult spinal deformity ( asd ) leads to good sagittal spinal balance if the range of correction is within lumbar lordosis ( ll)=pi9 , pelvic tilt ( pt)<20 , and sagittal vertical axis ( sva)<5 cm .
however , patients with post - fusion lumbar flatback are generally osteoporotic , elderly females who have poor paraspinal muscle condition due to fatty degenerative changes , atrophy of the back muscles , and thinning of the paraspinal and psoas muscles .
therefore , restoration of lumbar lordosis below the pi value might result in another ( or secondary ) postoperative or delayed sagittal imbalance due to the lack of the compensatory roles from these paraspinal muscles .
these studies suggested that ideal lumbar lordosis consists of 67% ll at l4s1 , 85% ll at l3s1 , and 97% ll at l2s1 .
the degree of pi was categorized into 6 groups ( i to vi ) , and theoretical ll and pt values were recommended for each group14 ) . in this study
, radiographical outcomes of post - fusion lumbar flatback patients treated with pedicle subtraction osteotomy ( pso ) or smith - petersen osteotomies ( spos ) with alifs were analyzed and compared at preoperative , postoperative 1 month , and at the final follow up and achieved mean pso angles were evaluated .
clinical outcomes by oswestry disability index ( odi ) , visual analog scale ( vas ) score of back pain / leg pain , and scoliosis research society-22 score ( crs-22r ) were analyzed and compared . at the final follow - up ,
all patients were divided into 2 groups ( sva 5 cm : normal sva , sva > 5 cm : positive sva ) and compared with both groups by according to radiological and clinical outcome parameters .
this was a retrospective study of patients with post - fusion lumbar deformity with sagittal imbalance .
twenty - eight patients ( 24 women and 4 men ) who underwent corrective osteotomies for post - fusion lumbar flatback deformity between 2008 and 2014 at our institution were enrolled .
the mean age at the time of surgery was 658 years ( range , 4775 years ) .
the mean follow - up period was 3018 months ( range , 1272 months ) .
the inclusion criterion was corrective fusion surgery that involved more than 4 intervertebral levels . to obtain adequate lumbar lordosis in the patients with post - fusion lumbar flatback deformity , pso or multilevel spos with lower lumbar alifs
all surgical treatments were performed with the staged posterior - anteriorposterior ( p - ap ) , anteriorposterior ( ap ) , or posterior ( p ) only approach .
upper instrumented vertebra ( uiv ) were at t7 ( 1 case ) , t8 ( 4 ) , t9 ( 4 ) , t10 ( 6 ) , t11 ( 7 ) , t12 ( 4 ) , and l1 ( 2 ) .
lower instrumented vertebra ( liv ) were at l5 ( 1 ) , s1 ( 10 ) , and the iliac ( 17 ) .
1 ) at l2 ( 4 ) , l3 ( 8) , and l4 ( 7 ) .
the achieved mean angle of pso was 30.14.8 ( 21 to 40 ) ( fig .
the other 9 cases underwent posterior lower lumbar polysegmental spos followed by anterior lower lumbar alifs ( fig .
2 ) ( table 1 ) . the data collected consisted of health - related quality of life ( hrqol ) questionnaires and demographic , radiographic , and clinical information .
sagittal spinal and pelvic parameters on whole spine standing x - rays were measured at baseline , postoperative one month , and final follow - up .
the following parameters were assessed : pelvic measurements , including pi , pt , and sacral slope ; regional measurements , including thoracic kyphosis ( tk : t5 to t12 ) , thoracolumbar cobb angle ( tl : t10 to l2 ) , and lumbar lordotic angle ( ll : l1 to s1 ) ; and global measures , including the sva , t1 pelvic angle ( t1pa ) , and pi - ll .
the pi , tk , tl , ll and sva , pi - ll , t1pa at preoperative , postoperative 1 month , and at the final follow up were assessed . achieved mean pso angles were also evaluated . at the final follow - up , all patients were divided into 2 groups ( sva 5 cm : normal sva , sva > 5 cm : positive sva ) and compared with both global sagittal and clinical outcome parameters .
clinical outcomes were evaluated with the odi and a vas for back and leg pain , and the srs-22r questionnaire .
complications were categorized into early perioperative or late complication , and revision surgeries were reviewed .
means and standard deviations were calculated and compared for continuous variables among preoperative , postoperative one month , and at the final follow - up periods .
comparison of parameters between the normal and positive sva groups was performed using mann - whitney test .
non parametric tests including wilcoxon signed rank test and frideman test were used for comparing radiological parameter over time and health - related quality of life between the preoperative and final .
all statistical analyses were performed using spss 18.0 ( ibm co , ny , usa ) .
this was a retrospective study of patients with post - fusion lumbar deformity with sagittal imbalance .
twenty - eight patients ( 24 women and 4 men ) who underwent corrective osteotomies for post - fusion lumbar flatback deformity between 2008 and 2014 at our institution were enrolled .
the mean age at the time of surgery was 658 years ( range , 4775 years ) .
the mean follow - up period was 3018 months ( range , 1272 months ) .
the inclusion criterion was corrective fusion surgery that involved more than 4 intervertebral levels . to obtain adequate lumbar lordosis in the patients with post - fusion lumbar flatback deformity , pso or multilevel spos with lower lumbar alifs
all surgical treatments were performed with the staged posterior - anteriorposterior ( p - ap ) , anteriorposterior ( ap ) , or posterior ( p ) only approach .
upper instrumented vertebra ( uiv ) were at t7 ( 1 case ) , t8 ( 4 ) , t9 ( 4 ) , t10 ( 6 ) , t11 ( 7 ) , t12 ( 4 ) , and l1 ( 2 ) .
lower instrumented vertebra ( liv ) were at l5 ( 1 ) , s1 ( 10 ) , and the iliac ( 17 ) .
1 ) at l2 ( 4 ) , l3 ( 8) , and l4 ( 7 ) .
the achieved mean angle of pso was 30.14.8 ( 21 to 40 ) ( fig .
the other 9 cases underwent posterior lower lumbar polysegmental spos followed by anterior lower lumbar alifs ( fig .
the data collected consisted of health - related quality of life ( hrqol ) questionnaires and demographic , radiographic , and clinical information . sagittal spinal and pelvic parameters on whole spine standing x - rays were measured at baseline , postoperative one month , and final follow - up .
the following parameters were assessed : pelvic measurements , including pi , pt , and sacral slope ; regional measurements , including thoracic kyphosis ( tk : t5 to t12 ) , thoracolumbar cobb angle ( tl : t10 to l2 ) , and lumbar lordotic angle ( ll : l1 to s1 ) ; and global measures , including the sva , t1 pelvic angle ( t1pa ) , and pi - ll .
the pi , tk , tl , ll and sva , pi - ll , t1pa at preoperative , postoperative 1 month , and at the final follow up were assessed .
achieved mean pso angles were also evaluated . at the final follow - up , all patients were divided into 2 groups ( sva 5 cm : normal sva , sva > 5 cm : positive sva ) and compared with both global sagittal and clinical outcome parameters .
clinical outcomes were evaluated with the odi and a vas for back and leg pain , and the srs-22r questionnaire .
complications were categorized into early perioperative or late complication , and revision surgeries were reviewed .
means and standard deviations were calculated and compared for continuous variables among preoperative , postoperative one month , and at the final follow - up periods .
comparison of parameters between the normal and positive sva groups was performed using mann - whitney test .
non parametric tests including wilcoxon signed rank test and frideman test were used for comparing radiological parameter over time and health - related quality of life between the preoperative and final .
all statistical analyses were performed using spss 18.0 ( ibm co , ny , usa ) .
as for the overall radiological outcome for total 28 patients , the mean pi was 539. the tk , ll , pt , pi - ll , sva , and t1pa were significantly improved both immediate postoperatively and on the final follow - up ( p<0.05 ) .
tk improved from 1017 kyphosis preoperatively to 319 at postoperative one month and was well maintained through the final follow - up ( 378 ) .
the ll improved from 223 kyphosis preoperatively to -608 lordosis at postoperative one month and maintained -5323 at the final . as for the changes in the pi - ll mismatch , sva , and
pt over the due clinical courses ( preoperative , postoperative one month , and final follow - up ) , the pi - ll improved from 5524 preoperatively to -66 at postoperative one month and this improvement was well maintained through the final follow up ( -47 ) .
the sva was 19481 mm preoperatively , 727 mm at postoperative 1 month , and 1935 mm at the final follow up .
the t1pa improved from 4314 preoperatively to 117 at postoperative one month and this improvement was maintained into 137 at final follow up .
similarly , the pt was 3011 preoperatively , 147 at postoperative one month , and 177 at the final follow up ( table 2 ) .
as for the clinical outcomes for this series between preoperative and final follow - up , all investigated parameters including odi , vas score , and srs-22r score were significantly improved ( p<0.05 ) .
vas of the back pain / leg pain were improved from 8.01.0/7.51.2 preoperatively to 2.51.4/2.31.5 at final .
mean srs-22r score was improved from 2.10.4 preoperatively to 3.60.4 at final ( function ; from 2.10.5 to 3.30.5 : pain ; from 2.10.5 to 3.90.5 : self - image ; from 2,00.6 to 3.50.5 : mental health ; from 2.30.6 to 3.70.5 ) ( table 3 ) .
the sva values at the final follow up were categorized as normal sva [ 50 mm , n=23(82% ) ] and positive sva [ > 50 mm , n=5 ( 18% ) ] .
the mean sva was 726 mm for the normal sva group and 7311 mm for the positive sva group , a statistically significant difference between groups ( p=0.001 ) .
the pi - ll was -56 for the normal sva group and 37 for the positive sva group ( p<0.05 ) .
the t1pa was 116 for normal sva group and 228 for the positive sva group ( p<0.05 ) .
however , the pt did not differ significantly between groups ( table 4 ) . on the contrary
, there were no significant differences in clinical outcome measures such as odi , vas score of the back pain / leg pain , and srs-22r score including 5 main domains between the two groups at the final follow - up .
all clinical outcome parameters were significantly improved for both groups on the final follow up from baseline ( table 5 ) .
perioperative complication was 29% ( 8 cases ) and early reoperation were 18% ( 5 cases ) . among them , 4 acute proximal junctional failures occurred ; 2 uiv fractures , one uiv+1 fracture , and one proximal screw loosening .
3 cases of deep vein thrombosis and one focal neurological deficit in spite of 3 times of neural decompression and exploration surgery of a left l3 nerve root compression after l3 pso occurred .
late complications were 46% ( 13 cases ) and late revision surgery were 29% ( 8 cases ) .
4 rod fracture were reoperated with rod change with / without additional 3 or 4 rods techniques
. 3 fusion extension due to delayed proximal junctional kyphosis and one nonunion at l5s1 level were reoperated . among 3 cases of proximal junctional kyphosis ,
one patient with delayed uiv+1 chronic compression fracture who had severe neurological deficit was addressed with neural decompression and fusion extension up to uiv+4 .
in addition , there were 5 cases of long - term complications ; one distal screw loosening , 3 proximal junctional kyphosis , and one asymptomatic rod fracture .
it is well known that post - fusion lumbar flatback deformity is caused by hypolordotic lumbar fusion , adjacent segment disease after lumbar fusion , non - union with or without insufficient anterior column support , and post - laminectomy fusion .
in addition to these conditions , atrophy or fatty degeneration in the paraspinal muscles might cause global sagittal imbalance in the lumbar spine that leads to increased fatigue and pain . to diagnose post - fusion lumbar flatback deformity , clinicians should review the patient 's history , ap and lateral whole spine standing x - rays , and ct and mri of the spine .
the patient 's history may include difficulty in standing upright and complaints of back or leg pain , difficulty in standing during large load bearing , difficulty bending , and frequently relying on the knee as buttress during gait or stair climbing . in whole spine standing x - rays , more than 5 cm of sva , loss of lumbar lordosis , pi - ll mismatch of more than 10 degrees , a large pt of more than 20 , and an increase in the t1-pelvic angle of more than 15 degrees can be measured , and paraspinal muscle atrophy and fatty infiltration , including thinning and stretching of the psoas muscles and back extensor muscle atrophy , can be detected on lumbar spine mri .
spine angiography 3-dimensional ct is very useful to confirm the condition of major abdominal vessels , including the abdominal aorta , inferior vena cava , and iliac vessels , as well as the previous bony fusion state , facet changes , intradiscal vacuum changes , and adjacent segment pathology .
accordingly , the initial radiological parameters included in this series of post - fusion lumbar flatback deformity demonstrated similar phenomena ; pi , 539 ; ll , 222 kyphosis ; pi - ll , 5524 ; sva , 19480 mm ; t1pa , 4313 , and pt : 3010. the aim of surgery for correction of post - fusion lumbar flatback deformity is to achieve a stable , well - balanced lumbar spine centered over the pelvis .
surgical treatment involves correction of lordotic spinal alignment in the flat or kyphotic lumbar spine by corrective osteotomies , such as pso or lower lumbar spos with alifs . in patients with post - fusion flatback
whose main pathology was hypolordotic lumbar fusion ( 19 cases ) , pso were performed at l2 in 4 cases , l3 in 8 cases , and l4 in 7 cases .
the posterior only approach , including pso , was done by one - stage operation , but lower lumbar spos with multilevel alifs were usually segmented as staged operations with initial posterior lower lumbar spos followed by anterior alifs and rod placement posteriorly ( 9 cases ) .
regarding surgical methods and indications , posterior only approach ( pso ) was performed in patients with evidence of satisfactory fusion at previous surgical levels on radiological follow ups whereas cases lacking the assured evidence of fusion , anterior approach such as alifs was combined for proper fusion achievement as well as restoration of lordosis .
similarly , in those cases in which satisfactory fusion up to s1 level was evident , liv was not extended to iliac level . on the other hand in patients with insufficient fusion at l45 and l5s1 level
most of these methods are geometrical and merely take into account the numerical amount of correction angles required to shift the location of the head properly over the pelvis .
however , recent studies have shown that sagittal spinopelvic parameters among patients with spinal deformity play a role in clinical outcomes .
these studies rated the severity of adult spinal deformity using the scoliosis research society ( srs)-schwab asd classification and compared the results to hrqol , pi - ll mismatch , sva , and pt values19 ) .
the radiological parameters are important , as they can predict the patients ' prognosis and satisfaction levels . among these parameters
, ll can be modified through surgical intervention , but pi is a constant
value that is fixed for each individual10 ) .
similarly , the pi - ll mismatch can adversely affect hrqol , regardless of actual surgical performance15161719 ) .
schwab et al.14 ) reported threshold values for sagittal spinopelvic alignment to achieve during reconstructive surgery for improved hrqol .
surgical outcomes are generally measured using pi - ll values . according to the srs - schwab classification ,
good surgical outcomes are achieved if the pi - ll value ranges within10. according to schwab , the equation ll = pi+9 ( 9 ) is a basic calculation in asymptomatic adults .
he suggested that realignment objectives in the sagittal plane should aim for a postoperative sva < 50 mm , pt < 20 , and ll = pi9 to achieve spinopelvic realignment14 ) .
lafage et al.5 ) have proposed that sva is associated with hrqol and pelvic retroversion .
berjano et al.2 ) have suggested that the rule ll=-(pi+10 ) can be used to plan for good alignment .
however , the authors note that is not easy to predict optimal alignment when correcting deformity13 ) . due to the variability of sva value according to the different degree of pelvic retroversion or knee flexion even in a single subject , the t1pa value has been frequently cited and well acclaimed as a reliable , consistent radiological parameter that could be correlated with surgical outcome recently .
this t1pa value accounts for both sva and pelvic tilt , and according to the several references , this might be reduced to less than 10 in order to achieve optimal surgical outcome both radiologically and clinically811 ) .
the representability of t1pa instead of sva was also reflected in our series ; 11 for the normal sva group and 22 for positive sva group even after correction .
the authors ' concept or suggestion according to past several years of surgical experiences in post - fusion lumbar flatback deformity would be that postoperative ll in these cases should be greater than pi to restore and maintain normal sagittal spinal balance . because asian patients with post - fusion lumbar flatback are generally osteoporotic , elderly females with poor paraspinal muscle condition due to fatty degenerative changes , atrophy of the back muscles , and thinning of the paraspinal and psoas muscles , the compensatory role from the paraspinal muscles is insufficient .
there is an obvious difference in the volume of the paraspinal muscles between asian and western populations and since its volume is usually smaller for asians , even if the initial or background pi - ll value might be similar , loading on the spine would be relatively higher .
therefore , restoration of lumbar lordosis below the pi value might result in another or secondary postoperative or delayed sagittal imbalance consequently .
roussouly110 ) and barrey et al.1 ) have also suggested theoretical values for pt and ll related to pelvic incidence .
they categorized pi into 6 groups ( i to vi ) and recommended theoretical ll and pt in each pi group . in this study , at the final follow - up , the normal sva group ( 82% ) had sva of 7 mm , while the positive sva group had sva of 73 mm , a significant difference between groups .
the pi - ll was -5 for the normal sva group and 3 for the positive sva group and t1pa was 11 for the normal sva group and 22 for positive sva group .
the odi score , vas scores for back and leg pain , and srs-22r score showed improvement in all 28 patients .
but between two groups for comparison , there were no significant differences in odi , vas score of the back pain / leg pain , and srs-22r score including 5 main domains . due to the intense , multilevel- involving , corrective procedure in revisional fashion , the expected occurences of untoward consequence could surge during the management of this post - fusion lumbar flatback deformity [ in our series ,
the reoperation rate was 46% ( 13 patients ) ] compared to other spinal procedures .
most of these necessity of revisional procedure were related to the proximal junctional problems ( uiv or uiv+1 fractures ) or instrument failure frequently brought up at the uiv level during early postoperative period except one case of severe neurological deficit .
fortunately , a positive prognosis through revision surgery was anticipated in most of compromised cases .
in addition , patients with all early revision and late revisions due to rod fractures or nonunion did not change significantly in the sva , only delayed pjk patients progressed positive sva . despite the slightly higher complication rate ,
most of patients were categorized as improved clinical outcomes with higher satisfaction , in accordance with radiological parameter improvements . due to the limited number of patients undergoing corrective osteotomy for post - fusion lumbar flatback deformity ,
therefore , future studies should focus on statistical analyses based on large study population . nevertheless this study has analyzed population with long follow up period and hence bears significance . additionally , concomitant presence of atrophic changes of the paraspinal muscles along with hypolordotic fusion , adjacent segment disease , and non - union with or without anterior column insufficiency probably resulted in post - fusion flatback deformity in our current study .
several studies have suggested that the volume of lumbar back muscles has a significant effect on the degenerative kyphosis of patients18 ) .
but we did not perform any evaluations regarding the muscle volumes and their effect which can be regarded as one of the limitations .
therefore , we believe that the volume of paraspinal muscles is an important determining factor and should be further evaluated to predict the patient 's condition or prognosis in post - lumbar fusion .
due to the limited number of patients undergoing corrective osteotomy for post - fusion lumbar flatback deformity , this series might not fulfill statistical power with probable yield of beta error .
nevertheless this study has analyzed population with long follow up period and hence bears significance . additionally , concomitant presence of atrophic changes of the paraspinal muscles along with hypolordotic fusion , adjacent segment disease , and non - union with or without anterior column insufficiency probably resulted in post - fusion flatback deformity in our current study .
several studies have suggested that the volume of lumbar back muscles has a significant effect on the degenerative kyphosis of patients18 ) .
but we did not perform any evaluations regarding the muscle volumes and their effect which can be regarded as one of the limitations
. therefore , we believe that the volume of paraspinal muscles is an important determining factor and should be further evaluated to predict the patient 's condition or prognosis in post - lumbar fusion .
pso or spos combined with multilevel alifs at the lower lumbar spine are significantly improves sagittal balance in post - fusion lumbar flatback patients with sagittal imbalance . for restoration and maintenance of normal sagittal spinal balance in these patients , pi - ll
might be made negative value and t1pa might be less than 11 even though positive sva group was also significantly improved clinical outcomes . | objectivesto review surgical results of post - fusion lumbar flatback treated with pedicle subtraction osteotomy ( pso ) or smith - petersen osteotomies ( spos).methodstwenty - eight patients underwent osteotomies .
radiological outcomes by sagittal vertical axis ( sva ) , and pelvic tilt ( pt ) , t1 pelvic angle ( t1pa ) , and pelvic incidence ( pi)-lumbar lordosis ( ll ) at preoperative , postoperative 1 month , and final were evaluated .
oswestry disability index ( odi ) , visual analog scale ( vas ) score of back pain / leg pain , and scoliosis research society-22 score ( srs-22r ) were analyzed and compared .
patients were divided into 2 groups ( sva 5 cm : normal , sva > 5 cm : positive ) at final and compared outcomes.resultsnineteen patients ( 68% ) had pso and the other 9 patients had spos with anterior lumbar interbody fusions ( alifs ) ( mean age : 65 years , follow - up : 31 months ) .
the pt , pi - ll , sva , t1pa were significantly improved at 1 month and at final ( p<0.01 ) .
vas score , odi , and srs-22r were also significantly improved at the final ( p<0.01 ) .
23 patients were restored with normal sva and the rest 5 patients demonstrated to positive sva .
sva and t1pa at 1 month and sva , pi - ll , and t1pa at final were significantly different ( p<0.05 ) while the odi , vas , and srs-22r did not differ significantly between the groups ( p>0.05 ) .
common reoperations were early 4 proximal junctional failures ( 14% ) and late four rod fractures.conclusionour results demonstrate that pso and spos with alifs at the lower lumbar are significantly improves sagittal balance .
for maintenance of normal sva , pi - ll might be made negative value and t1pa might be less than 11 even though positive sva group was also significantly improved clinical outcomes . |
in 1983 , barry j. marshall and j. robin warren discovered helicobacter pylori ( h. pylori ) in the gastric epithelium of patients with chronic active gastritis .
infection with h. pylori plays a key role in the pathogenesis of a number of gastroduodenal and other diseases [ 29 ] .
colonization of the gastric mucosa with h. pylori results in the development of chronic gastritis in all infected individuals , and in a subset of patients , the chronic gastritis progresses further to peptic ulcer disease , gastric neoplasms , and other specific extragastric disorders .
colonization of the stomach with h. pylori is found in about half of the human population worldwide .
there is general agreement that h. pylori should be eradicated in patients with peptic ulcers , but no consensus exists as to the optimal regimen [ 1016 ] . in the maastricht iii
consensus report , triple therapy using a ppi with clarithromycin and amoxicillin or metronidazole administered twice daily was the recommended treatment of first choice .
several factors such as smoking , age , antibiotic resistance , short duration of therapy , poor compliance and genetic polymorphism of cyp2c19 have been shown to influence the eradication rate of h. pylori after appropriate therapy [ 915,17 ] .
another factor that has been implicated in the failure of therapy is pretreatment with antisecretory drugs , especially proton pump inhibitors ( ppis ) [ 1315,1825 ] .
some data suggest that pretreatment with a ppi before the administration of h. pylori eradication therapy might decrease the efficacy of the treatment [ 1315 ] .
however , a meta - analysis investigating the influence of ppi pretreatment on triple and quadruple therapies for h. pylori eradication did not reveal any differences in the eradication rates between patients with and without pretreatment .
on the other hand , the effect of pretreatment with an h2 receptor antagonist ( h2ra ) , which is capable of suppressing acid secretion to a similar degree as ppis , on the efficacy rate of h. pylori eradication therapy has not yet been investigated .
this issue has important practical implications , because patients are often already receiving antisecretory therapy when they are diagnosed as having h. pylori infection , and it would be useful to ascertain whether such therapy should be withdrawn prior to the start of the eradication therapy .
we enrolled 310 consecutive outpatients ( 1987 years old ) who underwent upper gastrointestinal endoscopy at yokohama city university hospital , yokohama minami kyosai hospital or fujisawa shounandai hospital between august 1998 and december 2007 .
the diagnosis of h. pylori infection was made using the rapid urease test , bacterial cultures and histological examination of endoscopic biopsy specimens .
complete information about the patients , including the age , sex , and smoking habit at the time of the initial diagnosis , was obtained from the patients medical records .
patients with the smoking habit in this study referred to those who were current regular smokers .
patients were excluded if they were receiving continuous treatment with nsaids or if they were pregnant or breastfeeding .
other exclusion criteria included poor compliance , known penicillin allergy , previous eradication therapy , previous treatment with ppis , concomitant liver or kidney disease , severe cardiac or pulmonary disease , suspected or known malignancy , and the presence of zollinger - ellison syndrome or antral g cell hyperfunction . in this retrospective study conducted at three centers ,
the patients were assigned to receive an eradication regimen ( lansoprazole , clarithromycin , and amoxicillin ) with or without pretreatment with an h2ra ( famotidine , 20 mg or 40 mg , ranitidine , 150 mg or 300 mg , or lafutidine , 10 mg or 20 mg ) .
all the patients underwent a c - urea breath test ( c - ubt ) at least 4 weeks after the completion of the eradication therapy .
h. pylori eradication therapy was administered using the standard 7-day or 14-day ppi - based triple therapy protocol ( lpz , 30 or 60 mg b.i.d .
+ clarithromycin [ cam ] , 400 or 800 mg b.i.d . ) after obtaining informed consent from the patients .
the possible adverse effects of the drugs were explained to the patients , who were encouraged to complete the treatment course .
all the patients underwent a c - ubt at 4 weeks after the completion of the eradication therapy to confirm the eradication status of h. pylori .
c in excess of the cutoff value of 2.5% was considered to indicate active h. pylori infection , that is , failure of treatment . for the statistical analysis , a chi - square test or fisher
s exact test was used to compare the percentages and mann - whitney s u test was used to compare continuous data .
various risk factors were also evaluated simultaneously using multiple logistic regression . in all tests ,
the statistical analyses were performed using the stat view software ( sas institute , cary , n.c . ) .
this study was conducted in accordance with the declaration of helsinki and with the approval of the ethics committee of yokohama city university hospital .
we enrolled 310 consecutive outpatients ( 1987 years old ) who underwent upper gastrointestinal endoscopy at yokohama city university hospital , yokohama minami kyosai hospital or fujisawa shounandai hospital between august 1998 and december 2007 .
the diagnosis of h. pylori infection was made using the rapid urease test , bacterial cultures and histological examination of endoscopic biopsy specimens .
complete information about the patients , including the age , sex , and smoking habit at the time of the initial diagnosis , was obtained from the patients medical records .
patients with the smoking habit in this study referred to those who were current regular smokers .
patients were excluded if they were receiving continuous treatment with nsaids or if they were pregnant or breastfeeding .
other exclusion criteria included poor compliance , known penicillin allergy , previous eradication therapy , previous treatment with ppis , concomitant liver or kidney disease , severe cardiac or pulmonary disease , suspected or known malignancy , and the presence of zollinger - ellison syndrome or antral g cell hyperfunction .
in this retrospective study conducted at three centers , a total of 310 patients with h. pylori infection were treated .
the patients were assigned to receive an eradication regimen ( lansoprazole , clarithromycin , and amoxicillin ) with or without pretreatment with an h2ra ( famotidine , 20 mg or 40 mg , ranitidine , 150 mg or 300 mg , or lafutidine , 10 mg or 20 mg ) .
all the patients underwent a c - urea breath test ( c - ubt ) at least 4 weeks after the completion of the eradication therapy .
h. pylori eradication therapy was administered using the standard 7-day or 14-day ppi - based triple therapy protocol ( lpz , 30 or 60 mg b.i.d .
+ clarithromycin [ cam ] , 400 or 800 mg b.i.d . ) after obtaining informed consent from the patients .
the possible adverse effects of the drugs were explained to the patients , who were encouraged to complete the treatment course .
all the patients underwent a c - ubt at 4 weeks after the completion of the eradication therapy to confirm the eradication status of h. pylori .
c in excess of the cutoff value of 2.5% was considered to indicate active h. pylori infection , that is , failure of treatment .
for the statistical analysis , a chi - square test or fisher s exact test was used to compare the percentages and mann - whitney s u test was used to compare continuous data .
various risk factors were also evaluated simultaneously using multiple logistic regression . in all tests ,
the statistical analyses were performed using the stat view software ( sas institute , cary , n.c . ) .
this study was conducted in accordance with the declaration of helsinki and with the approval of the ethics committee of yokohama city university hospital .
a total of 310 cases ( 210 men and 100 women ; median age , 54.7 years ; range , 1987 years ) were enrolled in this study .
of the 158 patients who received h2ra pretreatment , 129 ( 81.6% ) were cured , while 118 ( 77.6% ) of the 152 patients who did not receive h2ra pretreatment were also cured ( p=0.3799 ) ( table 2 ) .
no significant differences in the age , sex , smoking habit , endoscopic results , duration of eradication treatment , dosage of lpz , dosage of cam , percentage receiving pretreatment with h2ra , or duration of pretreatment with h2ra were noted between the two outcome groups ( table 3 ) .
no significant difference in the outcome of eradication therapy was observed between the patients with and without h2ra pretreatment .
determining whether the timing of administration of antisecretory agents , such as ppis and h2ras , might have any influence on the efficacy rate of antibiotic treatment in patients with h. pylori infection is of clinical importance , because most patients with peptic ulcers have been treated with antisecretory agents prior to the initiation of h. pylori eradication treatment .
the aim of this study was to investigate the influence of pretreatment with h2ra on the efficacy of h. pylori eradication therapy .
our results indicated that pretreatment with h2ra does not influence the efficacy of h. pylori eradication therapy .
eradication therapy only for patients with gastroduodenal ulcer , gastric cancer after endoscopic therapy , gastric malt lymphoma , and itp with h. pylori positivity are covered by insurance in japan now . in this study , the majority of patients had received eradication therapy for the prevention of recurrence of peptic ulcer .
several studies have reported that pretreatment with antisecretory drugs was correlated with the failure of dual therapy with ppi and amoxicillin , with eradication rates being 3070% lower among patients who had received ppi pretreatment [ 1315 ] .
reported that pretreatment with ppi resulted in a marked reduction in the h. pylori eradication rate .
they speculated that treatment with a ppi directly or indirectly induced coccoid - persistent forms of h. pylori , resulting in lower cure rates .
the coccoid - persistent form of h. pylori is known to be less vulnerable to the actions of antibiotics . on the other hand , adachi et al .
reported that pretreatment with omeprazole might actually improve the eradication rate ( although there was no statistically significant difference ) . the strong antisecretory activity of ppis has been thought to play an important role in the eradication of h. pylori , as ppi - induced high gastric ph has been reported to increase the sensitivity of h. pylori to antimicrobial agents [ 24,2832 ] .
therefore , pretreatment with ppi or h2ra has been advocated based on the assumption that elevation of the gastric ph prior to the start of antibiotic administration would increase the cure rate . while this issue concerning pretreatment with ppis has remained controversial , a meta - analysis investigating the influence of ppi pretreatment on the efficacy of triple and quadruple h. pylori eradication therapies did not reveal any difference in the eradication rates between patients with and without ppi pretreatment .
this finding is of great practical importance when the immediate start of triple therapy is not possible , especially in cases where the antibiotic resistance must first be tested . since h2ras also have strong antisecretory activity , pretreatment with h2ras may improve the efficacy rate of h. pylori eradication therapy . however , ppis not only directly block the proton pump on the parietal cells of the stomach , they have also been demonstrated , both in vivo and in vitro , to exhibit antibacterial activity against h. pylori .
nevertheless , most studies comparing ppis and h2ras for h. pylori eradication have not shown any significant differences between regimens including either drug .
consequently , the adjuvant effect of antisecretory therapy is related more to the drugs ability to suppress acid secretion than to its antibacterial activity . because h2ras have sufficient antisecretory effect for pretreatment
, we hypothesized that h2ras might be suitable for pretreatment and examined the influence of h2ra pretreatment on the efficacy rates of h. pylori eradication therapy .
moreover , even if h2ras do not influence the form of existence of h. pylori , h2ra pretreatment might facilitate the eradication efficacy of triple / quadruple therapy against h. pylori .
however , no significant difference in the efficacy of eradication therapy was observed between patients with and without h2ra pretreatment .
the findings of this retrospective study indicated that pretreatment with h2ra does not significantly reduce the efficacy of eradication therapy in patients with h. pylori infection .
further prospective cohort studies are needed to confirm this finding . in the present study ,
other factors such as the age and smoking habit , which have been reported to influence the eradication rates of h. pylori in previous reports , were not found by multivariate logistic regression analysis to have any significant impact on the success rate of h. pylori eradication therapy ( table 4 ) .
one possible reason for this discrepancy is that clarithromycin resistance , which increases the risk of eradication failure [ 3638 ] , was not investigated in the present study . during continuous treatment with a proton pump inhibitor ( ppi ) , presence of significant nocturnal gastric acid secretion was reported .
this event , known as nocturnal gastric acid breakthrough ( nab ) , is defined as intragastric ph < 4 for more than one hour continuously at night .
addition of an h2-receptor antagonist ( h2ra ) at bedtime to a high - dose ppi is likely to improve the nocturnal gastric ph control and decrease nocturnal gastric acid breakthrough .
the intragastric ph during h. pylori eradication treatment has been shown to affect the eradication rates . therefore , a ppi - based regimen with h2ras at bedtime may improve the eradication rate of h. pylori , despite of contrary opinions .
and it was reported that use of h2ras during and after h. pylori eradication therapy may be effective for preventing the occurrence of reflux esophagitis , gastric erosions and duodenal erosions after h. pylori eradication .
in summary , this retrospective study indicated that pretreatment with h2ra does not significantly influence the efficacy of eradication therapy in patients with h. pylori infection .
further prospective cohort studies are needed to confirm this finding from the perspectives of economic benefit and quality of life . | summarybackgroundpretreatment with a proton pump inhibitor ( ppi ) reportedly decreases the efficacy of helicobacter pylori ( h. pylori ) eradication , however , the effect of pretreatment with an h2 receptor antagonist ( h2ra ) on h. pylori eradication has not yet been studied .
we compared the efficacy of eradication regimen ( lansoprazole / amoxicillin / clarithromycin ) in patients with h. pylori infection with or without h2ra pretreatment.material/methodsin this retrospective study conducted at three centers , 310 patients with h. pylori infection were treated .
the diagnosis of h. pylori infection was made using the rapid urease test , bacterial cultures and histological examination of endoscopic biopsy specimens .
the patients were assigned to receive an eradication regimen first or following pretreatment with h2ra .
eradication was assessed using the 13c - urea breath test more than 4 weeks after the completion of therapy.resultsoverall , h. pylori was eradicated in 79.7% of the cases : the eradication rate was 81.6% in the pretreatment group , and 77.6% in the eradication first group ( p=0.3799 , chi - square test ) .
no significant difference in the eradication rate was observed between the two groups.conclusionspretreatment with h2ra had no significant influence on the efficacy of h. pylori eradication therapy . |
it is a clonal myeloproliferative disorder , which is caused by the philadelphia ( ph ) chromosome that produces the constitutively active bcr - abl tyrosine kinase .
tyrosine kinase inhibitors ( tkis ) act by competitive inhibition of adenosine triphosphate ( atp ) that binds to bcr - abl .
imatinib is the first tyrosine kinase inhibitor to have achieved long term disease control in the majority of patients with cml . in an internationally randomized study of interferon versus
st1571 ( iris ) , 63% of all imatinib patients ' showed a complete cytogenetic response ( ccyr ) and the estimated event free survival ( efs ) was 83% .
the degree of treatment response at earlier time points in the cml correlates with efs and time to progression to accelerated phase/ blast crisis ( ap / bc ) . however , imatinib use is complicated by the development of resistance or intolerance , which leads to suboptimal responses or treatment failure .
the most common mechanisms for the development of this resistance are the occurrence of a mutation in the bcr - abl tyrosine kinase domain that inhibits imatinibs ability to bind to abl and poor patient compliance .
grade 3 neutropenia and thrombocytopenia were reported in 17% and 9% of the patients respectively , and most of them occurred within the first two years of imatinib treatment .
furthermore , most myelosuppression is dose - dependent , reversible , and can be managed by dose interruption or reduction . in the iris study ,
the second generation tkis - nilotinib or dasatinib - are more potent than imatinib and can overcome most imatinib resistant bcr - abl mutations .
in a two year follow up of a phase 2 study , nilotinib was given to patients with chronic phase ( cp ) or advanced phase cml who had failed , or were unable to tolerate , initial imatinib therapy.cross-intolerance with imatinib ( defined as occurrence of a grade 3 adverse event ( ae ) or persistence or recurrent grade 2 of the same ae that previously led to discontinuation of imatinib ) was observed in a sub - analysis of 121 out of 459 patients with cp and ap .
additional data suggests that patients who develop severe hematological toxicity with imatinib treatment , are at high risk of having a suboptimal response or hematological cross - intolerance with a second generation tki .
currently , the general assumption is that an initial trial of second - generation tkis , before proceeding to hematopoietic stem cell transplantation ( hsct ) , is reasonable in nearly all patients in whom imatinib treatment fails , particularly , because hsct is available only to a minority of patients and is associated with significant morbidity and mortality .
the hammersmith score helps predict the response to second generation tki 's and is based on calculating the cytogenetic response to imatinib , the sokal high risk group and the recurrence of neutropenia .
it is still unclear whether a small , select group of young cml patients in cp who fail imatinib treatment should undergo allogeneic hsct without being first treated with second - line tkis . here
we present a cp cml patient who developed severe thrombocytopenia during treatment with all three tki 's and we raise the question as to whether an early hsct should have been performed .
a 34-year - old man was diagnosed with ph - positive cml in may 2009 , after presenting the preceding year with weight loss and a white blood cell count ( wbc ) of 18810/l .
the patient received hydroxyurea for three weeks followed by imatinib 400 mg / day . within a month
the patient entered a clinical study that assessed therapeutic levels of imatinib and he was found to have a sub - therapeutic level of 640 ng ( the study estimated an optimal therapeutic level of approximately 1000 ng ) . after three months of imatinib treatment the bcr - abl transcript level decreased by only 0.2 logs as compared to the test performed at diagnosis . in november 2009 , the patient complained of flu like symptoms and developed severe thrombocytopenia with a platelet ( plt ) count of 2210/l , wbc of 4.410/l with absolute neutrophil count of 0.810/l , and hemoglobin of 12.8g / dl .
a peripheral blood smear showed atypical lymphocytes with toxic granulation , suggestive of acute viral infection .
the following tests were normal or negative ; herpes simplex virus , herpes zoster , cytomegalovirus , epstein barr virus , igm antibodies , hepatitis b surface antigen ( hbsag ) , hepatitis b surface antibody ( hbsab ) , hepatitis c virus antibody , antinuclear antibody and human immunodeficiency virus antibody .
bone marrow ( bm ) aspiration and biopsy revealed a hypoplastic marrow with a decreased number of megakaryocytes , no blast cells and no excess of reticulin .
cytogenetic studies demonstrated 93% mitotic cells with ph chromosome and fluorescent in situ hybridization ( fish ) showed 83% of the cells with bcr - abl .
the treatment with imatinib was stopped for two weeks and nilotinib 400 mg twice daily was started after the platelets rose above 50,000 l . within another two weeks , there was a decline in his blood count with plt 1510/l , normal levels of wbc ( 5.610/l ) , anc of 110/l and hemoglobin of 12.1g / dl .
of note , severe thrombocytopenia was also observed with a lower dose of nilotinib ( 200 mg twice daily ) .
the treatment with nilotinib was discontinued and the patient was treated empirically with prednisolone 80 mg daily . within 2 weeks , the plt level increased to 110,000 l .
after the treatment failure with nilotinib , dasatinib100 mg once daily was started , with a similar decrease in plt level to 2110/l . at this point , studies to evaluate the possibility of drug ( tki 's ) induced thrombocytopenia were performed .
tests for anti - human antibodies igm , igg and iga against plts were negative . however , after adding the tki drugs , drug - dependent antibodies against plt were found using an immunofluorescence test applying flow cytometry ( figure 1 ) .
moreover , the presence of these antibodies was dose dependent and was thus raised the possibility of drug induced thrombocytopenia .
the presence of antibodies against platelets was detected using platelet immunofluorescence test ( pift ) and the measuring unit was mean fluorescence intensity ( fi ) .
normal group o platelets from 6 different donors were washed and incubated with patient serum or control serum and with either pbs or tkis diluted in pbs from 1:1 up to 1:10000 . after washing platelets
were incubated with fitc labeled goat anti - human igg and analyzed using flow cytometer .
point zero demonstrates that in the absence of tkis there are not any antibodies .
the presence of antibodies against platelets was detected using platelet immunofluorescence test ( pift ) and the measuring unit was mean fluorescence intensity ( fi ) .
normal group o platelets from 6 different donors were washed and incubated with patient serum or control serum and with either pbs or tkis diluted in pbs from 1:1 up to 1:10000 . after washing platelets
were incubated with fitc labeled goat anti - human igg and analyzed using flow cytometer .
point zero demonstrates that in the absence of tkis there are not any antibodies . in august 2010 ,
cytogenetic studies showed 100% mitosis with philadelphia chromosome along with only one log reduction in bcr - abl load .
bone marrow aspiration and the peripheral counts revealed no blasts thereby implying that the patient was still in the cp of disease .
since the patient was intolerant to all tki 's , therapy with interferon alpha 3mu three times weekly was started before proceeding to hsct . during the interferon therapy
the patient underwent an uneventful , allogeneic , peripheral blood stem cell transplantation from a fully matched unrelated male donor after conditioning with bu - cy ( busulfan- cyclophosphamide ) and standard gvhd prophylaxis .
rt - qpcr for bcr / abl transcripts performed on day 28 showed 6 log decrease .
a repeated test on day 72 showed an increase of bcr - abl / abl ratio to 10 and cyclosporine was discontinued . during the following month the patient presented with skin rash , diarrhea and elevated liver enzymes , compatible with acute gvhd .
there was a concomitant decrease of bcr - abl / abl to 10 , 46xy karyotype and 100% donor chimerism .
his blood count is normal , with complete donor chimerism and undetectable bcr / abl .
our cml patient presented with an interesting combination of resistance and intolerance to tkis that was manifested mainly as grade 3 thrombocytopenia .
interruption of therapy , dose reduction and therapy with second generation tkis , did not prevent the recurrence of the adverse hematological events .
mutations in the bcr - abl tyrosine kinase domain were not found in our patient and he was compliant with medication . in cml , the hematopoiesis is mainly derived from the ph stem cells and myelosuppression is usually observed within the first weeks of tki treatment .
the reduced reserve of residual , healthy ph - bone marrow ( bm ) is incapable of reconstituting the peripheral blood count . in the case of our patient ,
the cytopenia occurred six months after the initiation of imatinib therapy and the ph clone was not suppressed , as shown by the 100% ph mitotic cells in the karyotype analysis .
the demonstration of anti - platelets antibodies in the patients ' serum only in the presence of tkis implies that the possible mechanism is indeed drug ( tkis ) induced thrombocytopenia .
however , the full significance of this finding is unclear since , unlike in itp ( immune thrombocytopenic purpura ) , there were decreased numbers of megakaryocytes in the bm , and since no tki induced immune thrombocytopenia has been previously reported .
hsct is effective therapy for patients with cp - cml but in the current era of second generation tki 's it is mainly indicated for patients who develop resistance .
less than 5% of patients develop repeated severe myelosuppression after only a few months of tki therapy .
late development of severe myelosuppression during imatinib treatment is probably an important indication for consideration of early hsct .
future studies are needed in order to understand the pathogenesis of recurrent severe myelosuppression during tki therapy . | the outcome and quality of life of chronic myeloid leukemia ( cml ) patients has remarkably changed with the treatment of tyrosine kinase inhibitors ( tkis ) . currently , hematopoietic stem cell transplantation ( hsct ) is considered mainly as a third line salvage therapy in cases of tkis resistance or intolerance . here
we describe a patient with chronic phase cml who developed both resistance and late occurrence of s severe thrombo - cytopenia on first and second generation tkis and eventually underwent hsct .
although the mechanism of the myelosuppression is not fully understood , we showed for the first time the development of dose dependent platelet antibodies in the presence of tkis , suggesting the possibility of tkis induced thrombocytopenia .
our case emphasizes that late development of severe myelosuppression during imatinib treatment is probably an important indication for consideration of early hsct . |
congenital hypothyroidism ( ch ) occurs in babies who are born without the ability to produce adequate amount of thyroid hormones .
the remaining 1020% of cases are due to thyroid dyshormonogenesis or defects in the intermediary steps of thyroid hormone biosynthesis .
studies have shown that thyroid dyshormonogenesis is associated with defects in genes that are involved in thyroid hormone synthesis .
mutations in the tpo are one of the most common causes of dyshormonogenetic ch disorder .
other candidate genes include dual oxidase 2 ( duox2 ) , solute carrier family 5 or sodium iodide symporter ( slc5a / nis ) , thyroglobulin ( tg ) , and thyroid stimulating hormone receptor ( tshr ) .
the tpo gene is located on chromosome 2p25 spanning at least 150 kb and contains 17 exons .
the full - length of the mrna transcript , tpo1 , is 3152 bp ( genbank accession number nm_000547.5 ) which encodes 933 amino acids .
tpo is tissue - specific and the mature tpo protein is organized into a large extracellular fragment , a short transmembrane segment and a short cytoplasmic tail .
tpo is a heme - containing protein that catalyzes the iodination of thyroglobulin and the coupling of some of the iodotyrosyl residues to generate active iodothyronines , t3 and t4 [ 12 , 13 ] . to date , more than 60 mutations in the tpo gene have been described .
the majority of the mutations were localized in exons 8 , 9 , and 10 of the gene that encode the catalytic heme binding domain of the protein [ 14 , 15 ] .
cases of ch due to defects in the tpo gene were usually associated with a recessive inheritance pattern .
however , an autosomal dominant inheritance pattern had also been reported . in the present study
, we characterized mutation(s ) in the tpo gene in two siblings with dyshormonogenetic ch and mng .
the effects of the mutation on protein function were predicted using in silico analyses as tpo protein synthesis is tissue - specific and thyroid tissues from both sisters were not available .
two sisters of malaysian - indian origin were diagnosed with ch during infancy , with both having no cord tsh screening performed at birth ( not introduced as a national policy at that time ) .
the proband , 11 - 1 , was diagnosed with ch at 4 months old when she presented with classical signs of hypothyroidism . at presentation ,
serum tsh was more than 160 iu / ml ( normal range : 0.35.0 uiu / ml ) and total t4 less than 13.0 nmol / l ( normal range : 64167 nmol / l ) , respectively .
her younger sister , ii-3 , was born at a private maternity centre in 1997 at 36 weeks of gestation with no cord tsh screening as well .
she was detected to have ch at 2 weeks old when she was screened during her hospital stay for birth asphyxia , with the background history of elder sister with ch . at diagnosis ,
her free t4 was 2.1 pmol / l ( normal range : 11.523.2 pmol / l ) .
technetium-99 m thyroid scintigraphy was performed when they were 3 years old when l - thyroxine was temporarily stopped for 4 weeks and thyroid function repeated .
however , the tsh rose markedly in both patients with decrease in free t4 confirming the need for life - long replacement .
in the course of their follow - up clinic visits , both patients 11 - 1 and 11 - 3 developed multinodular goiters at 11 years and 10 years , respectively .
however , the human thyroglobulin ( htg ) level of 11 - 1 was 137 ng / ml ( normal : 055 ng / ml ) while the htg level of ii-3 was markedly elevated to 1284 ng / ml . both had fine - needle aspiration and cytology ( fnac ) of their goiter , which revealed no evidence of malignancy .
patient 11 - 1 suffered as a consequence of the late treatment , affecting her growth and cognitive function with iq of 5055 ( normal iq = 85115 ) .
her younger sister , 11 - 3 , had normal physical growth and an iq of 80 which is slightly below the average iq range of 85115 .
their parents ( i-1 and i-2 ) who are consanguineous ( father is the maternal uncle ) and the other two proband 's siblings ( ii-2 and ii-4 ) were all healthy , euthyroid with the absence of congenital anomalies .
written informed consent was obtained from the parents for their blood and their children 's blood for genetic analysis of the tpo gene .
this study was approved by the university of malaya medical centre ( ummc ) ethical committee ( institutional review board ) in accordance to the ich - gcp guideline and the declaration of helsinki ( reference number , 654.16 ) .
peripheral venous blood samples were collected from the proband , the affected sister , and their healthy family members .
genomic dna was extracted from peripheral blood leukocytes using qiaamp dna blood mini kit ( qiagen , germany ) according to the manufacturer 's protocol .
the tpo gene was pcr - amplified with flanking intronic primers [ 18 , 19 ] covering all the exons including the hotspot mutation regions , exons 8 , 9 , 10 , 12 and 14 .
a 50 l of pcr reaction mixture containing 100250 ng of genomic dna , 1x taq buffer with kcl , 2.5 mm of mgcl , 200 m of dntp , 8% dimethyl sulfoxide ( dmso ) , 1 unit of taq dna polymerase ( fermentas , usa ) , and 20 pmol of each forward and reverse primers were prepared .
thirty five cycles of amplification were carried out with standard pcr protocol at annealing temperature of 55c for all coding exons for 30 seconds .
pcr products were purified using qiaquick pcr purification kit ( qiagen , germany ) following the manufacturer 's instructions .
the purified pcr products were sequenced using abi prism gene sequencer , model 3100 , version 3.7 ( research biolabs , singapore ) . to confirm that an alteration in the tpo gene is not a polymorphism ,
a total of 100 chromosomes from 50 unrelated healthy individuals were also screened for the same mutation .
thyroid tissue samples from the patients were not available ; therefore , in silico analyses were carried out to predict the impact of the mutation on protein structure and function .
protein sequence alignment across species was performed using clc sequence viewer 6.5.2 software ( clc bio , aarhus , denmark ) .
the potential functional impact of p.val501gly was predicted using polymorphism phenotyping 2 ( polyphen-2 ) , at http://genetics.bwh.harvard.edu/pph/ and sorting intolerant from tolerant ( sift ) , at http://sift.jcvi.org/www/sift_enst_submit.html .
both polyphen-2 and sift are online prediction tools which predict the degree of ( possible ) impact of an amino acid substitution on the structure and function of a human protein . this functional impact of the mutation was predicted as benign , possibly damaging , probably damaging , or unknown for polyphen and as tolerated or damaging for sift .
the crystal structure of human tpo is currently not available ; hence , in this study , its homology model was generated . using the online protein - protein blast program [ 22 , 23 ] from national center for biotechnology information ( ncbi ) website ( http://blast.ncbi.nlm.nih.gov/blast.cgi ) , the human tpo protein was found to have the highest amino acid homology to that of sheep lactoperoxidase , where fortunately , the crystal structure of the latter is available ( pdb i d : 2ikc ) .
thus , a homology model was generated with 2ikc as a template using modeller 9.10 software .
using the same software , a homology model of the human tpo containing the p.val501gly mutation was also generated for structural comparison . both models
were then subjected to molecular dynamics simulation using amber 10 software [ 25 , 26 ] with ff99sb force field , explicit water environment at 310 kelvin and run for 10 ns .
structures with the lowest potential energy were extracted , verified using ramachandran plots which were generated by procheck software ( http://nihserver.mbi.ucla.edu/saves ) to check the stereochemical quality of the protein , verify3d software to determine the compatibility of the 3d atomic model with its own 1d amino acid sequence , and errat software ( http://nihserver.mbi.ucla.edu/saves ) to analyse the statistics of nonbonded interactions between different atom types .
dna sequencing of exons 1 to 17 of the tpo gene in the index patient , ii-1 , revealed a novel homozygous mutation , c.1502t > g ( figure 1 ) .
the mutation is predicted to cause a change from valine to glycine at amino acid 501 ( p.val501gly ) .
in addition , other documented polymorphisms ( 12a > g , c.769g > t , c.1193g > c , c.2145c > t , c.2173a > c , and c.2540t > c ) in exons 1 , 7 , 8 , 12 , and 15 , respectively , were also detected in ii-1 .
the c.1502t > g mutation was not found in 100 chromosomes from 50 unrelated healthy individuals .
further dna sequencing analysis revealed that her affected sibling , ii-3 , was also homozygous for the same mutation . on the other hand , both parents and one of the index patient 's siblings ( ii-2 ) were found to be heterozygous for the mutation .
a family tree of the patient showing the mode of inheritance of the mutation is shown in figure 2 .
amino acid alignment of the human tpo proteins with mouse , rat , pig , dog , chicken , and frog indicated that val-501 residue is located within a highly conserved region ( figure 3 ) .
in silico analyses using polyphen-2 and
( sift score of 0 ) , respectively ( data not shown ) . the generated three - dimensional homology models based on 21kc template ( 47% sequence similarity ) covered 62% of the full sequence of tpo protein ( figure 4 ) .
ramachandran plots from procheck showed that the wild type and the mutant homology models had 2 and 7 amino acid residues located in the disallowed regions ( regions which are disallowed for all amino acids due to steric clashes , except for glycine which lacks a side chain ) , respectively .
verify3d showed that the wild type and mutant models had passed as being good models by having 87.2% and 90.8% of residues with an average 3d to 1d score of more than 0.2 , respectively .
errat 's results indicated that the two models had overall quality factor of about 88 over 100 ( the decrease in value is due to flexible loop regions which could not be aligned with available crystal structures ) .
comparison between predicted tertiary structures of the wild type and mutant proteins revealed that the h - bond between the backbone oxygen of leu-517 and amide hydrogen of mutant residue gly-501 ( figure 5(a ) ) contributed to the formation of a beta sheet in the mutated region ( figure 5(b ) ) , which requires beta strands to be connected laterally by hydrogen bonds .
significant structural alterations in the annotated binding pocket of heme ( glu-399 ) and iron binding site ( his-494 ) were also detected in the p.val501gly mutant ( figure 5(c ) ) .
mng associated with ch , which is more uncommon , is often caused by defective tpo gene [ 15 , 34 , 35 ] . in the present study
, we identified a novel , homozygous c.1502t > g mutation in the tpo gene in two siblings from consanguineous parents , with severe goitrous ch .
the mutation was not detected in 100 chromosomes from 50 normal unrelated individuals suggesting that the c.1502t > g alteration is not a polymorphism .
val-501 is conserved amongst many animals including mice and rats implying its importance in the structure / function of the tpo .
valine is seldom directly involved in protein function due to its very nonreactive side chain .
alteration of amino acid in this region is more likely to affect the protein conformation .
moreover , replacing valine with glycine might lead to less hydrophobic interactions as the former has a larger aliphatic side chain while the latter has h as its side chain .
nonetheless , tertiary modeling showed structural alterations in the tpo mutant , suggesting the mutation could cause structural instability of the protein .
a proposed iron ( heme axial ligand ) binding site , his-494 , is located six residues away from the mutation site .
the early step in the biosynthesis of thyroid hormones involves the oxidation of the iodide ( i ) to iodinium ( i ) ions by tpo in the presence of h2o2 . the heme iron in the tpo plays an important role in the redox reaction .
in this study , 3d analysis showed that the iron binding site of the p.val501gly mutant tpo is slightly exposed than that of the wild type ( figure 5(c ) ) .
it is very likely that the additional entry in the proposed binding pocket of mutant tpo would interfere with the binding of the gas ligand and/or cause improper conformational changing after binding .
alternatively , the absence of a relatively bulky aliphatic side chain , due to gly-501 lacking a side chain in p.val501gly mutant tpo , could alter the hydrophobic pocket and thus could interfere with the heme binding at glu-399 .
interestingly , rivolta et al . in 2003 reported that p.pro499leu mutation in the tpo which is also located within the hydrophobic pocket was associated with severe goitrous ch .
we therefore believed that this region is significantly important for the normal activity of the protein .
the mechanism by which p.val501gly in the tpo may induce mng remains to be elucidated , although the tertiary structure of the protein appeared to be modified .
considering that there is an increased risk of thyroid carcinoma in mng patients , it is important to have a careful surveillance for potential thyroid neoplasm in these patients .
in conclusion , we demonstrated a novel , homozygous c.1502t > g mutation ( p.val501gly ) in the tpo gene which could be associated with dyshormonogenetic ch and mng in members of a malaysian - indian family . | congenital hypothyroidism ( ch ) with multinodular goiter ( mng ) is uncommonly seen in children .
however , ch associated with goiter is often caused by defective thyroid peroxidase ( tpo ) gene . in this study , we screened for mutation(s ) in the tpo gene in two siblings with ch and mng and their healthy family members .
the two sisters , born to consanguineous parents , were diagnosed with ch during infancy and received treatment since then .
they developed mng during childhood despite adequate l - thyroxine replacement and negative thyroid antibody screening .
pcr - amplification of all exons using flanking primers followed by dna sequencing revealed that the two sisters were homozygous for a novel c.1502t > g mutation .
the mutation is predicted to substitute valine for glycine at a highly conserved amino acid residue 501 ( p.val501gly ) .
other healthy family members were either heterozygotes or mutation - free .
the mutation was not detected in 50 healthy unrelated individuals . in silico analyses using polyphen-2 and
sift predicted that the p.val501gly mutation is functionally damaging .
tertiary modeling showed structural alterations in the active site of the mutant tpo . in conclusion , a novel mutation , p.val501gly , in the tpo gene
was detected expanding the mutation spectrum of tpo associated with ch and mng . |
brucellosis is a worldwide zoonotic disease that accounts for huge loses to the livestock sector and poses a serious threat to public health .
the disease is caused by bacteria of the genus brucella , a member of the -2 proteobacteria .
brucellae are gram - negative , facultative , intracellular bacteria that can infect a wide range of domestic and wild animals as well as humans .
six classical species were initially recognized within the genus brucella , namely , b. abortus , b. melitensis , b. suis , b. ovis , b. canis , and b. neotomae .
brucella invades and replicates in professional phagocytic cells such as macrophages and dendritic cells as well as nonprofessional phagocytes such as trophoblasts [ 35 ] .
brucella mechanisms and virulence factors that mediate invasion and intracellular persistence have been poorly characterized .
brucellosis remains endemic and is a reemerging disease in many regions of the world including latin america , middle east , africa , central asia , and the mediterranean basin that affects human health and animal productivity .
the disease leads to a significant detrimental effect on local economies resulting in the perpetuation of poverty .
brucellosis is a serious veterinary and public health problem in india and the disease is reported in cattle , buffalo , sheep , goats , pigs , camel , dogs , and humans .
brucellosis is an endemic disease in india and the country experienced a sharply increasing rate of human brucellosis in recent years , and the species of main concern is b. melitensis .
b. melitensis , the most virulent species in humans , primarily affects goats and the zoonotic transmission occurs by ingestion of unpasteurized milk products or through direct contact with fetal tissues . genetic diversity and population structure of brucella spp .
multilocus sequence typing ( mlst ) has been considered as the robust tool for dissecting genetic diversity and population structure within the bacterial species .
the established mlst schema for brucella spp . employed nine highly distinct genomic loci . however , mlst resolution is limited and often fails to differentiate closely related strains . with the advent of next generation sequencing , whole
genome sequencing and analysis of brucella spp . from diverse hosts and geographical regions have been reported . until now
, 61 genomes of b. melitensis have been sequenced and made available in the genbank .
these sequenced species serve as vast resources for comparative genomics and understanding the evolutionary history .
comparative genomics will provide insights into the virulence mechanisms of the brucella spp . such as novel genomic islands and integration of prophages and
snps that regulate the expression of certain genes or affect the function of important virulence - associated proteins .
one of the previous studies demonstrated conservation of genes and genomic islands across the different brucella spp . .
in a recent study , the evolutionary relationship of b. melitensis on the basis of whole genome snps revealed spatial clustering of b. melitensis isolates into five genotypes .
all the asian isolates of b. melitensis clustered into genotype ii , whereas the isolates from europe and america clustered into genotypes iv and v , respectively .
we performed genotyping by mlst on b. melitensis strains isolated from naturally infected goats . to perform detailed genetic characterization and comparative genome analysis , we performed whole genome sequencing of one of the strains , b. melitensis ind1 , using the illumina platform .
the analysis revealed the extent of genetic variation of b. melitensis ind1 in comparison to b. melitensis isolates from other geographical locations .
data generated from our studies may help to develop new diagnostic assays based on stable markers such as snps ( single nucleotide polymorphisms ) and variable number of tandem repeats ( vntrs ) for molecular epidemiological studies .
identification of snps , indels , and novel phage integration sites will provide insights into the virulence mechanisms of this stealthy pathogen which could ultimately lead to the development of novel therapeutic and preventive strategies to control brucellosis .
for isolation of brucella , materials from four different sources are listed in the supplementary table 1 ( in supplementary material available online at http://dx.doi.org/10.1155/2016/3034756 ) .
samples were inoculated on sterile plates of brucella selective agar containing hemin and vitamin k1 media ( hi media , india ) and incubated at 37c for 48 hours .
the plates were observed at every 24 hours for the development of growth . after obtaining the growth ,
the colonies suspected for brucella on the basis of cultural characteristics were selected and streaked again on plates containing brucella selective agar with hemin and vitamin k1 and incubated at 37c for 2 days to obtain the pure culture .
cultures showing typical brucella characteristics were subjected to biotyping techniques such as h2s production , growth in the presence of thionin , and basic fuchsin ( 1040 g / ml ) dye incorporated into tryptic soy agar at different concentrations and co2 requirement immediately after the primary isolation as described .
lead acetate strips were used to identify the production of h2s during growth , and the growth was evaluated on media containing streptomycin ( 2.5 g / ml ) to discriminate the isolates from vaccine strain rev1 as described .
genomic dna of all five strains was isolated using the wizard genomic dna purification kit ( promega , usa ) .
isolated dna was used for polymerase chain reactions to amplify 16s rrna and the omp 31 gene for the confirmatory identification of brucella melitensis using the taq pcr master mix kit ( qiagen ) .
16s rrna is specific to the genus brucella while omp 31 is a species - specific gene to brucella melitensis [ 1416 ] . for mlst analysis ,
of the nine loci , seven belong to classical housekeeping genes , one locus derived from the outer membrane protein 25 gene , and one from an intergenic region .
genomic dna was isolated from bm ind isolates using the wizard genomic dna purification kit ( promega ) .
genomic fragments were amplified by pcr using the following cycling parameters : 94c for 2 min , 35 cycles of 94c for 30 sec , 53c for 30 sec , and 72c for 1 min and 72c for 5 min .
remaining pcr products were purified and subjected to sanger sequencing using the forward and reverse primers that were used for pcr amplifications .
editing of the sequences and generation of contigs from forward and reverse sequences was performed using lasergene 8 software ( dna star , usa ) . to perform the phylogenetic analysis , nine genomic fragments mentioned above
were fetched from representative brucella species and the loci were amplified in silico using the mlst primers .
all the sequences were concatenated to identify the allelic profile with the help of brucellabase and the concatenated sequences were subjected to multiple sequence alignment using mafft version 7.123b .
the complete genome of brucella melitensis ind1 ( bm ind1 ) was sequenced using illumina technology .
the sequenced data have been deposited at ddbj / embl / genbank under the accession number jmkl00000000 .
paired end data generated were filtered for low quality reads using in - house script .
after preprocessing , high quality data were used to make a scaffold level assembly using the soapdenovo version 2.01 assembler .
scaffolds were further mapped with raw reads using bowtie2 version 2.2.4 , and coverage at each base was calculated using samtools version 0.1.19 .
completely sequenced genomes of b. melitensis such as b. melitensis 16 m , b. melitensis m28 , b. melitensis atcc23457 , and b. melitensis ni were initially considered as candidates for the template to construct the chromosomal assembly of bm ind1 .
the raw data of bm ind1 were aligned onto all the genomes using bowtie2 and the snps were identified using samtools , bcftools , and vcftools ( http://vcftools.sourceforge.net/ ) .
highly confident snps were filtered out using scripts from ismu pipeline based on the criteria that the raw read depth is greater than 10 and there is no reference base in the alignment .
finally , b. melitensis atcc 23457 that showed the minimum number of snps with bm ind1 was used as the template for chromosome level assembly .
bm ind1 chromosomal level assembly was further manually curated using the blast output of bm ind1 scaffolds with the b. melitensis atcc 23457 genome .
we compared the syntenic relationships between bm ind1 , b. melitensis atcc 23457 , and b. melitensis 16 m using mauve version 2.3.1 .
the structural and functional annotations of bm ind1 genome were carried out by rast server .
first , we assessed the completeness of the assembly and annotations of each sequenced genome and filtered out the incomplete ones . to assess the core genome and single copy orthologs , orthomcl v.1.4 was used with default parameters .
we used 2319 single copy orthologs to construct a maximum - likelihood tree following the approach of wattam et al . .
raxml version 8.1.21 was used to generate a tree for all the dataset using the protgammalg model of evolution .
the genome was searched for prophage sequences and phage attachment site using phast ( phage search tool ) , available at http://phast.wishartlab.com/ .
a precompiled tandem repeat finder version 4.07b was downloaded from http://tandem.bu.edu/trf/trf.download.html and run on linux ( 64-bit ) platform using the parameter of a minimum alignment score of 80 .
we downloaded the genome sequences of b. melitensis 16 m , b. melitensis m28 , b. melitensis atcc 23457 , b. melitensis m5 - 90 , b. melitensis ni , b. melitensis ether , and b. abortus 2308 for snp analysis .
we established a pipeline for finding snps between two reference sequences using nucmer and show - snps program from the mummer3 package .
snps were extracted from each strain against bm ind1 and the data were further annotated using snpeff to predict snp effects in the genome . to find insertions and deletions in the coding region ,
vcf files generated against b. melitensis atcc 23457 using bm ind1 reads for template genome selection were annotated with snpeff .
indels in the coding regions and their corresponding functions were extracted from b. melitensis atcc 23457 using in - house perl script .
we isolated four strains of b. melitensis from naturally infected goat followed by mlst analysis to understand the genetic diversity among the b. melitensis ind strains . to perform mlst analysis , we amplified nine loci that included seven housekeeping genes , one locus from the outer membrane protein 25 ( omp25 ) , and one locus from an intergenic region ( supplementary figure 1 ) .
the inclusion of loci from omp25 and intergenic region was reported to have provided more discriminatory power in the phylogenetic analysis .
subsequently , we compared the allelic profiles of the four b. melitensis ind isolates with each other and with other reported brucella species .
all the b. melitensis ind isolates displayed identical sequences with an allelic profile of 3 - 2 - 3 - 2 - 1 - 5 - 3 - 8 - 2 , which belong to sequence typing- ( st- ) 8 .
the phylogram was rooted with b. microti and in the phylogram all the b. melitensis isolates clustered into one lineage ( figure 1 ) .
bm ind strains grouped with other asian strains , that is , b. melitensis m28 , b. melitensis m5 - 90 , and b. melitensis ni , whereas b. melitensis ether and b. melitensis 16 m branched separately .
this was anticipated as all the asian strains belong to st-8 and b. melitensis ether and b. melitensis 16 m falls into st-9 and st-7 , respectively .
in fact , the support value is very low for the branches of asian isolates in the phylogram owing to the same allelic profile ( st-8 ) of mlst loci that were considered for the phylogenetic analysis .
as expected , b. suis and b. ovis clustered into different clades in the phylogram ( figure 1 ) .
we performed the whole genome sequencing of bm ind1 to analyze the genetic divergence and genomic features in detail .
this provided 102 contigs that were further assembled into 29 scaffolds ( table 1 ) .
each scaffold base was covered more than 100 times ( 100x ) ; however , some scaffolds like scaffolds 9 , 14 , 18 , 20 , and 22 have shown high depth of coverage ( supplementary figure 2 ) .
this is likely due to the presence of duplicated or repeat regions of the genome . to generate chromosome level assembly ,
. generally , a reference genome of a closely related species is used as the template to align and order the scaffolds . to achieve this
, we considered genomes of b. melitensis 16 m reported from usa , b. melitensis ni from mongolia , b. melitensis ether from italy , b. melitensis atcc 23457 from india , and the b. melitensis m28 from china .
all these genomes are completely sequenced and well annotated and the genome assembly of few has previously been used as reference genomes for other strains [ 35 , 36 ] .
we aligned the raw data of bm ind1 on the genomes of above b. melitensis strains using bowtie2 and identified the snps ( supplementary table 2 ) .
since b. melitensis atcc 23457 displayed the least number of snps , which indicates minimum genetic divergence , we selected the genome of this strain as the reference genome for chromosomal assembly of bm ind1 . b. melitensis ind1 and b. melitensis atcc 23457 were isolated from india ; however , they belong to different biovars .
bm ind1 scaffolds were assembled into two chromosomes using b. melitensis atcc 23457 as the template .
total number of scaffolds assigned on chromosomes i and ii are 23 and 6 , respectively .
subsequently , assembly was manually curated with a focus on those scaffolds that showed higher physical coverage to fix the duplications .
we observed the duplications of scaffolds 20 , 22 , 27 , 28 , and 29 on chromosome i and scaffold 26 on chromosome ii with respect to the b. melitensis atcc 23457 genome .
however , no duplication in other scaffolds was observed , especially scaffolds 9 and 14 that showed high coverage ( supplementary figure 2(b ) ) .
we assume that the higher coverage in these scaffolds may be due to internal repeats or sequencing bias .
next , we aligned the genomes of b. melitensis atcc 23457 , b. melitensis 16 m , and bm ind1 and observed for macrolevel synteny and large genomic rearrangements .
in fact , they were highly syntenic with each other except for one segment of b. melitensis 16 m on chromosome ii which was in reverse orientation in b. melitensis atcc 23457 and bm ind1 ( figure 2(a ) ) .
all general features of the genome are summarized in figures 2(b ) and 2(c ) .
we annotated the genome of b. melitensis bm ind1 using rapid annotations with subsystems technology ( rast ) to obtain the coding and noncoding genes .
a total of 55 trna , 12 rrna , and 3191 protein coding genes with an average cds length of 874 bp were annotated ( table 2 ) .
in addition , rast annotates the genomic structures and assigns their functions on the basis of presence of subsystems in the genome .
this makes functional annotation of genes more accurate than simply assigning the functions on the basis of sequence similarity of known genes .
functions of 2562 genes were assigned while 629 genes were annotated as hypothetical ( table 2 ) .
a total of 1649 genes were assigned for different subsystems where maximum number ( 405 ) was assigned for metabolism of amino acids followed by carbohydrate metabolism ( 331 ) ( figure 3 ) .
determining the phylogenetic relationship in a bacterial population is essential to understand the population structure , evolutionary history , and host relationship and to develop diagnostic assays for molecular epidemiological studies . to perform comparative phylogenomics
, we downloaded all the currently available b. melitensis genomes ( 59 genomes ) from genbank and considered b. abortus 2308 as the outgroup species .
we evaluated the completeness of assembly and annotation of the genomes by assessing the number of orthologous genes , which are highly conserved among the strains .
any b. melitensis strain showing less number of orthologous genes than the number of orthologous genes present in b. abortus with respect to b. melitensis were ignored for the downstream phylogenomic studies .
this facilitates more accurate core genome estimation and identification of single copy orthologs present in the species , which improves resolution of the phylogenetic tree .
therefore , we ignored the genomes of 12 b. melitensis strains and considered the entire repertoire of coding genes of 48 b. melitensis strains including bm ind1 for the analysis ( supplementary table 3 ) .
a total of 151361 genes of b. melitensis strains were clustered in 3800 gene families , of which 25124 gene families present in all the 48 strains .
however , 2461 genes only showed exact single copy orthology in each strain that could be considered as the core genome of b. melitensis clade .
reported that 2,285 core genes are present in the brucella genus by analyzing the genomes of 40 brucella species . conceivably , the core genome of b. melitensis clade was higher than the total number of core genes present across the brucella genus .
we used b. abortus 2308 as the outgroup for whole genome phylogeny that increased the total cluster of genes to 3829 . after including the b. abortus strain ,
the total number of single copy orthologs decreased to 2319 genes . in the whole genome phylogram , bm ind1 clustered with other asian isolates of b. melitensis as observed in the mlst analysis ( figure 4 ) .
bm ind1 grouped with b. melitensis ni which was originated from mongolia and both the strains belong to biovar 3 ( figure 4 ) .
the phylogenetic relationship established here is in agreement with the earlier reports [ 10 , 11 ] .
tan et al . performed a comparative whole genome snp analysis of b. melitensis strains from around the world and reported clustering of b. melitensis isolates into five genotypes . in agreement with this observation
, our phylogenomic studies also revealed the clustering of b. melitensis isolates into five groups where group 1 formed the earliest diverging clade .
parallel to group ii , another lineage evolved which further branched into groups iii , iv , and v. group iii represents isolates from africa and groups iv and v constitute isolates from europe and america , respectively .
prophages that are integrated into the genome of bacteria can contribute many biological properties to their bacterial hosts such as virulence , biosynthesis , and secretion of toxins , genomic divergence , and evolution .
we analyzed the genome of bm ind1 for prophages using phast that was designed to identify and annotate prophage sequences in bacterial genomes .
the analysis identified 2 putative prophage integrations in chromosome i of bm ind1 ( table 3 and figure 5 ) .
region 1 is composed of a fragment of 13.7 kb size that encoded 18 genes , out of which 14 genes were phage specific and 4 genes were bacteria specific .
region ii is composed of 22.6 kb with 14 genes where 8 genes were phage specific and remaining 6 genes belonged to brucella .
notably , region 1 is considered as intact prophage upstream of qseb locus and the rast server could identify the genes in this region .
we identified two putative phage integrations in chromosome i of b. melitensis 16 m genome , which did not show any similarity to that of bm ind1 or b. melitensis m28 .
the analyses clearly indicate that the prophage integration events contribute to the genetic diversity of b. melitensis .
vntrs play an important role in evolution , gene regulation , genome structure , antigenic variation and virulence [ 3840 ] .
mutations in vntrs produce a wide range of allelic diversity and vntrs are considered as a powerful technique in molecular typing of bacterial species [ 4143 ] .
we have analysed the genome of bm ind1 and identified 78 vntrs with dna motif size ranges from 8 to 30 bps and the copy number ranges from 1.9 to 10.4 ( supplementary table 4 ) . the data generated in our analysis could be used for developing rapid diagnostic assays for high - resolution molecular epidemiological and clinical studies .
snps data will help to develop novel high - resolution molecular typing techniques for inter- and intraspecies discrimination of pathogenic microorganisms .
we compared the genome of bm ind1 with seven other b. melitensis strains , that is , b. melitensis 16 m , b. melitensis m28 , b. melitensis atcc 23457 , b. melitensis m5 - 90 , b. melitensis ni , b. melitensis ether , and b. abortus 2308 for snps
the highest number of snps was detected with b. abortus as it belonged to a different species ( table 4 ) .
four b. melitensis strains that are originated from asia , namely , b. melitensis m28 bv1 , b. melitensis m5 - 90 bv1 , b. melitensis atcc 23457 bv2 , and b. melitensis ni bv3 exhibited fewer snps ranging from 252 to 351 indicating their close genetic relatedness irrespective of their biovars .
this observation was in agreement with the reported snp - based phylogenetic analysis by tan et al .
however , the snps observed with different strains in additional file 4 were less than the snps detected from ngs raw reads for template genome selection .
this is because the polymorphisms extracted in these cases were derived from uniquely aligned regions between two genome sequences .
most of the identified snps were in the coding regions of the genomes that may be attributed to the high proportion of coding regions in bacteria . while analysing the distribution of snp locus among all asian strains , 142 snps were shared by four strains , namely , b. melitensis m28 , b. melitensis atcc 23457 , b. melitensis m5 - 90 , and b. melitensis ni ( figure 7 ) .
out of 142 snps , 141 are shared by all the 7 strains included in the polymorphism analysis ( table 5 ) .
therefore , these 141 unique loci in bm ind1 could be employed for genotyping and other molecular epidemiological studies .
b. abortus 2308 , b. melitensis ether , and b. melitensis 16 m shared the maximum number of snps ( 952 ) against bm ind1 .
these 952 loci are conserved in all asian strains , which may indicate that asian strains evolved from a common ancestor , and these loci mutated before its differentiation .
this finding is in agreement with whole genome phylogenetic analysis where b. melitensis m28 , b. melitensis m5 - 90 , b. melitensis ni , and bm ind1 clustered together as a separate clade ( figure 4 ) .
however , 4864 snp loci are highly specific to b. abortus 2308 , which are not shared by any of the six b. melitensis strains .
these loci with interspecific polymorphisms can differentiate these two species in clinical and epidemiological studies .
also , identified snps that are unique to each b. melitensis strain could be employed for in - depth molecular analysis and development of novel molecular typing tools .
we also categorized the genes containing snps based on their functions assigned by rast server ( supplementary figure 3 ) .
the subsystem category , which has shown the highest proportion of genes containing snps , is nitrogen metabolism followed by phosphorous , carbohydrate , and amino acid metabolism .
our findings are in agreement with differential utilization of carbohydrates and amino acids by closely related brucella species and biovars .
a biotyping system has recently been developed to discriminate brucella species and biovars based on their differential metabolic activity .
indels refers to deletions or insertions of nucleotides in one genome with respect to another , which could be employed as sequence signatures to characterize evolution of diverged organisms .
indels can have a drastic effect on a gene leading to frameshift , truncations , or extensions of an encoded protein .
we have identified 51 indels in the bm ind1 genome with respect to b. melitensis atcc 23457 , of which 25 are located in the coding regions ( table 6 ) .
one noted indel is in the glycerol-3-phosphate dehydrogenase gene ( glpd ) of bm ind1 .
insertion of a guanosine residue in the 3 end of glpd resulted in two amino acid mismatch followed by deletion of 69 amino acids from the c - terminus of this protein .
we verified this indel by pcr amplification and sequencing of corresponding regions from the glpd gene of b. melitensis ind strains including bm ind1 .
insertion of g was present in the glpd gene of all the four b. melitensis ind strains with respect to atcc23457 and 16 m ( figure 8) .
it has been reported that b. melitensis 16 m deficient in glpd was attenuated in human and mouse macrophages [ 47 , 48 ] .
however , our preliminary infection studies did not indicate attenuation of bm ind1 in mouse peripheral blood mononuclear cells .
our future studies will focus on in vitro and in vivo infections studies using bm ind1 to analyse its virulence properties with the objective of developing novel live attenuated vaccines for livestock brucellosis .
in conclusion , genomic characterization and comparative genome analysis of bm ind1 revealed genetic structure of b. melitensis from india as well as from other geographical locations .
comparative genome analysis identified the sources of genetic variation in the form of snps , vntrs , prophages , and indels .
these genetic markers could be employed for developing high - resolution epidemiological typing tools to understand the structure of brucella population and for outbreak analysis .
information on prophage integration events and indels in the virulence - associated genes will provide important leads for the further experimental characterization of virulence properties of bm ind1 .
this may ultimately lead to the development of efficient therapeutic and preventive strategies to control animal and human brucellosis . | brucellosis is the most frequent zoonotic disease worldwide , with over 500,000 new human infections every year .
brucella melitensis , the most virulent species in humans , primarily affects goats and the zoonotic transmission occurs by ingestion of unpasteurized milk products or through direct contact with fetal tissues .
brucellosis is endemic in india but no information is available on population structure and genetic diversity of brucella spp . in india .
we performed multilocus sequence typing of four b. melitensis strains isolated from naturally infected goats from india . for more detailed genetic characterization
, we carried out whole genome sequencing and comparative genome analysis of one of the b. melitensis isolates , bm ind1 .
genome analysis identified 141 unique snps , 78 vntrs , 51 indels , and 2 putative prophage integrations in the bm ind1 genome .
our data may help to develop improved epidemiological typing tools and efficient preventive strategies to control brucellosis . |
typical pulmonary carcinoids , in general , have good prognosis in contrast to atypical carcinoids .
it is important if this differentiation can be made in the preoperative stage as it has therapeutic as well as prognostic implications . to date ,
structural imaging findings are similar in both and differentiation between typical and atypical carcinoids is not possible .
[ f]fluorodeoxyglucose ( fdg)-positron emission tomography ( pet)/computed tomography ( ct ) has been found to be of limited use in the carcinoid tumours , especially the typical ones , due to their indolent growth and low glucose turnover .
these somatostatin receptors allow functional imaging of these tumours with radiolabelled somatostatin analogues like [ ga]1,4,7,10-tetraazacyclododecane - n - tetraacetic acid-(d)-phe - thy - octreotide ( dotatoc ) .
the density of these receptors is related to the degree of the tumour differentiation , the most well - differentiated ones ( i.e. typical carcinoids ) expressing the highest density .
this report analyses our experience with a combination of [ f]fdg - pet / ct and [ ga]dotatoc - pet / ct in 20 patients with pulmonary carcinoids to assess the possibility of differentiating the 2 histopathologic variants based on the uptake patterns .
ethical clearance was obtained from the institutional ethics committee for this retrospective evaluation of the patients records .
all the patients had a thorough clinical examination , haemogram , routine biochemistry , structural imaging by contrast - enhanced ct of the chest , [ f]fdg - pet / ct , [ ga]dotatoc - pet / ct , bronchoscopy ( with biopsy ) , followed by surgery ( except case 15 ) .
the results were analysed against the histopathologic classification ( typical or atypical ) , which was done as per the who criteria by a pathologist who had more than 20 years experience in this field .
the tissue diagnosis was obtained either by biopsy or histopathologic examination of the operative specimen ( in the rest of the cases ) .
the statistical analysis was done using spss statistical data editor ( version 17 ) . due to the non - parametric nature of the data ( as shown by independent sample t - test ) , median values of standardized uptake value ( suv)max was calculated . the mann
whitney test ( non - parametric test ) was used to calculate the statistical differences in suvmax values of the carcinoids on both [ f]fdg - pet / ct and [ ga]dotatoc - pet / ct
pet images were taken on a dedicated pet / ct scanner ( siemens biograph 64 , usa)with lutetium oxyorthosilicate ( lu2sio5):ce detectors with attenuation coefficient of 0.89 cm , photofraction of 30% , decay constant of 40 ns , and the energy resolution at 511 kev with spatial resolution of 6 mm .
after fasting for at least 4 h , verifying the serum glucose level ( which should be < 140 mg / dl ) and with patients in a resting state , in a quiet room , a dose of 370 mbq ( 10 mci ) of [ f]fdg was injected intravenously . after a 4560 min uptake period ,
ct acquisition was performed on spiral dual slice ct with a slice thickness of 4 mm and a pitch of 1 .
images were acquired using a matrix of 512 512 pixels and pixel size of about 1 mm .
after transmission imaging , three - dimensional pet acquisition was taken for 35 min per bed position for one or two bed positions .
pet images were reconstructed using an iterative method with ordered subset expectation maximization ( 2 iterations and 8 subsets ) with a 5-mm filter .
after completion of pet acquisition , the reconstructed attenuation corrected pet images , ct images and fused images of matching pairs of pet and ct images were available for review .
[ ga]dotatoc - pet / ct was obtained after intravenous injection of 74111 mbq ( 23 mci ) of radiotracer . during the pet / ct acquisition
all pet / ct parameters were kept the same as during [ f]fdg - pet / ct , except for the timing of the three - dimensional pet acquisition , which was 58 min per bed position .
after the dotatoc - pet acquisition , the reconstructed attenuation corrected pet images , ct images and fused images of matching pairs of pet and ct images were available .
a gap of at least 24 h was given between the [ f]fdg - pet / ct and [ ga]dotatoc - pet / ct scans to ensure clearance of the radiotracer and to avoid interference between the 2 radiotracers .
two experienced nuclear medicine physicians evaluated the findings independently and they were blinded to the clinical history , and the clinical and structural imaging findings .
the corresponding areas in the ct images and fused pet - ct images were corroborated .
the suvmax values of the tumour tissue were calculated . for semi - quantitative analysis ,
a region of interest ( roi ) was drawn around the site of the abnormal uptake in the consequent 46 pet / ct slices .
the slice with maximal uptake in the roi was chosen for measurement of metabolic activity of the tracer ( suv ) . from these rois ,
pet images were taken on a dedicated pet / ct scanner ( siemens biograph 64 , usa)with lutetium oxyorthosilicate ( lu2sio5):ce detectors with attenuation coefficient of 0.89 cm , photofraction of 30% , decay constant of 40 ns , and the energy resolution at 511 kev with spatial resolution of 6 mm .
after fasting for at least 4 h , verifying the serum glucose level ( which should be < 140 mg / dl ) and with patients in a resting state , in a quiet room , a dose of 370 mbq ( 10 mci ) of [ f]fdg was injected intravenously . after a 4560 min uptake period ,
ct acquisition was performed on spiral dual slice ct with a slice thickness of 4 mm and a pitch of 1 .
images were acquired using a matrix of 512 512 pixels and pixel size of about 1 mm .
after transmission imaging , three - dimensional pet acquisition was taken for 35 min per bed position for one or two bed positions .
pet images were reconstructed using an iterative method with ordered subset expectation maximization ( 2 iterations and 8 subsets ) with a 5-mm filter .
after completion of pet acquisition , the reconstructed attenuation corrected pet images , ct images and fused images of matching pairs of pet and ct images were available for review .
[ ga]dotatoc - pet / ct was obtained after intravenous injection of 74111 mbq ( 23 mci ) of radiotracer . during the pet / ct acquisition
all pet / ct parameters were kept the same as during [ f]fdg - pet / ct , except for the timing of the three - dimensional pet acquisition , which was 58 min per bed position .
after the dotatoc - pet acquisition , the reconstructed attenuation corrected pet images , ct images and fused images of matching pairs of pet and ct images were available .
a gap of at least 24 h was given between the [ f]fdg - pet / ct and [ ga]dotatoc - pet / ct scans to ensure clearance of the radiotracer and to avoid interference between the 2 radiotracers .
two experienced nuclear medicine physicians evaluated the findings independently and they were blinded to the clinical history , and the clinical and structural imaging findings .
the corresponding areas in the ct images and fused pet - ct images were corroborated .
the suvmax values of the tumour tissue were calculated . for semi - quantitative analysis ,
a region of interest ( roi ) was drawn around the site of the abnormal uptake in the consequent 46 pet / ct slices .
the slice with maximal uptake in the roi was chosen for measurement of metabolic activity of the tracer ( suv ) . from these rois ,
the patient characteristics , results of structural and functional imaging and the final histopathology are described in table 1 .
the mean age of the patients was 32.7 years ( range 1653 years ) .
table 1patient characteristics , structural and functional imaging , and final histopathologycase no.age ( years)ctsuvmax on [ f]fdg - pet / ctsuvmax on [ ga]dotatoc- pet / ctratio of suvmax of dotatoc / suvmax of fdghpetumour size ( mm)tumour site1403.2 1.8r int .
br1.323.217.8tc2161.5 0.9l main br3.15818.7tc3353.8 3.7r main br3.24313.4tc4442.5 5r ll br1.4117.8tc5401 0.7posterior basal segment of l ll1.08.88.8tc6236.8 5r hilar area2.83311.7tc7403.6 3.4r ll br2.66023.1tc8454 6l main br1.73319.4tc9251.8 1.5r main br0.82025tc10223 2.5r int .
br2.64517.3tc13223.2 2.8r main br3.06622tc14538 7r main br2.96.32.1ac15405 7.1r main br + calcification6.418.52.8ac16344 4.8r ll3.112.33.9ac17161.5 1.5l upper lobe bronchus3.913.93.6ac18313.4 4.7r hilar mass , ll bronchial division5.91.10.2ac19295.5 1.5l main br6.810.31.5ac20364.4 3.8r main br8.44.70.5acac , atypical carcinoid ; br , bronchus ; hpe , histopathological examination ; int . ,
intermediate ; l , left ; ll , lower lobe ; pe , pleural effusion ; r , right , suv , standardized uptake value ; tc , typical carcinoid .
patient characteristics , structural and functional imaging , and final histopathology ac , atypical carcinoid ; br , bronchus ; hpe , histopathological examination ; int . , intermediate ; l , left ; ll , lower lobe ; pe , pleural effusion ; r , right , suv , standardized uptake value ; tc , typical carcinoid . out of the 20 patients , 13 were fund to have typical carcinoid and 7 had atypical carcinoid . the mean age of the patients with typical carcinoids was 32 years while that of patients with atypical carcinoids was 34.1 years .
the suvmax of all carcinoids on [ f]fdg - pet / ct ranged from 0.8 to 8.40 ( median suvmax , 2.8 ) .
the suvmax of typical carcinoids on [ f]fdg - pet / ct ranged from 0.8 to 3.20 ( median suvmax , 2.6 ) .
the [ f]fdg - pet / ct images of a typical carcinoid are shown in fig .
six cases of typical carcinoids failed to reveal any significant radiotracer uptake on [ f]fdg - pet / ct .
thus , the overall detection rate of [ f]fdg - pet / ct for typical carcinoid tumours was 53.85% .
all the atypical carcinoids revealed uptake on [ f]fdg - pet / ct ( detection rate , 100% ) .
the suvmax ranged from 2.9 to 8.4 ( median suvmax , 5.9 ) ( table 2 ) . due to the 6 false - negative tumours , the overall detection rate of [ f]fdg - pet / ct in our series was 70% .
atypical carcinoids revealed significantly higher uptake values on [ f]fdg - pet / ct compared with the typical carcinoids ( p = 0.001 ) .
figure 1pet / ct ( a ) and whole - body projection images ( b ) of a 40-year - old woman ( case 1 ) with typical carcinoid ( arrow ) revealing low tumour uptake on [ f]fdg - pet / ct ( suvmax , 1.3 ) .
pet / ct image ( c ) and whole - body projection image ( d ) of the same patient revealing high tumour uptake on [ ga]dotatoc - pet / ct ( suvmax , 23.2 ) .
table 2suvmax values and ratios of carcinoids on [ f]fdg - pet / ct and [ ga]dotatoc - pet / cttumour histologysuvmax on [ f]fdg - pet / ct ( median and range)suvmax on [ ga]dotatoc - pet / ct ( median and range)ratio of suvmax on [ ga]dotatoc - pet / ct to that on f18 fdg - pet / ctall carcinoids2.8 ( 0.88.4)21.6 ( 1.166)12.5 ( 0.230)typical carcinoids2.6 ( 0.83.2)33 ( 8.866)17.8 ( 7.830)atypical carcinoids5.9 ( 2.98.4)10.3 ( 1.118.5)2.1 ( 0.23.9 )
pet / ct ( a ) and whole - body projection images ( b ) of a 40-year - old woman ( case 1 ) with typical carcinoid ( arrow ) revealing low tumour uptake on [ f]fdg - pet / ct ( suvmax , 1.3 ) . pet / ct image ( c ) and whole - body projection image ( d ) of the same patient revealing high tumour uptake on [ ga]dotatoc - pet / ct ( suvmax , 23.2 ) .
suvmax values and ratios of carcinoids on [ f]fdg - pet / ct and [ ga]dotatoc - pet / ct all but one carcinoid revealed significant uptake on [ ga]dotatoc - pet / ct , with the suvmax ranging from 1.1 to 66 ( median suvmax , 21.6 ) .
all the typical carcinoids revealed significant uptake on [ ga]dotatoc - pet / ct ( detection rate , 100% ) .
the [ ga]dotatoc - pet / ct images of a typical carcinoid are shown in fig .
the suvmax of typical carcinoids ranged from 8.80 to 66 ( median suvmax , 33 ) .
one atypical carcinoid did not reveal any significant uptake on [ ga]dotatoc - pet / ct ( case 18 , fig .
2 ) thus the detection rate of [ ga]dotatoc - pet / ct for atypical carcinoids was 85.7% .
the suvmax in atypical carcinoids ranged from 1.1 to 18.5 ( median suvmax , 10.3 ) ( table 2 ) .
typical carcinoids revealed significantly higher uptake values on [ ga]dotatoc - pet / ct compared with the atypical carcinoids ( p = 0.002 ) .
figure 2[ga]dotatoc - pet / ct image of case 18 ( atypical carcinoid ) revealing no significant uptake in the tumour ( arrow ) ( suvmax , 1.1 ) .
[ ga]dotatoc - pet / ct image of case 18 ( atypical carcinoid ) revealing no significant uptake in the tumour ( arrow ) ( suvmax , 1.1 )
. the ratios of suvmax on [ ga]dotatoc - pet / ct to that on [ f]fdg - pet / ct were calculated for all the patients .
the ratio ranged from 7.8 to 30 in typical carcinoids ( median suvmax ratio , 17.8 ) , and from 0.20 to 3.9 in atypical carcinoids ( median suvmax of 2.1 ) ( table 2 ) .
this difference in the ratios between typical and atypical carcinoids was statistically significant ( p < 0.001 ) .
the world health organization s histological classification defines them as malignant tumours of epithelial origin with growth patterns that suggest neuroendocrine differentiation .
the distinction between typical carcinoids and atypical carcinoids is clinically important , because it has therapeutic as well as prognostic implications .
histopathologic features favouring the diagnosis of atypical carcinoids include increased mitotic activity , pleomorphism , irregular nuclei with hyperchromatism and prominent nucleoli , areas of increased cellularity with loss of architecture and areas of necrosis within the tumour .
typical carcinoid tumours have been found to have a much better prognosis with a reported 5-year survival of 87100% in contrast to the atypical carcinoids , which have a reported 5-year survival rate of 2569% .
pulmonary carcinoids are usually detected on structural imaging modalities like ct , which demonstrate a space - occupying lesion but do not have any specific characteristics to differentiate between the typical and atypical carcinoids .
the uptake patterns of pulmonary carcinoids on [ f]fdg - pet / ct are variable .
rege et al . reported the [ f]fdg - pet / ct findings in a patient with a pulmonary lesion with history of ectopic adrenocorticotrophic hormone secretion .
the lesion was hypometabolic on [ f]fdg - pet and was found be an indolent carcinoid at histology .
reported a series of 7 cases of pulmonary carcinoids of which 3 were visually negative on [ f]fdg - pet ( all typical carcinoids ) , 3 were hypometabolic ( 2 typical and 1 atypical ) and were erroneously categorized as benign nodules . only 1 case ( typical carcinoid ) revealed visual positivity and suvmax of 6.6 .
cheran et al . evaluated 3 patients with pulmonary carcinoids by [ f]fdg - pet and found them to have minimal or no uptake .
most of the tumours evaluated in our study , revealed low uptake on [ f]fdg - pet .
six cases failed to reveal any significant radiotracer uptake on [ f]fdg - pet / ct .
the overall detection rate of [ f]fdg - pet / ct in our series was 70% .
some studies have shown that atypical carcinoids reveal high suvmax values on [ f]fdg - pet due to their aggressive nature and thus , high metabolism .
wartski et al . evaluated 2 cases of pulmonary carcinoid , one typical and another atypical .
the typical carcinoids were 3.2 cm and 4.2 cm in size and revealed suv of 3.2 and 3.4 , respectively , on [ f]fdg .
the atypical carcinoids revealed suvs ranging from 3.3 to 7.1 except one , which had an suv of 1.7 .
daniels et al . reviewed 16 patients with bronchial carcinoid who underwent [ f]fdg - pet imaging .
they demonstrated a trend toward higher [ f]fdg - pet sensitivity for atypical carcinoid tumours ( 80% ) compared with typical carcinoid tumours ( 72.7% ) .
reported that typical pulmonary carcinoids revealed lower uptake values on [ f]fdg - pet / ct ( suvmax range 1.716 , median suvmax 4.9 ) compared with atypical and other less differentiated neuroendocrine tumours ( suvmax range 11.720 , median suvmax 16 ) . in our study , 6 typical carcinoids showed no significant uptake on [ f]fdg - pet / ct and the ones that did reveal uptake were mildly positive ( suvmax 2.63.2 ) .
all the atypical carcinoids revealed statistically significant higher uptake on [ f]fdg - pet / ct compared with typical carcinoids ( table 2 ) .
carcinoid tumours are rich in somatostatin receptors ( ssr ) , so they typically show high uptake on [ ga]dotatoc - pet / ct .
various studies in the literature have demonstrated the utility of [ ga]dotatoc - pet / ct in the evaluation of carcinoids but the literature on its use in pulmonary carcinoids is scanty .
evaluated 8 patients with histologically proven metastatic carcinoid tumours by [ in]octreotide scintigraphy and [ ga]dotatoc - pet / ct ( 6 abdominal and 2 bronchial ) . [ ga]dotatoc - pet could identify all lesions in 8 patients whereas [ in]octreotide identified 85% of lesions identified primarily by the morphological imaging .
evaluated 15 cases of carcinoids , including 2 cases of pulmonary carcinoids using [ f]fdg - pet and [ ga]dotatoc - pet / ct and reported an overall sensitivity of 68.2% and 92% , respectively .
gabriel et al . evaluated 84 cases of neuroendocrine tumours comprising 5 bronchial carcinoids using [ ga]dotatoc - pet .
they reported a higher sensitivity for tumour detection by [ ga]dotatoc compared with single photon emission - computed tomography ( spect ) or ct with a sensitivity of 97% and a specificity of 92% .
the typical carcinoids have been reported to express the somatostatin receptors more abundantly compared with atypical carcinoids .
thus , it seems logical that the typical carcinoids show higher uptake of radiotracer on somatostatin receptor - based imaging studies .
this was confirmed in a study by kayani et al . who found that typical carcinoids revealed higher uptake values on [ ga]dotatate ( 1,4,7,10-tetraazacyclododecane - n , n , n , n - tetraacetic acid-(d)-phe - thy - octreotate)-pet / ct .
in our study , the typical carcinoids revealed higher suvmax on [ ga]dotatoc - pet / ct compared with atypical carcinoids ( statistically significant ) .
the only tumour that did not reveal any significant uptake was an atypical one ( table 2 , fig .
an interesting observation was made when the ratios of suvmax on [ ga]dotatoc - pet / ct and [ f]fdg - pet / ct were calculated .
the ratios were significantly and uniformly higher in typical carcinoids ( 7.830 , median 17.8 ) compared with atypical carcinoids ( 0.203.9 , median 2.1 ) , p < 0.001 . to the best of our knowledge
this is the only study that aims to co - relate the histology of pulmonary carcinoids with the uptake patterns of [ f]fdg - pet / ct and [ ga]dotatoc - pet / ct scans and their suv ratios .
our study tries to establish the important role that these imaging modalities can play in the management of pulmonary carcinoids .
the differential uptake patterns observed in our study can be of immense help for the clinician .
although histopathologic examination by either bronchoscopy or percutaneous biopsy remains the gold standard test for confirmation , nonetheless the information provided by these non - invasive imaging modalities can substantially aid in the prediction of the histopathologic variant of carcinoids and can reliably guide the investigator .
our study reveals that typical carcinoids had a lower uptake of [ f]fdg compared with the atypical carcinoids .
the ratio of suvmax on [ ga]dotatoc - pet / ct and [ f]fdg - pet / ct revealed higher values for typical carcinoids .
the ratio of suvmax was a better predictor of the histopathologic variety of the carcinoid tumour compared with the suvmax on [ ga]dotatoc - pet / ct and [ f]fdg - pet / ct scans , individually .
this differential uptake has potential use for preoperative assessment , possible prediction of the histology and prognostication of pulmonary carcinoids .
a larger study is indicated to validate this observation and to objectively determine a cut - off value for the possible differentiation . | abstractpulmonary carcinoids are histologically classified into typical and atypical .
it is important to identify these preoperatively for treatment planning and prognosis .
structural imaging can not conclusively differentiate between them .
the aim of this study was to assess the possibility of differentiating the 2 variants using [ 18f]fluorodeoxyglucose ( fdg)-positron emission tomography ( pet)/computed tomography ( ct ) and [ 68ga]1,4,7,10-tetraazacyclododecane - ni iiii - tetraacetic acid-(d)-phe1-thy3-octreotide ( dotatoc)-pet / ct . the imaging results of 20 patients with pulmonary carcinoids ( 13 typical , 7 atypical ) on [ 18f]fdg - pet / ct and [ 68ga]dotatoc - pet / ct were assessed retrospectively .
six typical carcinoids failed to reveal significant uptake on [ 18f]fdg - pet / ct .
all the atypical carcinoids revealed significant uptake on the [ 18f]fdg - pet / ct that was higher than that in typical carcinoids ( standardized uptake value ( suv)max , 2.98.4 , p = 0.001 ) .
the suvmax in typical carcinoids on [ 68ga]dotatoc - pet / ct was significantly higher ( suvmax , 8.866 ) compared with atypical carcinoids ( suvmax , 1.118.5 , p = 0.002 )
. ratios of suvmax on [ 68ga]dotatoc - pet / ct to that on [ 18f]fdg - pet / ct were significantly higher ( p < 0.001 ) in typical carcinoids compared with atypical carcinoids . the different uptake patterns on [ 18f]fdg and [ 68ga]dotatoc - pet / ct .
and the ratio of suvmax may be helpful in differentiating between typical and atypical carcinoids . |
ectopic pancreas is generally well - developed and normally organized pancreatic tissue located outside the pancreas , without anatomic or vascular connections with the true pancreas .
the incidence of ectopic pancreas at all sites has been estimated to be 0.5513.7% in an autopsy series .
the incidence of ectopic pancreas of all removed specimens is 0.25% , and in the gastrointestinal tract , with 2438% in the stomach , 936% in the duodenum , and 0.527% in the jejunum [ 2 , 3 ] .
patients with ectopic pancreas are asymptomatic or exhibit nonspecific symptoms , and the condition is often found secondary to other conditions , such as neoplastic transformation , intussusception , acute pancreatitis [ 6 , 7 ] and pseudocyst .
perforation of the stomach due to ectopic pancreas has been reported in only 2 cases , including our patient , in pubmed from 1963 to june 2012 .
the gastric ectopic pancreas is located in the antrum in 8595% of cases and categorized in the classification by heinrich .
histological examination revealed pancreatic tissue with acini , ducts , and islets of langerhans between the muscular layer and subserosa around the ulceration , categorized as type i of heinrich 's classification .
reports on surgery for perforated gastrointestinal tract due to ectopic pancreas are extremely rare . here
we report an extremely rare case of ectopic pancreas that developed into a perforated gastric ulcer .
a 68-year - old japanese woman with hypertension and hyperlipemia consulted a general practitioner after experiencing intermittent pain in the right upper abdomen .
she suddenly felt intense pain in the right flank of the abdomen and was taken to the nearest hospital .
upper gastrointestinal tract perforation was diagnosed by abdominal computed tomography , and she was transferred to our hospital . on initial medical examination ,
temperature , blood pressure and heart rate were 37.0 degrees , 137/70 mm hg and 77 beats / min , respectively .
complete blood count , coagulation test and laboratory analysis performed on admission revealed the following values ( normal range in parentheses ) : white blood cells , 12,770/l ( 3,2009,000 ) ; hemoglobin , 11.9 g / dl ( 11.315.2 ) ; hematocrit , 36.1% ( 33.045.0 ) ; platelets , 56.5 10/l ( 15.033.0 ) ; prothrombin time , 12.3 s ( 12.015.0 ) ; activated partial thromboplastin time , 28.0 s ( 26.036.0 ) ; fibrinogen , 484 mg / dl ( 150350 ) ; blood fibrin degradation product , 7.0 g / ml ( 0.05.0 ) ; d - dimer , 2.38 g / ml ( 0.025.0 ) ; total protein , 6.3 g / dl ( 6.58.1 ) ; albumin , 2.7 g / dl ( 3.85.1 ) ; blood urea nitrogen , 23.0 mg / dl ( 7.021.0 ) ; creatinine , 0.44 mg / dl ( 0.40.8 ) ; ast , 19 iu ( 1232 ) ; alt , 17 iu ( 1070 ) ; alkaline phosphatase , 311 iu ( 109335 ) ; -glutamyl transpeptidase , 135 iu ( 1248 ) ; serum solids , 133 meq / l ( 137147 ) ; serum chloride , 98 meq / l ( 99109 ) ; serum potassium , 3.5 meq / l ( 3.54.8 ) ; c - reactive protein , 1.84 mg / dl ( 0.000.30 ) .
abdominal computed tomography showed free air and ascites on the surface of the liver , and elevated levels of adipose tissue around the antrum and pylorus of the stomach ( fig .
intraoperative examination revealed that the site of perforation ( size 25 mm ) was the lesser curvature of the antrum of the stomach ( fig .
on macroscopic examination , the location of the ulceration with perforation ( dimensions 30 40 mm ) was confirmed as the lesser curvature of the antrum of the stomach .
microscopic examination revealed that pancreatic tissue within the ulceration showed islets of langerhans , acini , and ducts .
considering the circumstances mentioned above , the heterotopia pancreas was diagnosed as type i by heinrich 's criteria ( fig .
the postoperative course was uneventful , and the patient was discharged on day 13 and remains clinically healthy .
ectopic pancreas , first reported by shultz in 1727 , is also called heterotopic , heterotropic or aberrant pancreas , and is frequently found in the gastrointestinal tract .
ectopic pancreas most commonly develops in the gastrointestinal tract , with 2438% in the stomach , 936% in the duodenum , and 0.527% in the jejunum , and rare cases are reported in the common bile duct , gallbladder , esophagus , lung , mediastinum , vater 's papilla , mesentery , mesocolon and omentum [ 3 , 9 , 10 , 11 ] .
histopathological examination shows that gastric ectopic pancreas is located in the antrum in 8595% of cases , and its incidence in the submucosal , muscularis and subserosal layers is 73 , 17 and 10% , respectively .
type ii exhibits ectopic tissue with incomplete or lobular arrangement ( only a few acini and multiple ducts ) , and endocrine elements are absent .
type iii comprises ectopic tissue of proliferating ducts ( so - called adenomyoma ) , with neither acini nor endocrine elements .
the microscopic perspective of gurocak et al . revealed intramurally located dilated pancreatic ducts and acini ( type ii of heinrich 's classification )
. histological examination of our case showed an ulceration measuring 30 40 mm at the antrum of the stomach ; pancreatic tissue with acini , ducts , and islets of langerhans was found between the muscular layer and subserosa around the ulceration .
therefore , this ectopic pancreas was categorized as type i , and the gastric perforation was found to be caused by ectopic pancreatitis .
most patients with ectopic pancreas are asymptomatic or exhibit nonspecific symptoms such as abdominal pain , nausea , vomiting and hemorrhage .
ectopic pancreas is found rarely in association with neoplastic transformation , intussusception , acute pancreatitis [ 6 , 7 ] and pseudocyst . to date , as few as 4 cases of gastrointestinal perforation developing secondary to ectopic pancreas have been reported in pubmed ( key words : ectopic ( heterotopic , aberrant ) pancreas , perforation ; target period : 1963 to june 2012 ) .
perforation due to ectopic pancreas is found in the duodenal diverticulum and small intestine ; gastric perforation due to ectopic pancreas is quite rare .
the 5 patients , including our patient , required urgent surgery , and perforation due to ectopic pancreas was diagnosed on the basis of pathological findings .
it is not possible to prove through preoperative examination that a gastric perforation is caused by ectopic pancreas .
gastric perforation due to ectopic pancreas has occurred in only 1 of the 5 reported cases of perforation and is an extremely rare condition .
first reported gastric perforation by heterotopic pancreas . in that case , the size of ulceration in the gastric antrum and the perforation were 35 30 mm and 10 mm , respectively . in our case ,
the ulceration and perforation were located at the anterior wall of the antrum in the stomach and measured 30 40 mm and 25 mm , respectively .
the location and size of the ulceration were similar , but the size of the perforation was bigger in our case . in conclusion , diagnosis of ectopic pancreas
is extremely difficult during preoperative examination in patients with diffuse peritonitis due to perforated gastric ulcer . to our knowledge , this is just the second case of a patient with diffuse peritonitis due to perforated gastric ectopic pancreas . during preoperative diagnosis of a perforated gastrointestinal tract , it is suggested that ectopic pancreatic tissue be considered as a cause , especially in cases of gastric perforation .
| ectopic pancreas is frequently found in the gastrointestinal tract .
lesions comprise well - developed and normally organized pancreatic tissue outside the pancreas , without anatomic or vascular connections with the true pancreas .
most patients with ectopic pancreas are asymptomatic or exhibit nonspecific symptoms .
a 68-year - old japanese woman had been experiencing intermittent pain in the right upper abdomen .
suddenly , the abdominal pain changed to intense pain in the right flank of the abdomen 2 days later . on initial medical examination ,
the abdomen exhibited rebound tenderness and distension .
the results of laboratory tests revealed increased inflammatory reaction .
abdominal computed tomography showed free air and ascites on the surface of the liver and elevated levels of adipose tissue around the antrum and pylorus of the stomach .
perforation of the upper gastrointestinal tract was diagnosed and we performed urgent surgery .
the site of perforation , whose size was 25 mm , was the lesser curvature of the antrum of the stomach .
since it was not possible to perform omentopexy , we performed extensive gastric resection .
the reconstruction was a billroth ii operation .
microscopic analysis revealed pancreatic tissue within the ulceration , showing islets of langerhans , acini , and ducts ; the lesion was diagnosed as type i using heinrich 's criteria .
the postoperative course was uneventful .
the patient was discharged on day 13 and remains clinically healthy .
gastric perforation due to ectopic pancreas has been reported in 2 cases , including our patient , and is extremely rare .
once gastric perforation has been diagnosed , the presence of ectopic pancreas might be considered . |
there is a significantly higher prevalence of depression in patients with type 2 diabetes mellitus ( t2 dm ) than in the general population,1,2 and their depressive symptoms have high rates of recurrence and chronicity.3 furthermore , psychological disorders affect treatment choices , self - management , and short- and long - term outcomes in these patients.4 it is , therefore , vitally important to account for psychological factors in diabetes care .
patient self - efficacy and social support are two concepts central to the psychological aspects of diabetes care .
patient self - efficacy is defined as the belief in one s capabilities to organize and execute the courses of action required to produce given attainments.5 poor self - efficacy has been associated with increased depressive symptoms as well as poor glycemic control,6 and stronger self - efficacy has been associated with better self - management behaviors in diabetes , including control of dietary habits , exercise , blood sugar testing , and medication compliance.7,8 the evidence appears to be consistent that self - efficacy directly and indirectly influences diabetic therapeutic effectiveness and prognosis .
social support is defined as the assistance received from others , which is a known buffer of psychological distress.912 it is measured in three dimensions : subjective support , objective support , and support utilization .
subjective support is the level of support a person perceives , objective support is the level of support available , and support utilization is a measure of support used by the person .
previous studies showed that patients with lower levels of social support had poorer general health than those with higher levels of social support,9 and , among persons with diabetes , higher levels of social support were associated with better diabetes self - care behaviors , such as following a diabetes diet plan , caring for feet , exercising , monitoring blood glucose,10 and adhering to treatment.11 in addition , a high level of social support could mitigate the negative impact of cognitive impairment on glycemic control.12 type d personality has been recognized as a risk factor for a wide range of diseases.13 type d personality is the interaction of two stable personality traits : negative affectivity ( na ) , which is the tendency to experience negative emotions across time and situations , and social inhibition ( si ) , which is the tendency to inhibit the expression of emotions .
patients who score high on na frequently report feelings of dysphoria , worry , and tension , and patients who score high on si tend to avoid negative reactions from others.14,15 type d personality captures these two key personality traits and could be considered a potent and easier alternative scale to understand care for patients with diabetes and other diseases for which psychological factors play important roles .
previous studies showed that type d personality was predictive of poor health outcomes in patients with cardiovascular disease,13,1517 along with an increased risk of vital exhaustion18 and increased odds of impaired health status.19 however , research on the association between type d personality and t2 dm has been limited .
nefs et al20,21 found that t2 dm patients with type d personality experienced less social support and more stressful life events , loneliness , depressed mood , anhedonia , and anxiety .
milicevic et al22 found that t2 dm patients with type d personality were less compliant with visits to primary care physicians .
li et al23 discovered that type d personality predicted poor medication adherence in t2 dm patients .
more studies are needed to understand the impact of type d personality in relation to other important sociopsychological factors in t2 dm patients , such as self - efficacy and social support . in this study
, we tried to estimate the prevalence of type d personality in a sample of chinese patients with t2 dm and assess the relationship between type d personality and self - efficacy / social support . by establishing a link between type d personality and the two well - established and well - researched sociopsychological concepts ( ie , self - efficacy and social support )
, we intended to understand this personality type in the context of diabetic patients and study how to use this information to implement adequate diabetic care .
consecutive participants were recruited among the outpatients and inpatients who visited the endocrine clinics of two of the largest hospitals in guangzhou city , guangdong , between january 1 , 2014 , and july 31 , 2014 .
the participants were provided with both written and oral information regarding the study and were informed that they were free to withdraw from the study at any time .
the following inclusion criteria were applied to the study participants : the patients 1 ) who were diagnosed as having t2 dm according to the american diabetes association ( ada ) criteria ( 2010 ) ; 2 ) who had been receiving diabetic treatment for > 1 year ; 3 ) who had no concurrent malignant tumor , type 1 diabetes , gestational diabetes , vision impairment due to complications , limited physical activity due to advanced renal failure , or acute complications ; 4 ) who were local chinese inhabitants ; 5 ) who were at least 18 years of age ; and 6 ) who were able and willing to complete the questionnaire alone . once recruited and having provided written consent to participate , the subjects were first asked to complete the general questionnaire containing sociodemographic data in a quiet environment , and then three - scale questionnaires ( ie , personality , social support , and self - efficacy scales ) under the guidance of trained professionals according to the standard instructions of each scale .
the patient s medical records were reviewed to retrieve additional information on medical conditions and treatment history .
glycosylated hemoglobin ( hba1c ) , an integrated marker of glycemic control , was obtained from their medical records if it was measured in the past 3 months ; otherwise , blood samples were taken after the patients completed the questionnaires , and hba1c was tested in the laboratories of the participating hospitals .
there are 14 items in the ds14 , covering two domains , na and si . the former includes item 2 ( i often make a fuss about unimportant things ) , item 4 ( i often feel unhappy ) , item 5 ( i often feel irritated ) , item 7 ( i take a gloomy view of things ) , item 9 ( i am often in a bad mood ) , item 12 ( i often find myself worrying about something ) , and item 13 ( i am often down in the dumps ) . the latter includes item 1 ( i make contact easily when i meet people ) , item 3 ( i often talk to strangers ) , item 6 ( i often feel inhibited in social interactions ) , item 8 ( i find it hard to start a conversation ) , item 10 ( i am a closed kind of person ) , item 11
( i would rather keep other people at a distance ) , and item 14 ( when socializing , i do not find the right things to talk about ) .
the responses are measured on a 5-point likert scale ranging from 0 ( false ) to 4 ( true ) .
each of the two subscales includes seven items , with a total score ranging from 0 to 28 .
a patient was determined to have type d personality if both subscales score 10 points or higher .
both subscales have been shown previously to be internally consistent ( cronbach s =0.88 and 0.86 for na and si , respectively).14 the chinese version of the ds14 scale has been tested previously and found to have a similar level of internal consistency ( cronbach s =0.90 and 0.85 for na and si , respectively).24 we used the self - efficacy scale designed by lorig et al25 at stanford university during their research of self - management behavior in patients with chronic diseases .
it contains six items reflecting the multiple aspects of self - efficacy in patients with chronic diseases , including emotional control , communication with doctors , symptom management , and role function .
each item is scored from 0 ( no confidence at all ) to 9 ( full confidence ) .
the average score of the six items indicates the level of self - efficacy , with a higher score reflecting better self - efficacy .
the cronbach s value of this scale was previously estimated at 0.89.25 we used the 10-item social support rating scale to assess a patient s social support.26 this scale includes three subscales : subjective support ( four items ) , objective support ( three items ) , and the utilization of support ( three items ) , with items scored on either yes no or 4-point likert scale .
objective support score is the sum of scores from items 2 , 6 , and 7 , subjective support score is the sum of scores from items 1 , 3 , 4 , and 5 , support utilization score is the sum of scores from items 8 , 9 , and 10 , and the total social support score is the sum of scores across all 10 items .
a study showed that the cronbach s of this instrument is between 0.825 and 0.896.27 data were entered into a database using epidata 3.1 software .
simultaneous data entry was carried out by two persons ( hospital staff or graduate students ) with data entry experience , and the consistency check function of the epidata software was used to ensure accuracy .
once the data were collected and entered into the computer , the cronbach s values were calculated to assess the internal consistency of the three scales used .
differences in the sociodemographic characteristics between patients with type d and non - type d personalities were compared using the unpaired , two - sided t - test ( for continuous variables ) and the chi - square test ( for categorical variables ) .
bivariate analyses , including the t - test and correlation analysis , were used to assess the relationship between type d personality and self - efficacy / social support scores .
finally , regression analyses were conducted to examine the relationship between type d personality and self - efficacy / social support while controlling for confounding sociodemographic characteristics
all statistical analyses were performed using spss 15.0 ( ibm , armonk , ny , usa ) for windows .
consecutive participants were recruited among the outpatients and inpatients who visited the endocrine clinics of two of the largest hospitals in guangzhou city , guangdong , between january 1 , 2014 , and july 31 , 2014 .
the participants were provided with both written and oral information regarding the study and were informed that they were free to withdraw from the study at any time .
the following inclusion criteria were applied to the study participants : the patients 1 ) who were diagnosed as having t2 dm according to the american diabetes association ( ada ) criteria ( 2010 ) ; 2 ) who had been receiving diabetic treatment for > 1 year ; 3 ) who had no concurrent malignant tumor , type 1 diabetes , gestational diabetes , vision impairment due to complications , limited physical activity due to advanced renal failure , or acute complications ; 4 ) who were local chinese inhabitants ; 5 ) who were at least 18 years of age ; and 6 ) who were able and willing to complete the questionnaire alone .
once recruited and having provided written consent to participate , the subjects were first asked to complete the general questionnaire containing sociodemographic data in a quiet environment , and then three - scale questionnaires ( ie , personality , social support , and self - efficacy scales ) under the guidance of trained professionals according to the standard instructions of each scale .
the patient s medical records were reviewed to retrieve additional information on medical conditions and treatment history .
glycosylated hemoglobin ( hba1c ) , an integrated marker of glycemic control , was obtained from their medical records if it was measured in the past 3 months ; otherwise , blood samples were taken after the patients completed the questionnaires , and hba1c was tested in the laboratories of the participating hospitals .
there are 14 items in the ds14 , covering two domains , na and si . the former includes item 2 ( i often make a fuss about unimportant things ) , item 4 ( i often feel unhappy ) , item 5 ( i often feel irritated ) , item 7 ( i take a gloomy view of things ) , item 9 ( i am often in a bad mood ) , item 12 ( i often find myself worrying about something ) , and item 13 ( i am often down in the dumps ) .
the latter includes item 1 ( i make contact easily when i meet people ) , item 3 ( i often talk to strangers ) , item 6 ( i often feel inhibited in social interactions ) , item 8 ( i find it hard to start a conversation ) , item 10 ( i am a closed kind of person ) , item 11 ( i would rather keep other people at a distance ) , and item 14 ( when socializing , i do not find the right things to talk about ) .
the responses are measured on a 5-point likert scale ranging from 0 ( false ) to 4 ( true ) .
each of the two subscales includes seven items , with a total score ranging from 0 to 28 .
a patient was determined to have type d personality if both subscales score 10 points or higher .
both subscales have been shown previously to be internally consistent ( cronbach s =0.88 and 0.86 for na and si , respectively).14 the chinese version of the ds14 scale has been tested previously and found to have a similar level of internal consistency ( cronbach s =0.90 and 0.85 for na and si , respectively).24 we used the self - efficacy scale designed by lorig et al25 at stanford university during their research of self - management behavior in patients with chronic diseases .
it contains six items reflecting the multiple aspects of self - efficacy in patients with chronic diseases , including emotional control , communication with doctors , symptom management , and role function .
each item is scored from 0 ( no confidence at all ) to 9 ( full confidence ) .
the average score of the six items indicates the level of self - efficacy , with a higher score reflecting better self - efficacy .
the cronbach s value of this scale was previously estimated at 0.89.25 we used the 10-item social support rating scale to assess a patient s social support.26 this scale includes three subscales : subjective support ( four items ) , objective support ( three items ) , and the utilization of support ( three items ) , with items scored on either yes no or 4-point likert scale .
objective support score is the sum of scores from items 2 , 6 , and 7 , subjective support score is the sum of scores from items 1 , 3 , 4 , and 5 , support utilization score is the sum of scores from items 8 , 9 , and 10 , and the total social support score is the sum of scores across all 10 items .
a study showed that the cronbach s of this instrument is between 0.825 and 0.896.27
simultaneous data entry was carried out by two persons ( hospital staff or graduate students ) with data entry experience , and the consistency check function of the epidata software was used to ensure accuracy .
once the data were collected and entered into the computer , the cronbach s values were calculated to assess the internal consistency of the three scales used .
differences in the sociodemographic characteristics between patients with type d and non - type d personalities were compared using the unpaired , two - sided t - test ( for continuous variables ) and the chi - square test ( for categorical variables ) .
bivariate analyses , including the t - test and correlation analysis , were used to assess the relationship between type d personality and self - efficacy / social support scores .
finally , regression analyses were conducted to examine the relationship between type d personality and self - efficacy / social support while controlling for confounding sociodemographic characteristics . a p - value of < 0.05 was considered statistically significant .
all statistical analyses were performed using spss 15.0 ( ibm , armonk , ny , usa ) for windows .
three - quarters of the participants were recruited from the outpatient departments of the two hospitals .
of the 532 patients in the sample , 97 ( 18.2% ) had type d personality .
table 1 lists the sociodemographic characteristics of the patients with and without type d personality .
overall , there were no significant differences in the distributions of age , gender , education , body mass index ( bmi ) , family size , and income between the two groups , even though the groups with the highest education , largest family size , and highest income appeared to have the lowest percentages of type d personality .
notably , patients with type d personality had significantly higher levels of hba1c than those without type d personality .
the cronbach s values were 0.88 and 0.77 for the two type d personality subscales , na and si , respectively , and 0.89 and 0.60 for self - efficacy and social support scales , respectively .
these assessments based on the collected data demonstrate good internal consistency for all three scales in this study .
table 2 compares the self - efficacy and social support scales in t2 dm patients with and without type d personality .
as shown in the table , patients with type d personality had significantly lower scores in self - efficacy , total social support , subjective support , and support utilization than patients without type d personality , but objective support score did not significantly differ between the two groups .
table 3 further reveals the correlations between the various sociodemographic characteristics , na , si , self - efficacy , and social support . as shown in the table , total social support and subjective support
controlling for other characteristics , patients with type d personality reported significantly lower self - efficacy and social support .
in addition , patients who were older , had smaller family size , and had lower income had significantly lower social support , but such characteristics were not significantly associated with self - efficacy .
in this study , we found that 18.2% of the t2 dm patients had type d personality . this rate is lower than previously reported for healthy chinese adults ( 31.9% ) and chinese patients with coronary heart disease ( 31.4%).24 it is likely that our convenience sample underestimates the prevalence of type d personality among chinese patients with t2 dm .
it is also likely that , because the participants in our study were more advanced in age and had higher educational levels than the average chinese individuals , they may be more capable of dealing with their emotional problems , causing them to under - report na and si .
the current study shows that the presence of type d personality did not vary significantly by sociodemographic characteristics , which is consistent with the results from previous studies.14,18,20 this finding supports the general theory that type d personality is an inherent personal trait and does not vary according to sociodemographic factors.28 as given in table 1 , hba1c levels were significantly higher in the patients with type d personality than those with non - type d personality .
blood glucose is an important factor influencing diabetes progression , as evidenced by several previous studies .
one prospective study showed that poor blood glucose control led to diabetic nephropathy and diabetic retinopathy.29 a study conducted at 215 collaborating centers in 20 countries from asia , australasia , europe , and north america showed a one - fifth reduction in renal complications resulted from intensive control of glucose.30 another study found that , in t2 dm patients with blood glucose controlled to the normal level , complications of the eyes and renal injury decreased by 76% and 35%56% , respectively , and the incidence of microalbuminuria decreased by 33%.31 stratton et al32 estimated that , for every 1% reduction in hba1c , the risk of complications decreases by 21% , the incidence of myocardial infarction decreases by 14% , the incidence of microvascular complications decreases by 37% , the incidence of major vascular complications decreases by 40% , and the diabetes - associated mortality rate decreases by 21% .
most recently , van dooren et al33 showed that type d personality was associated with higher hba1c levels . in short ,
t2 dm patients with type d personality are more likely to have poor glycemic control and , therefore , are at a higher risk of complications and mortality .
overall , the results of our bivariate and multivariate analyses confirmed our hypothesis that type d personality predicts lower levels of self - efficacy and social support among t2 dm patients .
in other words , patients who were classified as having type d personality experienced lower levels of self - efficacy and social support . thus far , only a few studies have specifically investigated the connection between type d personality and self - efficacy .
mikkelsen and einarsen34 showed that self - efficacy is significantly associated with na , worry , irritability , coercion , and depressive symptoms , which are all salient traits of type d personality,14,20 and the relationship between this type of personality and depression has been clearly established.17,35 therefore , it is likely that the feelings of na cause such individuals to believe that they lack the confidence , strength , or patience to take control of their diabetic condition and implement appropriate care , and , as a result , they would score lower in self - efficacy .
more recent studies revealed that self - efficacy mediated the relationship between type d personality and physical activity,36 and the relationship between type d and medication adherence.37 our findings are consistent with these previous studies and highlight the negative impact of type d personality on patient self - efficacy in controlling their medical conditions .
our findings regarding the lower social support expe rienced by type d patients are also in line with the literature.3840 one of the defining features of type d personality is inhibitions in social interaction and acquisition of social support .
this study revealed that patients with type d personality reported significantly lower levels of subjective support and less support utilization , but similar level of objective support , compared to those of other patients .
this finding suggests that , because of si , patients with type d personality perceive lower social support ( ie , subjective support ) and are , therefore , less likely to use such support ( ie , support utilization ) , even though the social support from their families , friends , neighbors , colleagues , and communities available to them is the same as that for other patients .
first , the study sample was not representative of chinese patients with t2 dm and the sample size was small . therefore , our results should not be generalized to the normal population of patients with t2 dm or to patients with other chronic conditions in china .
in addition , our assessments were based on patient self - reports ; therefore , our findings may be subject to various reporting biases .
the results of the current study indicate that type d personality is predictive of reduced self - efficacy and social support among patients with t2 dm in china . because self - efficacy and social support play important roles in treatment compliance and self - care , our study highlights the special needs in diabetes patients with type d personality and extra efforts needed to ensure sufficient patient follow - up and management to achieve long - term glycemic control and prognosis on par with patients with non - type d personality . | objectivethe aim of this study was to estimate the prevalence of type d personality and assess the relationship between this personality type and self - efficacy / social support in chinese patients with type 2 diabetes mellitus ( t2dm).patients and methodsfrom january 1 , 2014 , to july 31 , 2014 , 532 consecutive patients with t2 dm were recruited from two hospitals in guangzhou , china .
the participants completed questionnaires containing questions about sociodemographic characteristics , type d personality , self - efficacy , and social support scales , and their medical records were reviewed for additional data.resultsof the 532 patients , 18.2% had type d personality .
patients with this personality type reported significantly lower levels of self - efficacy ( p<0.001 ) , total social support ( p<0.001 ) , subjective support ( p<0.001 ) , and support utilization ( p=0.003 ) , but similar level of objective support ( p=0.314 ) , compared to those of patients without type d personality .
negative affectivity and social inhibition , two intrinsic traits of type d personality , negatively correlated with self - efficacy and social support scores .
type d personality was significantly associated with less self - efficacy and social support ( p<0.001 ) , controlling for other sociodemographic factors .
glycosylated hemoglobin ( hba1c ) levels were significantly higher in t2 dm patients with type d personality than in patients with non - type d personality.conclusionthis study provides new evidence linking type d personality with self - efficacy , social support , and poor glycemic control , highlighting the special need for care among t2 dm patients with type d personality . |
pulpstones are a group of calcified masses in the dental pulp of healthy , diseased and unerupted teeth
. stones may exist either freely within the pulp tissue or be attached to or embedded in dentin .
the size may vary from a small microscopic particle to large masses that almost obliterate the pulp chamber .
two types of pulp stones have been described : denticles possessing a central cavity filled with epithelial remnants surrounded peripherally by odontoblasts and pulp stones being compact degenerative masses of calcified tissues .
they are reported to occur more often in the coronal region , but are also found in the radicular pulp .
pulp stones have been reported in patients with systemic or genetic diseases such as van der woude syndrome . despite several microscopic and histochemical studies ,
however , a number of conditions have been claimed to predispose to pulp stone formation such as pulp degeneration , inductive interactions between the epithelium and pulp tissue , age , caries , operative procedures , periodontal diseases , epithelial rests in the pulp tissue , orthodontic tooth movement , circulatory disturbances in pulp tissue , idiopathic factors and genetic predisposition .
recent literature still suggests that pulp stones are a feature of an irritated pulp , attempting to repair itself .
the true prevalence is likely to be higher because pulp stones with a diameter smaller than 200 m can not be seen on radiographs .
some researchers have investigated the prevalence of pulp stones or association of this condition with other dental conditions . however , very few studies have assessed the prevalence of pulp stones in indian population .
the aim of this study was to determine the occurrence of pulp stones using radiographs and to correlate their association with age , gender , systemic diseases such as hypertension , diabetes and gastritis ; tooth type , jaw type , caries and restored teeth .
a total of 2000 patients were selected randomly from the outpatient department of radio diagnosis of three different institutions belonging to three different regions of andhra pradesh for a period of 6 months .
patients below the age of 10 and above the age of 60 were excluded from the study . among these , 2000 patients a total of 4449 teeth were examined .
all the patients who had undergone a diagnostic radiograph of the premolar and molar region were considered for the study .
patient 's medical history was recorded for diseases such as diabetes , hypertension and gastritis .
patients with more than one systemic disease and patients with other medical problems apart from diabetes , hypertension and gastritis were excluded from the study .
patient 's dental status was thoroughly examined for dmf , which were recorded in the proforma .
diagnostic radiographs were taken using an analog iopa unit under icrp standards protecting the patient and the operator with constant exposure parameters ( 70 kvp , 6 ma and 0.6 s pulse ) .
all the radiographs were processed manually using visual method in a dark room under safe light .
the radiographs were evaluated for the presence or absence of pulp stones and were documented in the proforma .
statistical analysis of the data was done using the statistical package for the social sciences ( spss 15.0 ) using chi - square analysis .
the distribution of patients with pulp stones according to the age groups is shown in table 1 .
the occurrence of pulp stones was most significant in among the age groups of 31 - 50 years of age .
of the 2000 patients a total of 518 patients had pulp stones which accounts to 25.9% prevalence .
a total of 4449 teeth were examined of which 18% of teeth showed pulp stones .
age distribution and its correlation of presence of pulp stone the distribution of teeth with pulp stones in comparison to the medical history is shown in table 2 .
patients with diabetes , hypertension and gastritis showed higher prevalence of pulp stones with a p < 0.05 , which was statistically significant . medical history and its correlation of presence of pulp stone the distribution of teeth with pulp stones according to the location and tooth type is shown in table 3 .
when individual tooth type is taken into consideration , maxillary 1 and 2 molars showed a strongly positive significance with 33% and 31% prevalence respectively to the overall prevalence of 18% .
teeth distribution and its correlation of presence of pulp stone regarding the tooth pathology , of the 4449 teeth examined , 1870 teeth had dental caries out of which 224 teeth showed pulp stones which accounts to 11.9% prevalence and 105 teeth were restored out of which 18 teeth showed 17.1% prevalence .
the prevalence of pulp stones were statistically significant [ table 4 ] in teeth with dental caries and teeth without pathology .
detection of pulp stones using dental radiographs is only possible when the diameter is more than 200 m . most of the studies in the literature used paralleling technique to take the radiographs .
al - hadi hamasha and darwazeh in their study used periapical and bitewing radiographs , olak et al . used bitewing radiographs , whereas satheeshkumar et al . and
syrynska et al . used panoramic radiographs in their study for detection of pulp stones .
one study had proved that there is no significant difference in the technique to identify the pulp stones . in our study as we considered all the intraoral periapical radiographs taken in the out - patient department of radiology , we used bisecting angle technique to take the radiographs .
al - hadi hamasha and darwazeh examined patient records of 814 jordanian adults and found that pulp stones were present on radiographs in 51% of the patients and 22% of the teeth studied .
baghdady vs et al . assessed 515 iraqi subjects and recorded that 19% of the teeth had pulp stones .
examined the prevalence of pulp stones in australian population and found in 46% of the subjects and 10% of the teeth examined .
gulsahi et al . in their study found pulp stones in 12% of the subjects and 5% of the teeth examined .
examined bitewing radiographs of 469 turkish patients and found 57.6% prevalence of pulp stones in their patients and 15% prevalence among the teeth examined . in the present study , the prevalence of pulp stones in 26% of the subjects and 18% of the total teeth examined .
some investigators have reported that pulp stones were more common in females than in males .
some showed that there is no significance . in our study , we found a slight higher prevalence in females than in males . according to ranjithkar s et al pulp stones
these include tumoralcalcinosis , dentin dysplasia type ii , elfin facies syndrome , familial expansile osteolysis , elhers - danlos syndrome type i , osteogenesis imperfecta type i and otodental syndrome . in forensic dentistry ,
a radiographic matching of pulp stone configurations , along with other features recorded in dental records , may provide valuable information in the identification of a deceased person .
the correlation of pulp stones with cardiovascular diseases and other systemic diseases has been investigated .
found a significant relation between pulp stones and presence of arteriosclerosis and other cardiovascular diseases .
high incidence of calcification in the dental pulp of patients with coronary atherosclerosis upon radiographic examination were reported .
theyalso found calcification and lumen narrowing within extirpated dental pulp vessels , in both medium and small precapillary arteriole of cardiovascular patients .
nayak et al . in their study on indian population found the prevalence of pulp stone in hypertensive patients was 15.85% higher than normal subjects .
the present study also showed a higher prevalence of pulp stones in patients with hypertension , diabetes and gastritis when compared to the overall prevalence of pulp stones .
type ii diabetic patients , in the present study , were detected with pulp stones higher than the overall prevalence of pulp stones .
russell had investigated human pulp histologically on non - carious extracted teeth of seven patients suffering from diabetes for a long term duration and control group of 13 non - diabetics .
he concluded that calcification in angiopathies and thickened basement membrane were noted in both large and small blood vessels and vascular changes seemed more pronounced in the central area of the pulp .
calcifications in diabetics were frequent and often sickle - shaped . in another histopathological study , conducted by bissada and sharawy 's on 21 human dental pulps of diabetics and 20 matched controls , no vascular changes groups were found in the dental pulp of both .
dental pulp of patients who suffer from diabetes mellitus tend to age more readily because it of obliterative endarteritis and because it has limited or no collateral blood circulation in fully developed teeth .
the occurrence of pulp stones in our study was significantly higher in the younger age group patients and also in those groups of people who had underlying systemic problems . | aim : the aim of this study was to determine the occurrence of pulp stones in a group of the population of andhra pradesh .
this study also aims to determine the association of pulp stones in different sexes , tooth type , dental arches and sides and with systemic diseases.materials and methods : a total of 4449 teeth of 2000 patients were examined , comprising of patients with c.v.s .
disorders ; type ii diabetes mellitus and gastritis were examined .
patients were selected from the radiology department who came for diagnostic radiograph of posterior teeth . the presence or absences of pulp stones
were recorded.statistical analysis used : statistical analysis of the data was done using the statistical package for the social sciences ( spss 15.0-spss inc . , 233 south waker drive , 11th floor , chicago , il 60606 - 6412 . ) using chi - square analysis .
differences were considered as significant when p 0.05.results:pulp stones were found in 799 ( 17.9% ) of 4449 teeth detected .
significantly , higher numbers of pulp stones were recorded in patients with systemic diseases .
the occurrences of pulp stones were significantly higher in maxillary first and second molars .
no significant difference was found between sexes and sides.conclusions:positive correlation was found between systemic disorder and occurrence of pulp stones . |
mucopolysaccharidosis - i ( mps i ) is a lysosomal storage disorder inherited as an autosomal - recessive condition and is caused due to deficiency of the lysosomal enzyme 1-iduronidase , which results in the progressive accumulation of glycosaminoglycans ( gag ) within the lysosomes , subsequently leading to multiorgan dysfunction and damage .
patients affected with mps i are unable to degrade the gag , dermatan sulfate and heparan sulfate , which provide structural support to the extracellular matrix and cartilaginous structures such as joints and heart valves .
mps i has an estimated incidence of 1 case per 100,000 live births , and the attenuated type represented about 20% of the total mps i population .
mps i includes separate diseases on the basis of clinical presentation , hurler syndrome ( severe ) , hurler - scheie syndrome ( intermediate ) and scheie syndrome ( mild ) .
however , with mps i being recognized as a disease continuum due to variation in age of onset and rate of disease progression , an international panel comprising of 12 ( international ) experts on mps i revised and updated the initial guidelines in the year 2008 on the basis of additional clinical data and therapeutic advances . based on their recommendations , mps i has been classified into two broader groups , severe mps i ( hurler syndrome ) and attenuated mps i ( hurler - scheie and scheie syndromes ) .
the purpose of this article is to describe a rare case of a 15-year - old female child affected with mps i , hurler - scheie ( h / s ) .
a 15-year - old indian female with a known diagnosis of mps type i h / s was referred to our dental clinic by a pediatrician for dental care . on physical examination
, she appeared short statured ( stunted growth ) with lumbar kyphosis and presented coarse facial features [ figure 1 ] .
medical history revealed that she suffered frequent respiratory infections ( chronic sinus infections ) , stiffness of joints and hepatomegaly .
gradual hearing loss and vision impairment has been reported with evidence of corneal clouding [ figure 2 ] .
patient with stunted growth and coarse facial features picture showing cloudy corneas intraoral examination revealed macroglossia , high - arched palate , anterior open bite and decayed 11 , 12 , 21 , 16 , 26 , 36 and 46 ( fdi notation ) .
oral hygiene of the patient was good with no evidence of significant deposits of calculus [ figure 3 ] .
intraoral picture showing decayed teeth and macroglossia cardiological evaluation including echocardiogram revealed mild mitral regurgitation with good left ventricular function .
anteroposterior view of the chest showed widened metaphyses and oar - shaped ribs , which were suggestive of dysostosis multiplex [ figure 4 ] .
wrist radiographs revealed bullet - shaped phalanges with proximal pointing of the second to fifth metacarpals [ figure 5 ] .
chest radiograph showing oar - shaped ribs with narrowing at the vertebral ends and broadening at the sterna ends hand
wrist radiograph showing bullet - shaped phalanges with proximal pointing of the second to fifth metacarpals dental management needed multiple visits and included restoration of 16 , 26 , 36 and 46 with silver amalgam and 11 , 12 and 21 with composite restoration .
further , importance of home oral care and follow - up visits was emphasized to the parents .
the patient is currently under enzyme replacement therapy and regular medical evaluation involving the multidisciplinary team .
mps i , caused by deficiency of the lysosomal enzyme , alfa - l - iduronidase , has been mapped to the chromosome band 4p16.3 .
children with severe mps i usually die within the first decade of life as a result of cardiorespiratory failure and progressive neurological disease . on the contrary
however , cases of attenuated mps i show wide variation with respect to age of presentation , symptoms and disease course . the clinical presentation may be limited to growth failure , cloudy corneas , mild coarsening of facial features , hepatomegaly and micrognathism in mps i h / s .
the typical features appear at about the age of 12 years , including macrocephaly and coarse facies , and restriction of joint mobility with deformities becomes apparent at the age of 1215 years .
the combination of metaphyseal deformities and thickened joint capsules secondary to gag deposition and fibrosis has been related to joint function abnormalities .
frequent upper and lower respiratory tract infections are common and occur secondary to enlargement of tonsils and adenoids and enlarged tongue .
patients with mps i h / s may show dental abnormalities including enamel defects , carious teeth , dentigerous cysts and abscesses . however , our patient showed caries involving the permanent first molars in all the four quadrants and maxillary incisors , high - arched palate and open bite due to macroglossia and mandibular prognathism .
regular dental and radiological evaluation should be performed every 6 months and parents or caregivers should be educated about dental home care .
our patient presented good oral hygiene , which can be attributed to the supervision and involvement of parents in oral care and normal cognitive development of the patient .
it has been recommended to undergo cardiac evaluation every 1 or 2 years after an initial diagnosis .
monitoring of patients with mps i h / s by a multidisciplinary team should include regular assessments , supportive care and treatment of a variety of systemic complications . delayed diagnosis in patients with attenuated mps i may be attributed to lack of disease awareness among parents and physicians . with the advent of hematopoietic stem cell transplantation and ,
more recently , enzyme replacement therapy , there exists a need for early diagnosis , better disease recognition and management .
early detection of the disease and appropriate management through a multidisciplinary approach is recommended to improve the quality of life . | mucopolysaccharidosis i ( mps i ) is a rare inherited disorder that belongs to a group of clinically progressive disorders and is caused by the deficiency of the lysosomal enzyme , 1-iduronidase .
mps i has been recently classified into a severe ( hurler syndrome ) and an attenuated type ( hurler - scheie and scheie syndromes ) .
the purpose of this article was to describe a rare case of mps type i , attenuated type ( hurler - scheie ) affecting a 15-year - old indian child . |
most insulinomas are benign , single and small , measuring less than 2 cm in diameter .
differentiation between benign and malignant insulinomas by histologic finding was difficult , therefore malignant insulinomas were diagnosed only by metastasis to lymph nodes or other organs .
hence we report a patient of a large malignant insulinoma with peripancreatic lymph node metastases and characterize it s endosonographic , immunohistochemical and electron microscopic features .
a 53-year - old woman presented with recurrent dizziness and loss of consciousness upon skipping meals for several months .
the results of complete blood counts , tumor markers , thyroid function test , parathyroid hormone , calcium , gastrin , prolactin and brain computed tomography ( ct ) were normal , but the random glucose level was 39 mg / dl . on 72-hour fasting test , she demonstrated cold sweat , disorientation , and dizziness at 5 hours with 25 mg / dl of serum glucose level .
the plasma insulin - to - glucose ratio was 1.41 ( table 1 ) and symptoms were immediately relieved following glucose administration ( figure 1 ) .
a hypoechoic , heterogeneous echoic mass was discovered at the tail of the pancreas ( figure 2 , 3 ) .
the mass measuring 5.84.74.5 cm in dimension ( figure 4 ) was composed of uniform bland cuboidal cells with granular eosinophilic cytoplasm and round nuclei ( figure 5a ) .
immunohistochemically , cytoplasm of tumor cells were strongly immunoreactive to insulin ( figure 6a ) but not to somatostatin and glucagon .
on ultrastructural study , atypical secretory granules were easily found in the cytoplasm ( figure 7a ) .
amyloid deposits were showed both by electron microscopy ( em , figure 7b ) and congo red stain under polarizing microscopy ( figure 8) .
immediate after surgery , the levels of insulin and glucose were normalized and the postoperative course was uneventful and without any complication ( table 1 ) . during 18-months of follow - up , she remains with no evidence of recurrence .
insulinomas are the most common pancreatic endocrine tumors ( pets ) and only less than 10% are malignant .
malignant insulinoma could be suspected clinically if the size of the tumor is 3 to 6 cm or larger and confirmed bythe evidence of metastasis which is mostly found in the liver , lymph nodes , or both .
a large variety of imaging modalities for the preoperative localization have been used , but the average accuracy remains low .
recently , eus is a new convenient diagnostic method for the localization of a pancreatic mass .
differentiations between benign and malignant insulinoma by eus depend upon size , heterogeneity of internal echo , multinodular structure , cystic transformation or necrosis and vascular invasion . in our case
eus findings that demonstrated large - sized , low - echoic mass with heterogeneous internal echo evoked a possibility of malignancy .
the ki-67 li is known to be less than 5% in benign tumors , but it was estimated as 13% in this tumor which could be a predictable feature of a malignant tumor .
more than half of the pets secrete multiple hormones ( insulin , glucagon , somatostatin ) , nevertheless , the clinical manifestations are nearly always derived from hypersecretion of only one of these hormones . in our case ,
the tumor cells were only immunoreactive to insulin , therefore , seemed to be as a pure insulinoma .
ultrastructurally , our tumor was devoid of typical insulin - secreting granules and only showed a decreased number of atypical secretory granules comparing with normal islet cell .
because atypical granule could be regarded as an incomplete form in the maturation process and stored with a small amount of functional insulin , these findings might be correlated with long , insidious , endurable clinical symptoms and malignant potential .
however , high li , heterogeneous eus findings , capsular invasion with large tumor and pure atypical secretory granules might be other valid diagnostic clues for diagnosis of malignant insulinoma .
those were thought to be the result of uncontrolled insulin or proinsulin release with decreased storage capacity .
distal pancreatectomy with splenectomy was performed because of the unfavorable eus findings . for patients with benign insulinoma ,
the long - term cure rate is about 88.697.5% by surgical treatment , and patients with malignant insulinoma have a 4966% of 5-year survival with 7.5-year of overall survival .
the patient has remained well for 18 months after surgical excision of the tumor , despite some unfavorable prognostic factors such as lymph node metastases , capsular invasions and high li . | malignant insulinoma in the beta cells of the pancreatic islet is rare and usually presented as hypoglycemia .
we report a case of large malignant insulinoma in a 53-year - old korean woman .
a presumptive clinical diagnosis was made before surgery , based on the high plasma insulin - to - glucose ratio and a large solitary heterogeneous pancreatic mass by abdominal computed tomography and endosonography .
the tumor measured 5.84.74.5 cm in dimension and showed capsular invasions and metastases in two of four peripancreatic lymph nodes .
the tumor cells were strongly immunoreactive to insulin and had a high ki-67 labeling index ( 13% ) and atypical membranous electron - dense granules , ranging from 120 to 400 nm in diameter , in the cytoplasm on electron microscopy .
the patient was treated by distal pancreatectomy with splenectomy and rapidly recovered without neurohypoglycemic symptoms .
this case showed not only lymph node metastases , the most reliable parameter for malignancy in pancreatic endocrine tumors , but also other valid diagnostic clues , such as high ki-67 labeling index , heterogeneous enodosonographic findings , capsular invasions with large tumor and pure atypical secretory granules . |
post - operative pain is the main cause of morbidity following tonsillectomy . despite recent efforts , it is still very difficult to provide sufficient analgesia for most patients .
establishment of good analgesia is important in the postoperative period after adenotonsillectomy , and numerous drug comparison studies are being performed in this field .
the palatine ( faucial ) tonsils are closely associated with several muscles like palatopharyngeus , palatoglossus and superior constrictor of the pharynx , and surgical intervention in the site of the tonsils might affect local muscles directly or indirectly through circulus tonsillaris nervous plexus .
ligaments and muscles around the palatine tonsils are involved in deglutition and themselves are rich sources of pain receptors .
while pain is subjective in general , muscular reactions to pain are objective and electromyography ( emg ) can provide a practitioner with qualitative and quantitative data for their assessment .
in addition to that , edema contributes substantially to the postoperative discomfort and morbidity of tonsillectomy in some patients .
subjective visual analogue scales of different variations remained the main tool for pain assessment being a weak point of many otherwise solid studies on analgesic drug comparison [ 58 ] .
electromyography ( emg ) of swallowing , however , might supply us with data helpful for both qualitative and quantitative assessment of analgesia .
emg reactivity in relation to pain was previously studied among patients following tonsillectomy , as well as among facial pain patients , and in patients with myogenous temporomandibular disorders ( tmd ) [ 1214 ] .
the current research was designed to evaluate any changes in muscular reactions to postsurgical painful irritation that can be observed before and after administration of oxicodone or dexamethasone .
we hypothesized that if these medications induce changes in muscular reactions that can be qualitatively and quantitatively assessed by the surface emg , our understanding of analgesic effects of them might have a sound basis .
the research was designed as an ordinary randomized , double - blind , and placebo - controlled study .
the patients were randomly chosen among surgical patients in the hospital across a 10-month period according to a predetermined randomization code .
the surgeons , anesthesiologists , and recovery room staff members were all blinded with regard to study medications .
the group 1 of surgical patients who undergone tonsillectomy and received oxycontin ( oxycodone ) postsurgical treatment included 30 adults , 17 women and 13 men , ranging in age from 18 to 39 years ( mean = 21.6 years ) .
the group 2 of post - tonsillectomy patients received dexacort ( dexamethasone , 20 mg ) and included 30 adults , 16 women and 14 men , ranging in age from 18 to 34 years ( mean = 20.2 years ) .
the group 3 ( n=30 , 15f , 15 m , mean age 21.3 ) was taken as a control placebo group .
these groups were randomly chosen from 230 patients with recurrent tonsillitis who were scheduled for tonsillectomy .
patients flow chart is presented in the table 1 . before the study all subjects completed a questionnaire regarding their general health and their medical history and signed an informed consent .
subjects had no history of dysphagia or odynophagia due to reasons other than tonsillitis ( exclusion criterion ) , and no history of medical problems or medications that might affect swallowing and drinking ( exclusion criterion ) .
administration of the oxycodone , dexamethasone , and the placebo was as follows : each subject in the group 1 received an intravenous 7-hr infusion of oxycodone 2 mg / hr ( martindale complete drug reference , 35 ed . , 2007 )
starting from 16 hours after surgery ; each subject in the group 2 received an intravenous infusion of dexamethasone ( dexacort , 20 mg ) 20 hours after surgery ; each subject in the group 3 received an intravenous 7-hr infusion of normal saline ( placebo ) ( 1623 hours after surgery ) .
the 4-hour interval for group 2 was chosen because terminal half - time for dexamethasone is approximately 4 hours .
the initial surface emg testing itself was performed 16 ( groups 1 and 3 ) or 20 ( group 2 ) hours after surgery , the final testing was performed 24 hours after surgery .
after the emg test was performed , the patients from the both groups received further analgesia at as needed basis .
outcome measures were assessed 24 hours after surgery . these included patient rating of pain by using the visual analog pain 110 scale ( vas data ) which was recorded by a blinded nurse observer who was unaware of the treatment , and the surface emg investigation ( emg data ) .
dysphagia severity was also clinically evaluated by using the previously published rating scale ( table 2 ) .
edema was observed clinically and was evaluated by using throat edema severity rating scale ( 05 ) ( table 3 ) .
four muscle locations were examined : ( 1 ) m. masseter ( ms location ) , ( 2 ) the submental - submandibular muscle group ( sub ) , ( 3 ) laryngeal strap muscle group ( lsm ) , and m. trapezius ( tz ) .
the equipment used for the emg recordings was a neurodyne neuromuscular sys/3 four channel computer based emg unit with neurodyne medical software ( neurodyne , cambridge , ma , usa ) , and ae-204 active sensors attached to ae-131 electrodes .
the computer program indicates mean , sd , minimum , maximum , range of muscle activity during each trial , and its duration .
specific electrode positions were as follows ( figure 1 ) : ( 1 ) ms location : two bipolar stick - on surface electrodes were placed parallel to the masseter muscle fibers .
( 2 ) sub location : two surface electrodes were attached to the skin beneath the chin on the right or left side of midline to record submental myoelectrical activity over the platisma .
( 3 ) lsm location : two electrodes were placed aside of the thyroid cartilage to record from the infrahyoid ( laryngeal strap ) muscles .
the exact electrode positions ( figure 1 ) for each muscle group are known since the 19 century , and in addition were clarified following anatomical correlates .
electrical impedance at sites of electrode contact was reduced , as target areas were lightly scrubbed with alcohol gauze pads , followed by application of an electrode gel .
after the four pairs of surface emg electrodes were attached , subjects remained completely relaxed for a minute in order to establish the emg visual pattern for resting potential of facial and neck muscles .
subjects were permitted to move chin slightly up while swallowing if needed , as we traced no changes of the graphic and numerical baseline associated with this movement .
rectus capitis posterior minor and minor , as well as some other posterior neck muscles and does not affect signals from above mentioned electrode locations . ) after electrode placement , each participant performed two tasks : three trials of swallowing normal volume of tap water for a particular person .
the data were statistically evaluated by one - dimensional analysis of variance , spss , standard version 10.0.5 ( spss , chicago , il , 1999 ) , and correlation between pain score and semg data were analyzed using criterion with 95% confident interval and calculating of correlation coefficient .
normalization procedure was performed for electric amplitude records in order to change computer - calculated range and mean ( raw range and mean ) into real range and mean ( raw data minus the mean resting potential of an actual muscle group covered by skin ) .
the research was designed as an ordinary randomized , double - blind , and placebo - controlled study .
the patients were randomly chosen among surgical patients in the hospital across a 10-month period according to a predetermined randomization code .
the surgeons , anesthesiologists , and recovery room staff members were all blinded with regard to study medications .
the group 1 of surgical patients who undergone tonsillectomy and received oxycontin ( oxycodone ) postsurgical treatment included 30 adults , 17 women and 13 men , ranging in age from 18 to 39 years ( mean = 21.6 years ) .
the group 2 of post - tonsillectomy patients received dexacort ( dexamethasone , 20 mg ) and included 30 adults , 16 women and 14 men , ranging in age from 18 to 34 years ( mean = 20.2 years ) .
the group 3 ( n=30 , 15f , 15 m , mean age 21.3 ) was taken as a control placebo group .
these groups were randomly chosen from 230 patients with recurrent tonsillitis who were scheduled for tonsillectomy .
patients flow chart is presented in the table 1 . before the study all subjects completed a questionnaire regarding their general health and their medical history and signed an informed consent .
subjects had no history of dysphagia or odynophagia due to reasons other than tonsillitis ( exclusion criterion ) , and no history of medical problems or medications that might affect swallowing and drinking ( exclusion criterion ) .
administration of the oxycodone , dexamethasone , and the placebo was as follows : each subject in the group 1 received an intravenous 7-hr infusion of oxycodone 2 mg / hr ( martindale complete drug reference , 35 ed . , 2007 )
starting from 16 hours after surgery ; each subject in the group 2 received an intravenous infusion of dexamethasone ( dexacort , 20 mg ) 20 hours after surgery ; each subject in the group 3 received an intravenous 7-hr infusion of normal saline ( placebo ) ( 1623 hours after surgery ) .
the 4-hour interval for group 2 was chosen because terminal half - time for dexamethasone is approximately 4 hours .
the initial surface emg testing itself was performed 16 ( groups 1 and 3 ) or 20 ( group 2 ) hours after surgery , the final testing was performed 24 hours after surgery .
after the emg test was performed , the patients from the both groups received further analgesia at as needed basis .
outcome measures were assessed 24 hours after surgery . these included patient rating of pain by using the visual analog pain 110 scale ( vas data ) which was recorded by a blinded nurse observer who was unaware of the treatment , and the surface emg investigation ( emg data ) .
dysphagia severity was also clinically evaluated by using the previously published rating scale ( table 2 ) .
edema was observed clinically and was evaluated by using throat edema severity rating scale ( 05 ) ( table 3 ) .
four muscle locations were examined : ( 1 ) m. masseter ( ms location ) , ( 2 ) the submental - submandibular muscle group ( sub ) , ( 3 ) laryngeal strap muscle group ( lsm ) , and m. trapezius ( tz ) .
the equipment used for the emg recordings was a neurodyne neuromuscular sys/3 four channel computer based emg unit with neurodyne medical software ( neurodyne , cambridge , ma , usa ) , and ae-204 active sensors attached to ae-131 electrodes .
the computer program indicates mean , sd , minimum , maximum , range of muscle activity during each trial , and its duration .
specific electrode positions were as follows ( figure 1 ) : ( 1 ) ms location : two bipolar stick - on surface electrodes were placed parallel to the masseter muscle fibers .
( 2 ) sub location : two surface electrodes were attached to the skin beneath the chin on the right or left side of midline to record submental myoelectrical activity over the platisma .
( 3 ) lsm location : two electrodes were placed aside of the thyroid cartilage to record from the infrahyoid ( laryngeal strap ) muscles .
the exact electrode positions ( figure 1 ) for each muscle group are known since the 19 century , and in addition were clarified following anatomical correlates .
electrical impedance at sites of electrode contact was reduced , as target areas were lightly scrubbed with alcohol gauze pads , followed by application of an electrode gel .
after the four pairs of surface emg electrodes were attached , subjects remained completely relaxed for a minute in order to establish the emg visual pattern for resting potential of facial and neck muscles .
subjects were permitted to move chin slightly up while swallowing if needed , as we traced no changes of the graphic and numerical baseline associated with this movement .
rectus capitis posterior minor and minor , as well as some other posterior neck muscles and does not affect signals from above mentioned electrode locations . ) after electrode placement , each participant performed two tasks : three trials of swallowing normal volume of tap water for a particular person .
the data were statistically evaluated by one - dimensional analysis of variance , spss , standard version 10.0.5 ( spss , chicago , il , 1999 ) , and correlation between pain score and semg data were analyzed using criterion with 95% confident interval and calculating of correlation coefficient .
normalization procedure was performed for electric amplitude records in order to change computer - calculated range and mean ( raw range and mean ) into real range and mean ( raw data minus the mean resting potential of an actual muscle group covered by skin ) .
tables 46 display comparative data beteeen group 1 ( oxycontin group ) and group 2 ( dexacort group ) , and group 3 ( placebo group ) .
it is clearly observed that oxycontin significantly changes both vas pain score and muscle reactions to analgesia ( amplitude ) , dexamethasone insignificantly changes both vas pain score and muscle reactions , while placebo changes vas pain score and tz muscle reaction only .
table 4 shows that both applications did not change the timing of deglutition , however , due to different reasons
real mean is displayed ( the mean resting potentials of an actual muscles covered by skin were 2.808 v = for the sub location and lsm location , 2.495 v = for the ms location , 3.826 v = for the tz location ) .
a typical single water swallow of a healthy individual between 18 and 70 years of age was observed graphically at the rectified and low - pass filtered semg as a normal wave with upward deflections and a sharp apex when recorded from the ms , sub and lsm locations .
when the surface emg record of a swallow was performed after oxycodone had been administered , it was clearly seen that the sharp apexes were gone and the swallow waves looked smoth ( figure 2 ) .
the dexamethasone group ( group 2 ) emg records indicated that dexamethasone did not affect muscles the same way as oxycodone did but nevertheless improved the swallow pattern towards its normalization ( figure 3 ) .
this positive effect , however , was observed only in 43% of cases ( 13 out of 30 ) .
clinical data of these 13 patients indicated significant edema around the operated site that was reduced after dexamethasone was administered . for these patients ,
the throat edema severity rating scale ( table 3 ) indicated mean 4.1 before and 2.3 after the treatment ( p<0.05 ) .
the records taken from the patients of the placebo group ( group 3 ) did not show even these changes ( figure 4 ) .
changes toward improvement in dysphagia severity rating scale were significant for group 1 , and insignificant for group 3 .
for group 2 , these changes were significant for patients with edema ( the same 43% of cases ) and insignificant for the others .
there were no significant differences between the three study groups with respect to age , sex , height , weight , or duration of surgery .
there was no significant difference in blood loss between the oxycodone group ( 2.31.6 ml / kg ) , the dexamethasone group ( 2.51.6 ml / kg ) , and the placebo group ( 1.90.9 ml / kg ) .
numerous reflexes , like glottic closure reflex , gag reflex , jaw reflex , are associated with the oral - pharyngeal area .
even the pharyngeal phase of swallowing itself is defined as the most complex reflex elicited by the nervous system .
it is important for a placebo - controlled study , because a medication can change the true involuntary reflex response and a placebo can not .
the current study clearly shows that oxycodone changes deglutition pattern and that this change can be well observed on the semg records ( figure 1vs.figure 2 ) .
in addition to graphic changes , quantitative amplitude changes also were well detected and the difference between group 1 and group 3 ( placebo ) was clearly seen .
dexamethasone also might change deglutition pattern while not as vividly as oxycodone , and this change is observed on the semg records ( figure 1vs.figure 3 ) .
thus , we might suggest that semg is a valid technique to study drugs like analgesics that affect muscular reactions .
while oxycodone reduces pain due to its effect on opioid receptors , that affect muscles as well , dexamethasone does not affect muscles .
steroids have a multiplicity of physiological actions : effects on metabolism , electrolyte and water balance , skeletal muscle , the cardiovascular system , the central nervous system , formed blood elements , and anti - inflammatory and immunosuppressive actions but otolaryngologists use them mainly because of their anti - inflammatory actions . in the present study
, it appears that the use of the steroid dexamethasone reduced pain significantly in some patients but only slightly in other patients .
the statistics was performed for the whole cohort of 30 patients and the mean data were obtained . if , however , only 43% of cases ( 13 patients ) with visible emg changes are examined , the results of dexamethasone administration appear more impressive .
while these 13 patients had significant clinically proven postoperative edema , we may think that the primary action of dexamethasone was the reduction of the edema and the reduction of pain was secondary to this effect .
the absence of statistically significant changes of timing patterns of deglutition might have an explanation .
it is known that postsurgical dysphagia / odynophagia increases time needed for a single swallow . it could be suggested ( an additional study on healthy subjects is needed to prove this ) that oxycodone due its opioid nature affects temporal relationships of the events of the swallowing cycle the same way .
thus , despite the fact that a patient is relieved from pain , his / her swallowing remains slow . in case of dexamethasone
the semg test was set 24 hours after surgery because 12 hours after surgery or earlier , the majority of the patients were unable to drink and presented a spasm of submental - submandibular muscles recorded from the sub location .
this fact , perhaps , might indicate a weakness of the semg method in cases when rapid 12 hours after surgery evaluation is needed .
otherwise , for the research purposes in drug comparison it seems to be very suitable and convenient technique .
application of dexamethasone after tonsillectomy is advisable because of the reduction of postoperative morbidity while the reduction of the postoperative pain is secondary to the reduction of edema .
semg might be used as an adjunctive measure of pain behavior via assessment of muscular reactions to pain and to analgesia . | summarybackgroundsurface electromyographic ( semg ) study of post - tonsillectomy swallow - evoked muscular reactions was performed in order to evaluate the efficacy and safety of oxycodone and dexamethasone in pain management after tonsillectomy.material/methods90 randomly chosen operated adults were divided into three groups .
group 1 ( n=30 ) was treated with oxycontin ( oxycodone ) injections ; group 2 ( n=30 ) was treated with dexacort ( dexamethasone ) , and group 3 ( n=30 ) was a placebo group .
pain assessment included visual analogue scale ( vas ) pain score and the emg data like the timing , electric amplitude and graphic patterns of muscular activity during deglutition .
we investigated masseter , infrahyoid and submental - submandibular muscles .
records from trapezius muscle were used for control .
the results were compared with previously established normative database .
the patients were tested 24 h after surgery .
the semg data were compared with vas pain score with regard to changes in clinical condition of the patients.resultsanalgesia with oxycodone smoothed the recorded semg swallow peaks and increases time of deglutition .
dexamethasone normalized muscular activity in deglutition in cases with edema as detected by the emg records .
statistically significant difference in muscle reactions was detected between the two groups and the placebo group.conclusionsapplication of oxycodone significantly reduces the postoperative pain .
application of dexamethasone after tonsillectomy is advisable because of the reduction of postoperative morbidity while the reduction of the postoperative pain is secondary to the reduction of edema .
semg might be used as an adjunctive measure of pain behavior via assessment of muscular reactions to pain and to analgesia . |
global regulatory networks control most cellular decisions , including differentiation , stress response , and environmental adaptation .
these networks frequently have a specific bow - tie organization where multiple inputs and outputs are connected to a core process or a regulator ( csete and doyle , 2004 ) . in bacteria ,
one of the most common global regulatory motifs comprises a sigma factor a promoter - specific subunit of the bacterial rna polymerase and an anti - sigma factor that binds and inhibits the sigma factor ( sterberg et al . , 2011 , paget , 2015 ) .
the multi - input structure of these motifs allows them to integrate a variety of stimuli that control stress responses ( hengge , 2009 ) and developmental checkpoints ( rudner and losick , 2001 ) .
one of the best - studied sigma / anti - sigma factor checkpoints is involved in the control of flagellar biosynthesis ( chilcott and hughes , 2000 , mukherjee and kearns , 2014 ) . in escherichia coli and other bacterial species ,
flagellar genes are organized in a hierarchical regulatory network of three classes based on their order of expression and common transcriptional regulation ( aldridge and hughes , 2002 , chevance and hughes , 2008 , chilcott and hughes , 2000 , kalir et al . , 2001 )
altogether , this network encodes more than 40 proteins that are necessary for flagellar motility and chemotaxis ( minamino and imada , 2015 , typas and sourjik , 2015 ) . at the upper level of this regulatory hierarchy is the class i master regulator that is formed by two polypeptides , flhd and flhc .
it activates class ii genes that encode structural components and assembly factors of the flagellar hook - basal body ( hbb ) complex and of the export apparatus required for biogenesis of flagellar filament .
two other class ii genes encode the sigma factor ( flia ) and the anti- factor ( flgm ) . at first
, both polypeptides are coexpressed , but is inhibited by binding to flgm . upon hbb completion ,
however , flgm is secreted from the cell through the flagellar export apparatus to release free . this activates expression of -controlled ( class iii ) genes that encode the subunit of the flagellar filament ( flic ) , stator components of the flagellar motor ( mota and motb ) , as well as the chemotaxis pathway ( hughes et al . , 1993 ) .
notably , flgm , flia , and a number of genes that are required for flagellar assembly possess both class ii and class iii promoters , and are therefore referred to as class ii+iii genes ( chevance and hughes , 2008 , chilcott and hughes , 2000 ) .
motility and chemotaxis of e. coli are among the most extensively studied biological processes ( sourjik and wingreen , 2012 , typas and sourjik , 2015 ) , and the chemotaxis pathway has been used as a model to study evolutionary network optimization ( alon et al . , 1999 ,
experimental evolution is increasingly applied to investigate predictions of evolutionary theory , particularly in microorganisms ( barrick and lenski , 2013 , elena and lenski , 2003 , hindr et al . , 2012 ,
for example , it has been extensively used to study the evolution of antibiotic and stress resistance ( chait et al .
, 2007 , maclean et al . , 2010 ) or catabolism of specific nutrients ( blount et al
. , 2012 ) , typically yielding mutations in a single or a small number of target genes ( blount et al .
such experimental evolutionary analyses have helped to better understand several features of the evolutionary process , such as epistatic interactions between multiple mutations , evolutionary trajectories , and evolution in changing environments ( taute et al . , 2014 ) . however , understanding the evolution of complex behaviors and of the underlying regulatory networks still remains a challenge ( hindr et al . , 2012 ) .
our results enabled us to characterize the evolvability of the regulatory network that controls bacterial motile behavior .
surprisingly , the observed evolutionary enhancement of motility was achieved through adaptive mutations in a number of functionally very different genes , encoding components of flagellar motor and export apparatus , as well as transcriptional and translational factors .
nevertheless , we showed that most of these mutations acted through a common adaptive mechanism , namely remodeling of the sigma / anti - sigma factor checkpoint , thus leading to common phenotypic changes .
this remodeling of the checkpoint apparently adjusts cell motility under selection both during laboratory evolution and in natural isolates of e. coli .
our findings suggest that bow - tie topology can generally enhance evolvability of networks , thus indicating a previously unrecognized evolutionary function of this widespread network motif .
we performed experimental evolution of motile behavior of e. coli using selection for enhanced spreading in tryptone broth ( tb ) soft agar ( tbsa ) , a porous medium containing a mixture of amino acids that act both as nutrients and as chemoattractants .
consumption of amino acids generates steep local attractant gradients that are subsequently followed by bacteria , which requires motility , chemotaxis , and growth ( berg and turner , 1979 , wolfe and berg , 1989 ) . in our experiment ,
a cell population was allowed to spread for 12 hr on a tbsa plate , at a growth rate of approximately one cell generation per hour , and cells from the edge of the outer spreading ring were taken to inoculate a new plate ( figure 1b ) .
we observed that spreading of the evolved strains greatly improved within the first 510 rounds of selection ( figure 1c ; figure s1a ) , although the extent of improvement differed between independently evolved lines ( l1l7 ; figure 1d ) .
consistently , evolved strains had a strong competitive advantage in spreading on tbsa plates compared with the parental strain rp437 ( figure s1b )
. evolved e. coli lines showed similarly increased spreading in minimal medium soft agar ( sa ) supplemented with serine ( figure 1e ) , confirming that the observed enhancement of spreading is not specific to a particular composition of the medium . as noted ,
spreading in sa that initially contains a uniform distribution of attractant requires not only motility and chemotaxis , but also consumption of attractant to create a gradient . to distinguish contributions of motility and metabolism , we tested behavior of evolved strains in gradients of a non - metabolizable analog of aspartate , -methyl - d , l - aspartate ( measp ) , established either in sa or in liquid ( krembel et al . , 2015 ) .
evolved strains showed enhanced chemotactic spreading up the gradient in both assays ( figures s1c and s1d ) , confirming specific selection for improved motility and chemotaxis in our experiments . to elucidate the mechanisms that underlie the observed enhancement in motility and chemotaxis , we first examined how experimental evolution affected e. coli motility ( figure s2a ) .
the most apparent phenotypic change in all evolved strains was increased swimming velocity in liquid : whereas velocity of the parental strain was comparable with that observed previously ( morehouse et al .
2011 ) , it nearly doubled in some of the evolved strains . moreover , the majority of evolved strains also exhibited an increased frequency of reorientations ( tumbling rate ) .
both of these features were strongly correlated with spreading in tbsa , although the significance of the correlation with increased tumbling rate was weaker .
as judged from the chemotactic bias , i.e. , chemotactic drift normalized by the swimming velocity , higher velocity alone could mostly account for the increase in the chemotactic drift in liquid .
our analysis further showed that evolved strains have higher frequency of flagellar motor rotation , as observed for individual motors in immobilized cells , as well as longer and , in the case of l1 , also more numerous flagellar filaments ( figure s2b ) .
the statistical analysis of these data suggest that increased flagellar length is the main determinant of higher velocity .
in contrast , higher tumbling rate shows only weak correlation with increased length and number of flagella ( figure s2c ) .
consistent with the observed increase in flagellar length , levels of extracellular flagellin were elevated in all strains ( figure s2d ) .
we next investigated whether the observed enhancement of motility may result from changes in gene expression .
for that , we measured transcriptional activity at all three levels of hierarchy of flagellar genes , using reporters for representative class i ( flhd ) , class ii ( flie ) , and class iii ( flic ) promoters .
interestingly , we observed that the activity of pflic was upregulated in all evolved strains , whereas the activity of pflhd and pflie was unchanged or even downregulated ( figure 2a ) .
this increase in pflic activity showed strong correlation with cell spreading in tbsa ( figure 2b ) and with swimming velocity ( figure 2c ) , indicating that specific upregulation of class iii gene expression is a major determinant of the behavioral enhancement .
consistent with this assumption , an increased expression of ( flia ) or titration of anti- ( flgm ) from a plasmid resulted in a similar co - regulation of pflic activity with spreading in tbsa and with swimming velocity ( figures 2b and 2c ) . among the other parameters , flagellar length showed highly significant correlation with pflic ( and anticorrelation with pflie ) activity ( figure 2d ; figure s3a ) , whereas flagellar number and motor speed did not show significant correlation with gene expression ( figures s3b and s3c ) .
however , overexpression of either flagellin flic or stator components mota and motb was not sufficient to increase spreading ( figure s3d ) , suggesting that concerted upregulation of multiple class iii genes is necessary to enhance motility .
these results were further confirmed using genome - wide transcriptome profiling of the evolved strains ( figure 2e ) .
here , we also considered that during experimental evolution changes in gene expression could result not only from selection for enhanced motility , but also from adaptation to growth in tb at 30c . in order to distinguish between these two types of selection
, we subjected all lines to growth in liquid tb medium at 30c for another 20 culture passages .
this selection indeed either partly or entirely reversed the enhancement of spreading in tbsa ( figure s3e ) , and these strains were included in the transcriptome analysis .
transcriptomics results confirmed strong correlation between enhanced spreading in soft agar and selective upregulation of class iii ( and class ii+iii ) flagellar genes , consistent with higher activity of . interestingly , expression of several genes related to cell wall biosynthesis ( murd , mure , and murg ) showed significant correlation with enhanced motility , whereas genes for atp synthase ( atpa , atpb , atpc , atpd , atpe , atpf , atpg , atph , and atpi ) , amino sugar metabolism ( nane , nank , naga , nagb , and nage ) , and succinyl - coenzyme a ( coa ) synthetase ( succ and sucd ) were weakly counter - regulated .
furthermore , a small number of genes were consistently up- or downregulated upon selection in both tbsa and liquid tb ( figure s3f ) , possibly associated with general growth selection .
subsequently , we used genome sequencing of the evolved strains to map mutations that emerged during selection for enhanced spreading in tbsa .
this analysis yielded point mutations ( snps ) in 14 genes , insertions of insertion sequence 1 ( is1 ) transposable sequence in 5 genes , as well as 1 gene deletion ( table s1 ) .
no mutations were found in non - coding regions , including promoter sequences . to our surprise , in spite of changes in flagellar gene expression in all evolved strains , only two of these mutations affected known transcriptional regulators of flagellar genes : one in flgm ( anti- ) ( l3 ) and another in sspa ( l1 ) that encodes a starvation - response factor ( hansen et al . , 2005 ) .
in addition , strain l1 carries mutation in a trna - encoding gene sert that has been previously shown to affect translation of flgm ( chevance et al . , 2006 ) . instead , multiple mutations mapped to genes that encode functional or structural components of flagellar apparatus . five strains
( l1 , l2 , l4 , l5 , and l7 ) have mutations in flii , the atpase component of the flagellar export machinery ( fan and macnab , 1996 ) .
two of these strains ( l1 and l4 ) have additional mutations in the switch components of the flagellar basal body , flim and flig .
these proteins are required for rotation of the motor and for its control by the chemotaxis system ( bren and eisenbach , 1998 , toker and macnab , 1997 ) , but they are also involved in the function of the export apparatus ( gonzlez - pedrajo et al . ,
furthermore , strain l3 carries , in addition to a mutation in flgm , a mutation in chez , the phosphatase of the chemotaxis system , and strain l2 has an additional mutation in the aspartate chemoreceptor tar . a significant number of mutations mapped to genes that had no previously established relation to the flagellar regulon .
these genes encoded a translation factor ( ycih ) , enzymes involved in cell envelope biogenesis ( ldtb , mdoh , and idi ) or in metabolism ( hisa , argh , glpk , and ydfg ) , transporters ( cuss and lacy ) , and proteins of unknown function ( yebo and yceo ) .
it is well established that not all mutations arising in evolutionary experiments must be adaptive ( rosenberg , 2001 ) . to distinguish contributions of different mutations to the observed enhancement of motility
, they were introduced individually into the wild - type strain and assayed for their effects on spreading in tbsa ( figure 3a ) .
we observed that all mutations in flagellar genes , as well as in genes encoding cell envelope biogenesis enzymes , increased spreading .
moreover , spreading was also enhanced by mutations in genes encoding the transcriptional ( sspa ) and translational ( ycih ) regulators , as well as the uncharacterized protein yebo .
other mutations had little or no effect or even decreased spreading . despite these very different functionalities of mutated proteins
, we observed that the effects of individual mutations on spreading in tbsa are highly correlated with increased swimming velocity and with increased tumbling rate , and similar correlation could be observed for the evolved strains or for varying expression of or flgm ( figures 3b and 3c ; figures s2a , s2b , and s4b ) .
this confirms that increased swimming velocity and tumbling rate can account for the enhancement in spreading .
consistent with our analysis for the evolved strains , individual mutations that enhanced spreading in tbsa also conferred an advantage for following measp gradients in soft agar ( figure 3d ) or in liquid ( figure 3e ) .
finally , essentially all individual adaptive mutations , including those in functional and structural flagellar proteins , led to elevated expression of class iii genes ( figure s4a ) .
these changes showed strong correlation with increased spreading in tbsa ( figure 3f ) and with swimming velocity ( figure 3 g ) , as observed already for the evolved strains ( figures 2b and 2c ) , confirming that adaptive changes in class iii gene expression are the major determinant of increased motility .
interestingly , the mutation in sspa showed a disproportional enhancement of motility ( figure 3 g ) and not only longer , but also more numerous flagella ( figure s4c ) , similar to l1 that carries it .
how could mutations in flagellar basal body and export apparatus mediate adaptive changes in class iii gene expression ?
a potential mechanism linking these proteins to gene expression is secretion of the anti- factor flgm .
as discussed above ( see introduction and figure 1a ) , flgm - mediated inhibition ensures that is activated only upon assembly of the basal body that allows flgm to be secreted .
however , because flgm is steadily expressed even after the initial checkpoint exit , its cellular levels continue to control expression of class iii genes ( kollmann et al . , 2005 ) .
therefore , mutations that change the efficiency of flgm secretion may also affect gene expression . to verify the involvement of this checkpoint control , we deleted flgm in all evolved strains as well as in strains that carry individual mutations . indeed ,
deletion of flgm entirely abolished the enhancement of spreading in tbsa in three of the evolved strains ( l4 , l5 , and l7 ) and reduced it in three other strains ( l1 , l3 , and l6 ) ( figure 4a ; figure s5a ) .
similarly , in strains carrying individual mutations in flagellar genes , spreading enhancement was either abolished ( for flii or flim mutations ) or reduced ( for flig mutation ) by the deletion of flgm ( figure 4b ; figure s5b ) .
this confirms our hypothesis that increased secretion of the anti- factor flgm is the main mechanism of adaptive motility enhancement via mutations in flagellar proteins .
interestingly , deletion of flgm also reduced the effects of the sspa and ycih mutations , although these gene products have no established connection to the regulation of flgm secretion .
nevertheless , the effects of ycih , ldtb , mdoh , yebo , and sspa mutations on motility appear to be at least partly independent of the flgm secretion , which may explain ( residual ) flgm - independent enhancement of spreading that is observed in strains l1 , l2 , l3 , and l6 that carry these mutations . in order to directly confirm the connection between the checkpoint remodeling and enhanced expression of class iii genes , we quantified efficiency of flgm secretion .
consistent with our explanation , in all evolved strains flgm secretion efficiency was significantly enhanced , leading to a lower cellular level of flgm relative to its level in the supernatant ( figure 4c ) .
moreover , there was a strong correlation between the flgm secretion efficiency and the level of class iii gene expression , both in the evolved strains and in strains containing individual mutations that enhanced motility ( figure 4d ) .
such correlation was not observed for secretion efficiency of flic ( figure s5c ) , suggesting that the adaptive enhancement of secretion is specific to flgm .
it is well established that evolution frequently needs to optimize different conflicting functions , thus leading to adaptive trade - offs ( schuetz et al . , 2012 ,
2012 ) . for expression of highly abundant flagellar and chemotaxis proteins , the most likely fitness trade - off may be associated with growth .
indeed , when relative growth fitness of the evolved strains in liquid tb was plotted as a function of pflic promoter activity , clear anticorrelation between the two was observed ( figure 5a ) . a similarly apparent trade - off
could be observed between the growth fitness and swimming velocity ( figure 5b ) .
consistent with that , growth rate increased upon disruption of flhc ( eliminating expression of the entire flagellar regulon ) or flia ( eliminating expression of class iii genes ) , and it was severely reduced by deletion of flgm ( upregulation of class iii genes ) .
disruption of flic gene increased growth rate nearly as much as flia deletion , confirming that a large part of fitness cost associated with class iii gene expression is due to production of flagellin .
indeed , deletion of flic largely alleviated the fitness burden in strain l1 ( figure 5c ) .
the remaining cost might arise from expression of other class iii genes or from the energy expenditure for flagellar rotation .
however , mutations in sspa and ycih seem to generally incur disproportionally high cost compared with their fitness increase , likely because they affect not only motility , but also other cellular functions .
we hypothesized that the observed mechanism of motility enhancement might also play a role in the evolutionary plasticity of natural isolates of e. coli .
consistent with that , we observed a highly significant correlation between activity of pflic promoter and spreading of motile natural isolates that were randomly selected from the ecor collection ( ochman and selander , 1984 ) ( figure 6a ) .
in contrast , correlation with the activity of pflie or pflhd promoters was either much weaker or non - significant ( figures 6b6d ) .
this confirms our hypothesis that variation in motility of the natural isolates is mainly determined by expression of class iii genes rather than of the entire flagellar regulon .
the underlying cause again appears to be the plasticity of checkpoint efficiency , because secretion of flgm is highly correlated with the spreading in tbsa and with the pflic activity in most natural isolates ( figures 6e and 6f ) .
taken together , our findings demonstrate that mutations in multiple flagellar proteins modulate motility through a common control of flgm secretion ( figure 7a ) .
we also obtained similar mutations in flagellar genes in another evolutionary experiment , where selection for motility was performed on minimal medium sa plates ( table s2 ) .
we thus hypothesized that the multi - input motif present in the flagellar checkpoint control , but also common in many other cellular networks , might play a general role in evolution . to test this hypothesis , we simulated in silico evolution of a simple bow - tie network where multiple input nodes ( x1 to x3 ) affect activity of a common intermediate node ( y ) that in turn activates the output node ( z ) , and compared it with evolution of linear networks where either one or multiple input nodes act independently ( figure 7b ) . in these simulations ,
the fitness increase for the bow - tie network under selection was markedly faster than for the linear networks ( figure 7c ) , even though the multi - input linear network can in principle attain the same maximal fitness as the bow - tie network ( see supplemental experimental procedures ) .
it is commonly assumed that as a consequence of long - term selection , living organisms have evolved to perform different tasks optimally ( de vos et al . , 2013 , flamholz et al . , 2013 , ibarra et al . , 2002 ,
however , proving this assumption is difficult , not least because details of the past evolutionary selection pressure and the underlying trade - offs between different functions are typically unknown ( hindr et al . , 2012 ) .
moreover , establishing the genotype - to - phenotype mapping during evolutionary adaptation remains a challenge , given the difficulty in distinguishing between neutral and adaptive mutations and specifically linking a mutation to one and unlinking it from other traits ( lehner , 2013 ) .
although experimental evolution can address some of these questions by subjecting ( micro-)organisms to selection under defined laboratory conditions and monitoring ensuing changes in their phenotype and genotype ( hindr et al . , 2012 ) , it is most frequently applied to simple traits that involve only one or a few proteins ( blount et al . , 2012 ,
, 2015 , toprak et al . , 2011 , weinreich et al . , 2006 ) . here , we used selection for e. coli motility in a porous environment to investigate how adaptive evolution under a specific selection pressure shapes this complex bacterial phenotype and what are the internal constraints and underlying mechanisms of genotype and phenotype remodeling .
we observed that enhanced motility evolved in all our experiments , consistent with high selective pressure at the edge of the spreading colony caused by nutrient depletion . on the phenotypic level ,
the enhancement of e. coli motility in soft agar was primarily driven by increased swimming velocity , but also by increased tumbling rate .
this phenotypic adaptation can be well understood in the context of bacterial chemotaxis between traps created by agar ( wolfe and berg , 1989 ) . here ,
similar to the chemotaxis in liquid , bacteria must swim up the gradient to detect changes in chemical concentration and to bias their motility by reducing tumbling rate ( sourjik and wingreen , 2012 ) .
higher swimming velocity should thus generally enhance spreading by accelerating both the random movement and chemotaxis .
however , in the porous environment , higher swimming velocity alone would also lead to more frequent trapping of bacteria in the agar pores , which would limit spreading .
the observed increase in tumbling rate may alleviate this hindrance by enabling bacteria not only to cover distances between pores faster , but also to escape them more efficiently ( wolfe and berg , 1989 ) . on the molecular level
, this phenotypic adaptation could be accounted for by upregulated expression of class iii flagellar genes .
this upregulation led to increased flagellar length and motor rotation rate , whereby the former was apparently the main determinant of faster swimming .
such specific upregulation of class iii genes in all of the independent evolutionary experiments was surprising , because the established transcriptional regulation of flagellar genes in e. coli occurs primarily at the upper ( class i ) level of the regulatory hierarchy and affects expression of all three gene classes ( fahrner and berg , 2015 ) ( figure 7a ) .
moreover , this is seemingly in contrast to previous studies showing that poorly motile e. coli strains could activate motility through insertion of insertion sequence 5 ( is5 ) element into the regulatory region of the flhdc operon ( barker et al . , 2004 , wang and wood , 2011 ) .
on the other hand , we observed no transcriptional regulation of individual class iii genes or changes in the network topology .
our results thus demonstrate that concerted upregulation of multiple class iii genes is required , and also sufficient , to enhance motility , likely because not only flic , but also other products of class iii ( and class ii+iii ) genes are required for assembly and energizing of longer flagella , and thereby for faster swimming .
consistent with that explanation , proton channel components mota and motb , as well as chaperones ( e.g. , flis ) that are required for flic filament assembly ( auvray et al . , 2001 ) , are encoded by class iii or class ii+iii genes .
we therefore propose that , besides its importance for sequential ordering of expression ( chilcott and hughes , 2000 , kalir et al . , 2001 )
, the topology of flagellar gene regulation may be a consequence of evolutionary selection for high plasticity of flagellar motility .
this way , all class iii genes that are required for increased motility are under common regulatory control , which enables concerted evolutionary tuning of their levels .
similarly unexpectedly , we observed that the main mechanism of the adaptive upregulation of class iii genes relies on enhanced secretion of the anti - sigma factor flgm rather than on other possible mechanisms , such as upregulation of the expression or activity of . the role of flgm secretion as a checkpoint control for the intermediate stage of flagella assembly in e. coli and other bacteria is well established .
the observed recurrent utilization of this checkpoint in evolutionary enhancement of cell motility suggests that it also serves another , entirely different adaptive function , namely in enabling bacteria to efficiently adjust motility dependent on environmental selection .
this adjustment occurs through modulation of flgm secretion , and it can be mediated by mutations in a number of proteins that form flagellar secretion machinery or regulate secretion in other ways ( figure 7a ) .
consistent with its general function in evolutionary plasticity , the /flgm checkpoint remodeling appears to explain the tuning of motility not only under laboratory selection , but also in natural isolates of e. coli .
interestingly , although the exact mechanisms through which mutations in different motor proteins enhance secretion remain to be investigated , our analysis suggests that this enhancement is specific to flgm as a substrate .
future studies of these mutations might thus provide further insights into specificity and evolution of the export machinery of type iii secretion systems .
we believe that the observed adaptive mechanism may be indicative of a previously unrecognized general role of global checkpoint control mechanisms , and of bacterial sigma / anti - sigma factors in particular , in the evolutionary plasticity of gene expression .
most such global control networks possess a specific bow - tie structure , where multiple regulatory inputs control the activity ( or levels ) of a key factor that in turn regulates multiple downstream targets , e.g. , expression of different genes .
previous adaptive explanations for such network structure suggested that it might enable independent evolution of the input and output functions without affecting the regulatory core ( csete and doyle , 2004 ) and also compress cellular input information ( friedlander et al . , 2015 ) .
our results strongly suggest that such multi - input design also enhances evolutionary plasticity of the regulatory networks , by substantially increasing the number of possible mutations that affect activity of the common central process ( the /flgm checkpoint in our case ) , and therefore adjust regulation of the downstream targets .
although it is difficult to assess the relative importance of selection for these individual properties , the enhanced evolvability is likely to be a major factor in the overall evolutionary enrichment of the bow - tie motifs in cellular networks . why did phenotype remodeling not occur at the level of class i gene expression , which is controlled by multiple transcriptional inputs ( figure 7c ) ?
for one , the transcription factors involved are not unique to the flagellar regulon , meaning that changes in their activity are likely to affect other cellular functions .
moreover , instead of increasing flagellar length , the upregulation of the entire flagellar regulon might increase flagellar number , which was apparently less critical for the enhancement of motility in our experiments .
we thus hypothesize that although previously reported activation of the flhdc operon by is5 insertion ( barker et al . , 2004 , wang and wood , 2011 ) can naturally act as a binary on - switch of motility , subsequent gradual tuning of motility under selection is more efficiently achieved through specific class iii gene upregulation . further supporting the adaptive advantage of such regulation
, we observed that even mutations resulting in the flgm - independent enhancement of motility led to higher expression of class iii , but not of other flagellar genes .
finally , we observed that selection for increased motility carries a cost of reduced growth fitness , meaning that evolution can not simultaneously maximize both functions . such conflict between optimization objectives is common in evolution ( garland , 2014 , roff and fairbairn , 2007 ) , and it might explain why many laboratory e. coli strains , as well as natural isolates , have attenuated motility .
interestingly , recent theoretical analysis has shown that for multiple conflicting optimization tasks , the existing phenotypes fall on a specific pareto optimality front connecting both extremes of optimization , which in case of two tasks is a line ( shoval et al . , 2012 ) .
in apparent agreement with this prediction , all strains in our analysis mapped roughly along the same line when growth fitness was plotted against swimming velocity .
this was even true for strains that carry individual mutations , which is not trivial because of possible epistatic interactions between multiple mutations in evolved strains .
we believe that such universal alignment points to remodeling of flgm secretion as a general mechanism for optimal tuning of the motility - growth trade - off , so that any change in flgm export would move the phenotype along the same pareto optimization line . in conclusion , experimental evolution demonstrated that bacteria can rapidly adjust such a complex trait as motility under selection pressure , following a defined trade - off line with conflicting optimization for growth .
this high phenotypic plasticity appears to be inherent in the design of the hierarchical flagellar gene regulatory network , where genes modulating motility and chemotaxis are under the common control of a sigma / anti - sigma factor checkpoint that can be rapidly tuned under selection .
thus , besides its well - established function in the control of timing of flagellar gene expression , this checkpoint plays a key role in the evolutionary plasticity of e. coli motility .
this latter function seems to be aided by its bow - tie architecture , and we propose that the similar design of other global regulatory networks might generally promote evolutionary adaptation .
all strains used in this study were derived from e. coli k12 strain rp437 that is commonly used as a wild - type for chemotaxis and motility studies ( parkinson , 1978 ) and has constitutively activated motility because of is5 insertion in the promoter region of flhdc operon ( barker et al . , 2004 ) .
details of strain and plasmid construction can be found in the supplemental experimental procedures .
cells were grown at 30c in tb medium [ 1% tryptone and 0.5% sodium chloride ( nacl ) ] supplemented with appropriate antibiotics ( ampicillin : 100 gml , kanamycin : 50 gml ) .
microevolution of e. coli strain rp437 was performed in 0.3% soft agar plates supplemented with tb .
after an overnight incubation at 30c , cells from the edge of the spreading ring were collected and re - inoculated onto a new soft agar plate .
the same procedure was repeated 2030 times , as indicated . for reverse evolution , evolved strains were repeatedly grown overnight in 3 ml liquid tb at 30c . for motility analysis , e. coli cells were grown in 10 ml tb medium at 34c in a rotary shaker at 180 rpm .
cells ( 1 ml ) at mid - log phase ( od600 [ optical density at 600 nm ] = 0.6 ) were collected and re - suspended in 1 ml tethering buffer ( 10 mm k2hpo4 , 10 mm kh2po4 , 100 m edta , 1 m l - methionine , 10 mm lactic acid [ ph 7.0 ] ) . bacterial spreading was measured by inoculating 2 l cell suspension at the top of 0.3% soft agar containing either tb or minimal a medium supplemented with 0.1 mm aspartate or serine as indicated , after 78 hr incubation in tb or 2022 hr incubation in minimal a soft agar at 30c . for measurements of chemotactic bias in soft agar , gradient of measp
was established in minimal a soft agar containing glucose for 14 hr at 4c before cell inoculation as described previously ( krembel et al . , 2015 ) .
swimming velocity and tumbling rate were measured by cell tracking in a glass chamber using phase - contrast microscopy ( nikon ti eclipse , 10 objective , na = 0.3 , cmos camera eosens 4cxp ) .
drift velocity and chemotaxis efficiency in measp gradient were tested in a microchamber made of poly - dimethylsiloxane ( pdms ) using differential dynamic microscopy ( ddm ) ( wilson et al . , 2011 ) and phase differential microscopy ( dm ) ( colin et al . , 2014 ) .
the cells were subjected to a linear gradient of measp of 100 m per 2 mm , and average concentration at the measurement segment was 50 m . all data were analyzed using imagej ( https://imagej.nih.gov/ij/ ) with custom - written plugins for swimming velocity , drift velocity , and tumbling rate analysis ( colin et al . , 2014 ) .
e. coli cells were grown in tb as described above and harvested at mid - log phase ( od600 = 0.6 ) .
dna samples were prepared using commercial kits ( qiagen ) , and rna samples were prepared using hot acid / phenol rna extraction method ( maniatis et al . , 1982 ) .
genome sequencing was performed at gatc biotech ; rna sequencing was performed at the bioquant sequencing facility ( university of heidelberg ) .
all sequencing data were analyzed using dnastar lasergene software with e. coli strain mg1655 as reference template . for promoter activity assays , e. coli strains transformed with reporter plasmids were grown in tb supplemented with kanamycin in 96-well plates at 30c in a rotary shaker at 180 rpm .
cell fluorescence was measured using flow cytometry on bd lsrfortessa sorp cell analyzer ( bd biosciences ) . for growth fitness measurements ,
equal amounts of a tested strain expressing cfp ( pvs129 ) and the wild - type rp437 strain labeled with yellow fluorescent protein ( yfp ) ( pvs132 ) were inoculated into 1 ml of tb medium supplemented with ampicillin and 20 m isopropyl--d - thiogalactopyranosid ( iptg ) .
after growth in a shaker at 30c for 20 hr , 10 l of cell suspensions was transferred into 1 ml pbs ( ph 7.4 ) , and the numbers of cfp- and yfp - labeled cells were measured by flow cytometry as described above .
the culture was then reinoculated and the dynamics of the two cell populations was so monitored for 5 days .
protein secretion was assayed using c - terminal fusions of a hemagglutinin ( ha ) tag to flgm or to 175-amino - acid n - terminal fragment of flic .
flgm - ha expression from a plasmid pbn4 was induced by 100 m iptg ; flic - ha expression from a plasmid pbn5 was induced by 50 m iptg .
protein levels in the pellet and supernatant , respectively , were analyzed by immunoblotting as described in the supplemental experimental procedures . for measurements of flic levels , density of cell culture
was adjusted to od600 = 1 , and extracellular flic filaments were sheared by precellys homogenizer ( bertin technologies ) at 4,500 rpm for 20 s. afterward , bacterial cells were collected by centrifugation ( 13,000 rpm for 1 min ) at 4c .
anti - flic antibody at a 1:10,000 dilution and secondary anti - rabbit igg antibody labeled with peroxidase at a 1:5,000 dilution were used for detection .
motor rotation assay was conducted as described previously ( ryu et al . , 2000 ) with minor modifications .
cells grown in tb medium were collected , re - suspended in tethering buffer , and flagella were sheared by 60 passages through 26 g syringe needle .
cells were allowed to settle on glass coverslips treated with poly - l - lysine ( sigma ) and incubated for 5 min with a suspension of 1.1 m polystyrene beads ( sigma ) .
rotation of beads was monitored using phase - contrast microscopy ( 40 magnification ) at 1,000 hz for 20 s. the beads were tracked using imagej with a custom - written plugin , and the traces were analyzed using matlab to extract the frequency of rotation .
carbon - coated copper grids ( 400 mesh ) were hydrophilized by glow discharging ( pelco easiglow ; ted pella ) .
five microliters of a cell suspension ( grown to od600 = 0.50.6 as described above ) was applied onto the hydrophilized grids and stained with 2% uranyl acetate after a short washing step with water .
samples were analyzed using a jeol jem-2100 transmission electron microscope with an acceleration voltage of either 80 or 200 kv .
details of statistical data analysis and computational simulations are described in the supplemental experimental procedures .
| summaryregulatory networks play a central role in the relationship between genotype and phenotype in all organisms . however , the mechanisms that underpin the evolutionary plasticity of these networks remain poorly understood . here , we used experimental selection for enhanced bacterial motility in a porous environment to explore the adaptability of one of the most complex networks known in bacteria .
we found that the resulting phenotypic changes are mediated by adaptive mutations in several functionally different proteins , including multiple components of the flagellar motor .
nevertheless , this evolutionary adaptation could be explained by a single mechanism , namely remodeling of the checkpoint regulating flagellar gene expression . supported by computer simulations ,
our findings suggest that the specific bow - tie topology of the checkpoint facilitates evolutionary tuning of the cost - benefit trade - off between motility and growth .
we propose that bow - tie regulatory motifs , which are widespread in cellular networks , play a general role in evolutionary adaptation . |
currently , the medicare outpatient pps pays a fixed amount for a limited bundle of routine , dialysis - related services , known as the composite rate ( cr ) .
dialysis facilities are allowed to bill medicare separately for other covered services furnished to esrd patients .
congress has required cms to develop an expanded pps for outpatient dialysis services that includes as many drugs and diagnostic procedures provided to esrd patients as possible ( section 422(c ) of public law 106 - 554 , the medicare , medicaid , and the state children 's health insurance program ( schip ) , benefits improvement and protection act of 2000 [ bipa ] ) .
one objective of the bipa is to determine the appropriateness of creating a more comprehensive dialysis bundle that would include routinely furnished drugs and diagnostic procedures that are currently billed separately from the cr . under a new system , the bundle of services covered by the pps
would then be expanded to include as many of the services provided to these patients as possible . for services included in the current cr ,
previous studies have found only weak evidence that case mix plays a large role in determining dialysis facility costs ( dor , held , and pauly , 1992 ; hirth et al . , 1999 ) . from a policy perspective , the lack of a strong relationship between case - mix factors and cr costs reduces the motivation for developing a case - mix adjustment mechanism to ensure access to quality care for patients with costly characteristics and to provide equitable reimbursement for the facilities that treat them . however , these previous studies have had access to only a limited range of case - mix descriptors .
further , the relationship between case mix and the costs of providing separately - billable services has not been studied yet .
the vast majority of separately - bill - able costs are incurred for the delivery of drugs , with recombinant human erythropoietin ( epo ) alone accounting for almost three - quarters of payments for items billed separately by dialysis units .
if these variations are systematic ( i.e. , can be predicted by observable patient characteristics ) , a case - mix adjustment system may be possible .
indeed , incorporation of these separately billable costs into an expanded pps bundle may make such a system necessary to minimize incentives to avoid treating patients with high expected drug usage or who incur other high costs .
the objectives of this study were to : ( 1 ) characterize costs and medicare payments per dialysis treatment for cr services and separately - billable components under the existing dialysis payment system ; ( 2 ) characterize the extent of variation in these costs and payments across dialysis providers ; and ( 3 ) determine the relationship between patient characteristics and these costs and payments . accomplishing these objectives would help to establish the utility of a case - mix adjustment for an expanded esrd outpatient pps , to identify the case - mix factors that are predictive of costs and payments , and to estimate the percentage of variation in spending accounted for by such an adjustment system .
first , the medicare cost reports with cost reporting periods , ending in calendar year 2000 , were obtained for each medicare certified , freestanding dialysis provider .
they have been used in previous research ( dor , held , pauly , 1992 ; hirth et al . , 1999 ) to estimate statistical cost functions , assessing the impact of dialysis modality mix , patient case mix , facility size , location , and practice patterns on medicare - allowable costs for cr services .
hospital - based facilities , which comprise 18 percent of dialysis facilities nationally ( forum of esrd networks clearinghouse office , 2000 ) , were excluded from the present study because their cost reports have a different format without certain data fields , which would impede comparability of the data .
in addition , only a small number of fy 2000 cost reports for hospital - based facilities are currently available from cms .
a potential weakness of the cost report data is that the level of auditing is not high because it is not used to determine payment amounts for individual facilities .
however , the two models for total facility costs ( dor , held , pauly , 1992 ; hirth et al . , 1999 ) had excellent explanatory power , which suggests that reporting errors were not substantial , since reporting errors would tend to reduce explanatory power .
further , several fields in the cost report have been verified against independent data sources with high levels of agreement ( e.g. , facility - reported numbers of medicare dialysis sessions correspond closely to medicare sessions in the billing data discussed later .
second , cms standard analytical files ( safs ) of medicare - paid claims provide the means to detail medicare spending on dialysis - related care for medicare - eligible esrd patients .
the data available from cms billing are limited by the rules of eligibility for medicare .
medicare eligibility begins 3 months after the onset of esrd unless that patient is already entitled to medicare based on age or other disability ; medicare remains the secondary payer for up to 30 additional months for patients with employer - sponsored health insurance .
another limitation of the billing data is that itemization is not available for patients currently enrolled in a medicare health maintenance organization ( hmo ) .
the outpatient saf provides the primary source of information about current payments received by dialysis facilities for treatment of esrd patients .
the data used here are limited to services provided between july 1 , 2000 and december 31 , 2000 to take advantage of changes made by cms in the format of the safs from version h to version i. version h recorded payments only for the claim as a whole , but it did not indicate which items were paid by medicare . nor
in contrast , the new version i format identifies payments by the hcfa common procedure coding system ( hcpcs ) code and revenue center .
thus , version i allows an accurate disaggregation of medicare payments for specific services without affecting the allocation of total spending to patients .
outpatient dialysis claims were identified as all claims for dialysis patients with the provider type code for dialysis facilities ( code 72 : clinic hospital based or independent renal dialysis facility ) .
identifying dialysis claims via provider type , rather than the presence on the claim of at least one item with a dialysis revenue center , yields a more complete set of dialysis - related claims .
for example , a claim including only ancillary services such as epo , other drugs , or laboratory tests may not indicate any dialysis revenue center , but it would be correctly identified as dialysis - related , based on the type of provider .
third , the only available source of comorbidity data for all esrd patients was the end stage renal disease medical evidence report ( cms form 2728 ) database .
form 2728 provides information on the cause of esrd and about 20 comorbidities for the first esrd treatment and selected baseline pre - esrd laboratory measures , including creatinine , albumin , and hematocrit . in 1995 ,
the reporting of this form was expanded from medicare - eligible patients only to all esrd patients .
the primary weaknesses in the form 2728 data are a lack of completeness in many of the forms and the fact that comorbidities are only reported at the start of esrd .
the reported comorbidities are not the primary purpose of the form , which is to establish medicare eligibility on the basis of chronic renal failure .
renal data system , 1999 ) and abstractions of medical records ( ashby et al .
, 1999 ; wolfe , ashby , and port , 2000 ; and longenecker et al . , 2000 ) have verified underreporting .
nonetheless , comorbidities reported on this form have been found to be useful predictors of mortality , suggesting that the most salient comorbidities are reported ( wolfe et al . , 2000 )
; ashby et al . , 1998 ; roys et al . , 1999 ) .
because completion of this form is required only at the onset of esrd , there is no followup reporting of newly developed comorbidities or of cured , former comorbidities .
additional sources of data include the cms hospital wage index and the database used to generate cms unit - specific reports for each dialysis facility .
the hospital wage index applicable for fy 2000 is used to adjust for area labor costs ( federal register , 1999 ) .
the unit - specific report database was the source of demographic information and the primary cause of renal failure for each facility 's set of patients .
the analysis sample of dialysis facilities was derived from all freestanding facilities with cost reports for fy 2000 ( n=2,498 ) .
several facilities with implausible values for costs per session ( less than $ 50 or more than $ 400 ) were excluded .
patient demographic and comorbidity data were linked to the facility at which they were treated on january 1 , 2000 for prevalent patients or on day 91 of chronic dialysis for patients incident during fy 2000 .
these matching protocols follow those employed in unit - specific performance reports prepared annually by the ( university of michigan kidney epidemiology and cost center , 2002 )
. facilities with fewer than 10 patients with available medical evidence forms were excluded to improve reliability of the comorbidity data . for payment analyses , each analysis ( dependent ) variable was restricted to facilities with values less than the 99th percentile in order to reduce the influence of outliers in the highest 1 percent . finally , facilities that did not appear in the 1999 cms annual facility survey ( u.s .
renal data system , 1999 ) and those with missing data for key variables were excluded . the final analysis sample included 2,115 facilities ( 85 percent ) with a subsample of 748 facilities that provided only incenter hemodialysis was also analyzed ( table 1 ) .
billing data were summarized at the facility level for all patients for whom medicare was the primary payer for the dialysis session .
we report results describing the relationship between patient case mix and several measures of dialysis facility cost and payment per hemodialysis ( hd)-equivalent session .
all dialysis modalities are included in the analysis : hd , peritoneal dialysis ( pd ) , hd and pd training . due to reporting conventions in billing data ,
pd treatments are measured in days and are reimbursed an amount that is three - sevenths of the rate paid for hd treatments . therefore , reported counts of pd treatments were multiplied by three - sevenths to produce hd - equivalent sessions .
therefore , reported treatment weeks were multiplied by three to yield hd - equivalent sessions . in these analyses , we measure costs as reported in the cost reports and payments received by dialysis facilities as medicare - allowable charges as reported in patients ' bills .
medicare - allowable charges are the sum of medicare payments from the billing data ( 80 percent of allowable charges ) and patient obligations or copays ( 20 percent of allowable charges ) . to the extent that some patient obligations are not collected from patients or their secondary insurers , medicare - allowable charges
however , to the extent that patients with private insurance pay higher rates than medicare - allowable charges , the average payment received by facilities would be underestimated by focusing on dialysis sessions delivered to medicare primary payer patients .
these measures are listed in table 1 , which summarizes the distribution of cost and payment per session by facility .
per - treatment total facility cost and total facility payments , medicare payment , and patient copay , were separated into cr and separately - billed services .
separately - billed services were further disaggregated into epo and non - epo . for all freestanding facilities in the analysis sample , we report results for the measures of cost and payment previously discussed . for those freestanding facilities providing only incenter hd services , we report results for a subset of these measures .
table 2 lists these variables , along with some results to be discussed later . included on the list are the usual demographic characteristics : age , sex , and race .
the next set of variables describes the main disease - related characteristics of the patient , including primary diagnosis , other ( non - medicare ) insurance coverage , and duration of esrd .
each of these variables is available for the census of patients treated at the facility .
characteristics of a facility 's patients based on cms 2728 ( and , hence , unavailable for patients incident prior to 1995 ) include 17 variables .
these variables indicate the percentage of patients for whom a particular comorbid condition is present and average values for six continuous measures ( laboratory values and weight ) , all measured at the beginning of chronic dialysis treatment . at the facility level , the fraction of patients with each comorbidity was computed , based on form 2728 for patients receiving treatment at each facility in calendar year 2000 .
the comorbidity measure at the facility level is , thus , not a measure of the comorbidity of all the patients receiving dialysis at each facility , but represents instead the comorbidity level of patients incident after 1995 when reporting of comorbidity data began on form 2728 .
one - half of all facilities in this study have baseline comorbidity information for at least 62 percent of their patients .
in addition to the measures of patient case mix displayed in table 2 , the analyses include six variables that reflect the facility 's mix of dialysis modalities : hd , hd training , pd , and pd training .
the training treatments are further classified into those paid at $ 20 above the cr ( hd training and continuous cycling pd training ) versus $ 12 above the cr ( continuous ambulatory pd training ) .
finally , our analyses control for the wage rates prevailing in the metropolitan area of the facility and for urban versus rural classification .
the first describes the distribution of cost and patient characteristics across dialysis facilities , using percentiles ( tables 1 and 2 ) .
this presentation gives a sense of the extent of variation in cost and case mix across facilities .
second , linear regression analyses were used to evaluate the relationships of average patient demographic and comorbidity measures to per session costs and payments at the facility level ( table 3 ) .
as previously mentioned , the dependent variables in these analyses were the average levels of cost ( 2000 cost report ) and payments ( july - december 2000 ) per hd - equivalent dialysis session for several categories of services .
analyses of per session dialysis facility costs and medicare payments were performed using linear regression analysis .
these analyses show how the average cost or payment per session differs from case mix and other predictors measured at the facility level . for each dependent variable , we estimated a series of linear models .
the third model adds all of the variables describing patient case mix , including demographics , main treatment characteristics , comorbidities , and clinical measures .
the first two models include those factors ( wages and modality mix ) that are likely to be accounted for by a new payment system even if case - mix adjustment is not implemented .
thus , our approach is to determine the extent to which adding measures of patient case mix helps to explain variation in dialysis cost and payment beyond the variation explained by factors that would be incorporated in ratesetting regardless of the implementation of case - mix adjustment .
we report results describing the relationship between patient case mix and several measures of dialysis facility cost and payment per hemodialysis ( hd)-equivalent session .
all dialysis modalities are included in the analysis : hd , peritoneal dialysis ( pd ) , hd and pd training . due to reporting conventions in billing data ,
pd treatments are measured in days and are reimbursed an amount that is three - sevenths of the rate paid for hd treatments . therefore , reported counts of pd treatments were multiplied by three - sevenths to produce hd - equivalent sessions .
therefore , reported treatment weeks were multiplied by three to yield hd - equivalent sessions . in these analyses , we measure costs as reported in the cost reports and payments received by dialysis facilities as medicare - allowable charges as reported in patients ' bills .
medicare - allowable charges are the sum of medicare payments from the billing data ( 80 percent of allowable charges ) and patient obligations or copays ( 20 percent of allowable charges ) . to the extent that some patient obligations are not collected from patients or their secondary insurers , medicare - allowable charges
however , to the extent that patients with private insurance pay higher rates than medicare - allowable charges , the average payment received by facilities would be underestimated by focusing on dialysis sessions delivered to medicare primary payer patients .
these measures are listed in table 1 , which summarizes the distribution of cost and payment per session by facility .
per - treatment total facility cost and total facility payments , medicare payment , and patient copay , were separated into cr and separately - billed services .
separately - billed services were further disaggregated into epo and non - epo . for all freestanding facilities in the analysis sample , we report results for the measures of cost and payment previously discussed . for those freestanding facilities providing only incenter hd services , we report results for a subset of these measures .
table 2 lists these variables , along with some results to be discussed later . included on the list are the usual demographic characteristics : age , sex , and race .
the next set of variables describes the main disease - related characteristics of the patient , including primary diagnosis , other ( non - medicare ) insurance coverage , and duration of esrd .
each of these variables is available for the census of patients treated at the facility .
characteristics of a facility 's patients based on cms 2728 ( and , hence , unavailable for patients incident prior to 1995 ) include 17 variables .
these variables indicate the percentage of patients for whom a particular comorbid condition is present and average values for six continuous measures ( laboratory values and weight ) , all measured at the beginning of chronic dialysis treatment . at the facility level , the fraction of patients with each comorbidity was computed , based on form 2728 for patients receiving treatment at each facility in calendar year 2000 .
the comorbidity measure at the facility level is , thus , not a measure of the comorbidity of all the patients receiving dialysis at each facility , but represents instead the comorbidity level of patients incident after 1995 when reporting of comorbidity data began on form 2728 .
one - half of all facilities in this study have baseline comorbidity information for at least 62 percent of their patients .
in addition to the measures of patient case mix displayed in table 2 , the analyses include six variables that reflect the facility 's mix of dialysis modalities : hd , hd training , pd , and pd training .
the training treatments are further classified into those paid at $ 20 above the cr ( hd training and continuous cycling pd training ) versus $ 12 above the cr ( continuous ambulatory pd training ) .
finally , our analyses control for the wage rates prevailing in the metropolitan area of the facility and for urban versus rural classification .
the first describes the distribution of cost and patient characteristics across dialysis facilities , using percentiles ( tables 1 and 2 ) .
this presentation gives a sense of the extent of variation in cost and case mix across facilities .
second , linear regression analyses were used to evaluate the relationships of average patient demographic and comorbidity measures to per session costs and payments at the facility level ( table 3 ) .
as previously mentioned , the dependent variables in these analyses were the average levels of cost ( 2000 cost report ) and payments ( july - december 2000 ) per hd - equivalent dialysis session for several categories of services .
analyses of per session dialysis facility costs and medicare payments were performed using linear regression analysis .
these analyses show how the average cost or payment per session differs from case mix and other predictors measured at the facility level . for each dependent variable , we estimated a series of linear models .
the third model adds all of the variables describing patient case mix , including demographics , main treatment characteristics , comorbidities , and clinical measures .
the first two models include those factors ( wages and modality mix ) that are likely to be accounted for by a new payment system even if case - mix adjustment is not implemented .
thus , our approach is to determine the extent to which adding measures of patient case mix helps to explain variation in dialysis cost and payment beyond the variation explained by factors that would be incorporated in ratesetting regardless of the implementation of case - mix adjustment .
table 1 describes the distribution of costs and payments per dialysis session across dialysis facilities .
considerable variability is evident in the data , both for cr and separately - billable services .
for example , the 95th percentile of reported costs for cr services is $ 191.21 versus $ 106.53 at the 5th percentile .
similarly , the 95th percentile of reported costs for separately - bill - able services is $ 80.87 versus $ 38.37 at the 5th percentile .
table 2 describes the distribution of outpatient characteristics across dialysis facilities . for each characteristic ,
the table reports the value at the 10th , 50th , and 90th percentiles of facilities . clearly , there are substantial differences in case mix across facilities . for example , the average patient age was 57 at the 10th percentile of facilities versus age 67 at the 90th percentile of facilities .
there are also substantial reported differences with regard to other factors , including duration of esrd , race , and the availability of other insurance at onset of esrd ( private or medicaid ) .
the 10th percentile of facilities has only 13 percent of patients starting esrd treatment within the last year versus 33 percent at the 90th percentile of facilities .
similarly , the percentage of black patients is 2 percent at the 10th percentile of facilities versus 81 percent at the 90th percentile .
the distribution of medicaid coverage ranges from 11 percent at the 10th percentile of facilities to 49 percent at the 90th percentile
. only 8 percent of patients received epo before esrd at the 10th percentile of facilities versus 46 percent for 90th percentile of facilities .
there also exists substantial variability in primary diagnosis across facilities . for each of the diagnosis categories ,
there is roughly a three - fold difference in percent of patients from the lowest 10th to the 90th percentile . in terms of the prevalence of comorbidities
, there is particularly wide variability in cardiac arrest , heart disease , peripheral vascular disease , and cerebrovascular disease .
given the substantial variation in both costs and case mix , a case - mix adjustment system would have potential value in an expanded outpatient pps bundle .
table 3 summarizes our analyses of the extent to which the variation in patient case mix documented in table 2 is related to dialysis facilities ' costs and payments described in table 1 .
the table shows the r values for the series of models previously discussed in the methods section .
the r statistic measures the fraction of the variation in per session costs or payments that can be attributed to the predictive factors in the model .
a higher value of r indicates that the costs or payments are more accurately predicted by the values of the predictor measures .
we focus on the r statistics to emphasize the overall explanatory power of case mix , rather than on the estimated coefficients for any particular case - mix factor .
the table also shows the average value for total per treatment cost and payment plus several important components of cost and payment .
the average cost per session across all 2,115 freestanding dialysis facilities is $ 199.11 , while the average payment is $ 202.08 .
hence , on average , dialysis treatment is slightly profitable for providers as a whole ( assuming that providers collect patient copays ) .
the cr accounts for roughly 70 percent of total cost or $ 141.16 . for cr services ,
in contrast , separately - billable services cost an average of $ 57.95 while associated payments are $ 77.06 . hence , as has been noted elsewhere ( medicare payment advisory commission , 2002 ) , while the cr payment does not cover cr costs , the separately - billable payment exceeds separately - billable costs by enough to more than make up the shortfall . among separately - billable services ,
almost three - quarters of the cost arises from the use of epo , which costs an average of $ 43.11 per treatment and generates an average payment of $ 53.74 .
total facility per treatment cost for the 2,115 freestanding facilities ' models , including area wage index and modality , explain roughly 5 percent of variation in costs across facilities .
once the patient case - mix measures are added , the explanatory power of the model triples to 15 percent . however , for per treatment costs associated with separately - billable services , inclusion of case mix boosts the explanatory power from 2 to 19 percent . among components of the separately - billable services , case mix is especially important in explaining variation in epo costs . without case - mix measures ,
table 3 summarizes the results of our analyses of the 748 dialysis facilities providing in - center hd only .
the average per treatment cost and payment amounts for these facilities are very similar to those for all freestanding facilities .
the product of hd - only facilities is somewhat more homogeneous than that for all freestanding facilities .
hence , one source of variation in cost is eliminated and , not surprisingly , the relative explanatory power of case mix appears even higher for these facilities .
the r for total per treatment cost increases nearly four - fold , from 5 to 19 percent .
for separately - billable services , case mix explains 23 percent of the variation in cost across dialysis facilities .
the necessary conditions for the development of a case - mix adjusted payment system for esrd include : ( 1 ) demonstrated variability in costs and case mix across facilities ; ( 2 ) a significant and substantial relationship between case mix and facility cost ; and ( 3 ) an economically feasible system for collecting data , measuring case mix .
the analyses presented in this article document the existence of substantial variability in measures of both costs and patient case mix across dialysis facilities .
the extent of cost and case - mix variability indicates that the failure to adjust for case mix in an expanded outpatient renal pps could place facilities at substantial financial risk .
any resulting underpayment of facilities that treat high - cost patients could eventually encourage facilities to avoid such patients altogether .
we find that these case - mix measures do explain a substantial fraction of the variation among facilities in per session costs and payments . as a group
, they explain a greater fraction of the variation in costs and payments than the wage index and dialytic modality , factors almost certain to be adjusted for by an expanded pps .
certain variables in the model ( e.g. , race / ethnicity ) ; coefficients of individual variables are not reported here to focus on overall predictive value of the models , though predictive of costs , may not be considered appropriate for case - mix adjustment .
although three of the four race / ethnicity variables ( asian , black , native american ) are significant at the 5 percent level relative to the reference group ( white persons ) , race / ethnicity contributes only a small proportion of the explained variation in costs per session ( r of the full model including race is 0.147 ; r excluding the race variable is 0.131 ) .
thus , about 90 percent of the model 's explanatory power can be attributed to other factors .
this analysis demonstrates the feasibility of at least beginning to build a case - mix adjustment system , using existing administrative data ; limitations and suggestions for further research are discussed later .
the multivariate models reported here are intended primarily to show that certain groups of predictors ( wage index , dialysis modality , and case mix ) have the potential to explain variation in costs and payments .
the r values reported here are interpretable for such purposes . however , for a variety of technical reasons , the detailed results from these models should be interpreted with great caution .
notably , correlated measures of patient conditions are included in the models and such redundancies can make the interpretation of individual predictive factors difficult .
for example , the list of comorbidities includes a variety of cardiac conditions which are positively and significantly correlated with each other . in these analyses ,
a comprehensive list of potential predictors of costs and medicare payments was considered , with no attempt made to develop parsimonious models for these outcomes .
thus , the coefficients of specific predictive factors ( not reported here ) should be interpreted recognizing the potential for multicolinearity among the factors .
ideally , case - mix measurements would be based on accurate , objective , current measures of all relevant patient characteristics ( with appropriate severity scales for comorbidity measures ) for all patients treated at each facility .
while accurate , current , and objective demographic and treatment - related data were available for all patients , comorbidity data were restricted to patients with a medical evidence form ( cms 2728 ) .
in addition , clinical conditions indicated in billing data from hospitals , physicians , and other non - dialysis providers should be studied to determine the extent to which these baseline comorbidity data change over time .
an alternative source of current comorbidity data could be based on annual administration of form 2728 .
however , that would raise other issues due to the possibility that certain comorbidities may themselves be outcomes of the quality of dialysis care received ; that is not an issue with the current form 2728 's administration at the start of dialysis .
another issue that must be faced before implementing a case - mix adjustment system is the potential that the expanded pps and any associated case - mix adjustment system could change practice patterns or reporting practices .
for example , adding epo to the prospective bundle would likely reduce dosages relative to the existing system under which epo can be billed separately . facilities may choose to administer epo subcutaneously , which involves discomfort for the patient , but might involve a lower dose than the intravenous route of administration that is prevalent today ( hynes et al . ,
both the average payment level in an expanded pps and the design of a case - mix adjustment system must anticipate , or at least monitor and adjust for , any such changes in practice patterns .
likewise , the completeness of comorbidity reporting will undoubtedly rise if these data are used to set payments .
this will necessitate some recalibration of the adjustment formula to account for the new data collection environment .
in addition , each case - mix indicator should be evaluated in terms of its potential for gaming .
factors that are especially subjective , amenable to manipulation , or difficult to verify in an audit , should require substantial justification before they are included in a case - mix adjustment formula .
further , other factors for which data are not currently available , might account for a portion of the remaining , unexplained variation in costs .
if such factors can be identified , efforts to collect additional case - mix data on a systematic basis might be justified .
our study was limited to services currently billed by dialysis providers . to the extent that services now billed by other types of providers ( e.g. ,
most separately - billable laboratory tests , vascular access procedures ) are included in an expanded service bundle , further analyses , relating case mix to existing billing data , will be necessary . finally , as a refinement to the models presented here
, it would be useful to estimate separate models for hd and pd patients to determine if each dialysis modality requires a unique case - mix adjustment system .
overall , we conclude that there is adequate justification for further exploring the development of a case - mix adjustment system for consideration in conjunction with an expanded outpatient renal pps .
ideally , case - mix measurements would be based on accurate , objective , current measures of all relevant patient characteristics ( with appropriate severity scales for comorbidity measures ) for all patients treated at each facility .
while accurate , current , and objective demographic and treatment - related data were available for all patients , comorbidity data were restricted to patients with a medical evidence form ( cms 2728 ) .
in addition , clinical conditions indicated in billing data from hospitals , physicians , and other non - dialysis providers should be studied to determine the extent to which these baseline comorbidity data change over time .
an alternative source of current comorbidity data could be based on annual administration of form 2728 .
however , that would raise other issues due to the possibility that certain comorbidities may themselves be outcomes of the quality of dialysis care received ; that is not an issue with the current form 2728 's administration at the start of dialysis .
another issue that must be faced before implementing a case - mix adjustment system is the potential that the expanded pps and any associated case - mix adjustment system could change practice patterns or reporting practices .
for example , adding epo to the prospective bundle would likely reduce dosages relative to the existing system under which epo can be billed separately .
facilities may choose to administer epo subcutaneously , which involves discomfort for the patient , but might involve a lower dose than the intravenous route of administration that is prevalent today ( hynes et al .
, 2002 ) . both the average payment level in an expanded pps and the design of a case - mix adjustment system must anticipate , or at least monitor and adjust for , any such changes in practice patterns
. likewise , the completeness of comorbidity reporting will undoubtedly rise if these data are used to set payments .
this will necessitate some recalibration of the adjustment formula to account for the new data collection environment .
in addition , each case - mix indicator should be evaluated in terms of its potential for gaming .
factors that are especially subjective , amenable to manipulation , or difficult to verify in an audit , should require substantial justification before they are included in a case - mix adjustment formula .
further , other factors for which data are not currently available , might account for a portion of the remaining , unexplained variation in costs .
if such factors can be identified , efforts to collect additional case - mix data on a systematic basis might be justified .
our study was limited to services currently billed by dialysis providers . to the extent that services now billed by other types of providers ( e.g. ,
most separately - billable laboratory tests , vascular access procedures ) are included in an expanded service bundle , further analyses , relating case mix to existing billing data , will be necessary .
finally , as a refinement to the models presented here , it would be useful to estimate separate models for hd and pd patients to determine if each dialysis modality requires a unique case - mix adjustment system .
overall , we conclude that there is adequate justification for further exploring the development of a case - mix adjustment system for consideration in conjunction with an expanded outpatient renal pps . | congress has required cms to expand the medicare outpatient prospective payment system ( pps ) for dialysis services to include as many drugs and diagnostic procedures provided to end stage renal disease ( esrd ) patients as possible .
one important implementation question is whether dialysis facility case mix should be reflected in payment .
we use fiscal year ( fy ) 2000 cost report and patient billing and clinical data to determine the relationship between costs and case mix , as represented by several patient demographic , diagnostic , and clinical characteristics .
results indicate considerable variability in costs and case mix across facilities and a significant and substantial relationship between case mix and facility cost , suggesting case mix payment adjustment may be important . |
the obturator nerve arises from the anterior division of the ventral rami of the second , third , and fourth lumbar nerves in the lumbar plexus .
the branch originating from the third lumbar nerve is the largest and is distributed in the skin of the abductor muscles and thigh .
it descends through the psoas major muscle forming a bundle , and enters the thigh through the upper part of the obturator foramen along the inner wall of the lesser pelvis , accompanied by the obturator artery .
the anterior branch is responsible for the sensory innervation of the hip joint and mid - thigh , as well as for the motor innervation of the superficial adductor muscles .
the posterior branch provides sensory perception to the skin on the posterior side of the knee joints and motor function to the deep adductor muscles26 ) .
obturator neuropathy ( obturator nerve entrapment syndrome ) is caused by pressure or injury to the obturator nerve on the upper part of the obturator foramen , which presents as pain or loss of sensation over the upper medial thigh with or without hip adduction weakness9 ) .
if untreated , hip adductor weakness will result in a functional disorder of the hip joint14 ) .
the majority of obturator neuropathy cases result from trauma , iatrogenic injuries such as orthopedic surgery ( hip arthroplasty , urologic surgery , and spine surgery via the retroperitoneal cavity ) , sports hernias , and gynecological problems such as ectopic pregnancies11217 ) .
moreover , obturator neuropathy may also be associated with compressive lesions such as cysts , neurofibroma , and lipoma78 ) . diagnosing and
treatment of obturator neuropathy is difficult , as it is a rare condition with which many neurosurgeons are unfamiliar .
knowledge of the obturator nerve anatomy is essential for adequate surgical planning and success . with an aim to improve the accuracy of diagnosis and to prevent complications
, we performed a cadaveric morphometric study to investigate the anatomical features and relationships between the obturator nerve and its surrounding anatomical landmarks .
twenty - eight lower limbs from fourteen ( 12 males and 2 females ) adult cadavers with an age range of 25 - 95 years ( mean , 61.6 years ) were dissected .
the cadavers were fixed with a mixture of formalin , phenol , alcohol , and glycerin .
the skin was removed in the supine position to expose the pectineus muscle by carefully detaching the soft tissue from the fascia lata and the femoral artery .
after the pectineus muscle was dissected , the obturator nerve was identified within the fascial layer ( fig .
anatomically important structures used in this study include the anterior superior iliac spine ( asis ) , the pubic tubercle , the inguinal ligament , the femoral artery , and the adductor longus .
different variables were measured in 28 specimens of 14 cadavers , as follows : point a : the distance from the asis to the obturator nerve exit zone of the foramen , and point b : the distance from the pubic tubercle to the obturator nerve exit zone of the foramen ( fig .
2 ) ; point c : the horizontal distance from the pubic tubercle to the obturator nerve exit zone of the foramen , and point d : the vertical distance from the pubic tubercle to the obturator nerve exit zone of the foramen ( fig .
3 ) ; point e : the shortest distance from the inguinal ligament to the obturator nerve exit zone of the foramen , point f : the length of the inguinal ligament from the asis to point e , point g : the length of the inguinal ligament from the pubic tubercle to point e ( fig .
4 ) , point h : the length of the obturator nerve exposed between the obturator nerve exit zone of the foramen and the adductor longus , and point i : the shortest distance from the obturator nerve exit zone of the foramen to the femoral artery ( fig . 5 ) .
distances and lengths between the obturator nerve exit zone of the foramen and anatomical landmarks were measured using a standard meter and a goniometer .
after exiting the obturator foramen , the obturator nerve passes below the inguinal ligament and traverses the medial side of the femoral artery . to expose the obturator nerve in the supine position ,
it is important that the pectineus muscle is identified following removal of the fascia lata and soft tissue .
the obturator nerve is located beneath the pectineus muscle and runs medially and downwards between the adductor longus and adductor brevis muscles .
table 1 shows the distance from the asis / pubic tubercle to the obturator nerve , while table 2 shows the vertical and horizontal relationships between the obturator nerve and the pubic tubercle .
the mean distance from the asis to the obturator nerve exit zone of the foramen was 113.46.5 mm in the right thigh and 114.27.4 mm in the left thigh .
the mean distance from the pubic tubercle to the obturator nerve exit zone was 30.54.4 mm in the right thigh and 30.35.4 mm in the left thigh .
the mean horizontal and vertical distances from the pubic tubercle to the obturator nerve exit were 18.23.2 mm in the right thigh and 16.53.0 mm in the left thigh , and 27.32.2 mm in the right thigh and 26.34.2 mm in the left thigh , respectively .
no statistically significant differences between the measurements obtained from the right and left thighs were obtained . the shortest distance ( point e ) between the obturator nerve and the inguinal ligament as well as the relationship and distance between the inguinal ligament and the asis / pubic tubercle and point e
the shortest mean distance between the obturator nerve and the inguinal ligament was 18.53.2 mm in the right thigh and 19.75.2 mm in the left thigh .
the mean length of the inguinal ligament from the asis to point e was 1064.2 mm in the right thigh and 99.67.7 mm in the left thigh , while the mean length of the inguinal ligament from the pubic tubercle to point e was 24.95.1 mm in the right thigh and 23.74.1 mm in the left thigh .
when the measurements between the asis and the pubic tubercle were compared based on point e from the inguinal ligament to the obturator nerve exit zone , the shallowest point of the obturator nerve was distributed approximately one - fifth from the medial inguinal ligament .
no statistically significant differences between the measurements obtained from the right and left thighs were obtained .
table 3 shows the shortest distance between the obturator artery and the femoral artery , as well as the length of the obturator nerve exposed between the obturator foramen and the adductor longus .
the mean length of the obturator nerve exposed between the obturator foramen and the adductor longus was 41.26.9 mm in the right thigh and 41.03.8 mm in the left thigh .
the shortest mean distance between the femoral artery and the obturator nerve exit zone was 30.05.4 mm in the right thigh and 25.83.9 mm in the left thigh .
no statistically significant differences between the measurements obtained from the right and left thighs were obtained .
after exiting the obturator foramen , the obturator nerve passes below the inguinal ligament and traverses the medial side of the femoral artery . to expose the obturator nerve in the supine position ,
it is important that the pectineus muscle is identified following removal of the fascia lata and soft tissue .
the obturator nerve is located beneath the pectineus muscle and runs medially and downwards between the adductor longus and adductor brevis muscles .
table 1 shows the distance from the asis / pubic tubercle to the obturator nerve , while table 2 shows the vertical and horizontal relationships between the obturator nerve and the pubic tubercle . the mean distance from the asis to the obturator nerve exit zone of the foramen was 113.46.5 mm in the right thigh and 114.27.4 mm in the left thigh .
the mean distance from the pubic tubercle to the obturator nerve exit zone was 30.54.4 mm in the right thigh and 30.35.4 mm in the left thigh .
the mean horizontal and vertical distances from the pubic tubercle to the obturator nerve exit were 18.23.2 mm in the right thigh and 16.53.0 mm in the left thigh , and 27.32.2 mm in the right thigh and 26.34.2 mm in the left thigh , respectively .
no statistically significant differences between the measurements obtained from the right and left thighs were obtained .
the shortest distance ( point e ) between the obturator nerve and the inguinal ligament as well as the relationship and distance between the inguinal ligament and the asis / pubic tubercle and point e are shown in table 3 .
the shortest mean distance between the obturator nerve and the inguinal ligament was 18.53.2 mm in the right thigh and 19.75.2 mm in the left thigh .
the mean length of the inguinal ligament from the asis to point e was 1064.2 mm in the right thigh and 99.67.7 mm in the left thigh , while the mean length of the inguinal ligament from the pubic tubercle to point e was 24.95.1 mm in the right thigh and 23.74.1 mm in the left thigh .
when the measurements between the asis and the pubic tubercle were compared based on point e from the inguinal ligament to the obturator nerve exit zone , the shallowest point of the obturator nerve was distributed approximately one - fifth from the medial inguinal ligament .
no statistically significant differences between the measurements obtained from the right and left thighs were obtained .
table 3 shows the shortest distance between the obturator artery and the femoral artery , as well as the length of the obturator nerve exposed between the obturator foramen and the adductor longus .
the mean length of the obturator nerve exposed between the obturator foramen and the adductor longus was 41.26.9 mm in the right thigh and 41.03.8 mm in the left thigh .
the shortest mean distance between the femoral artery and the obturator nerve exit zone was 30.05.4 mm in the right thigh and 25.83.9 mm in the left thigh .
no statistically significant differences between the measurements obtained from the right and left thighs were obtained .
obturator nerve injuries are a direct result of nerve sectioning , stretching , crushing , electrocoagulating , or ligating .
identifying an isolated obturator nerve lesion can be difficult based on history and physical examination alone , because other nerve injuries in the lower abdomen and pelvic region also present with groin and thigh pain .
magnetic resonance imaging ( mri ) is helpful to detect atrophy of the adductor brevis and adductor longus muscles and to identify the anatomical structures of the inguinal and surrounding areas16 ) .
longer fibrillation potentials , high amplitude , and complex exercise units in the adductor brevis and adductor longus muscles are observed in patients with obturator neuropathy ; however , the iliopsoas and quadriceps muscles of the lower limb do not reveal any abnormal findings .
thus , obturator neuropathy should be differentiated from lumbar plexopathy , diabetic polyneuropathy , and lumbar neuropathy .
treatment for obturator neuropathy or related nerve injuries include medication , physical therapy , massage therapy , restricted exercise , and rehabilitation .
surgical procedures may be considered depending on the severity of the injury , recovery , and response to conservative therapy . moreover
several authors have reported that obturator block is effective for the relief of pelvic and thigh pain , as well as for the management of adductor muscle spasticity caused by spinal cord injury , traumatic brain injury , stroke , cerebral palsy , and multiple sclerosis351315 ) .
the pubic approach involves the insertion of a needle perpendicular to the skin , 15 mm lateral and 15 mm inferior to the tubercle , while in the inguinal approach , the needle is inserted at the midpoint of the inguinal crease between the femoral arterial pulse and the inner border of the adductor longus tendon4 ) . in our study , the median distances between the obturator foramen and the pubic tubercle , the femoral artery , and the adductor longus muscle were 17 mm , 28 mm , and 41 mm , respectively . regarding arterial variations and postural alterations ,
ultrasound guidance allows for better nerve identification , and recent studies utilizing this technique for obturator nerve block have reported success rates of greater than 90% . while ultrasound guidance can be useful for diagnosis , its usefulness is limited in cases of surgical exploration .
previous studies have investigated the anatomical features of the obturator nerve . according to locher et al.11 ) , the median distances between the projection of the obturator nerve on the skin to the pubic tubercle and to the symphysis were 23 mm and 51 mm , respectively , based on anatomical investigation and mri analysis of 10 cadavers .
a similar value of 27 mm was obtained as the median distance between the obturator nerve and the pubic tubercle in our study .
the pubic tubercle is an objective landmark and can be useful for understanding anatomical structures . understanding the relationships between various anatomical structures is essential for surgery .
different critical sites that risk compressing the obturator nerve include the obturator canal , the obturator membrane , and the obturator externus muscle10 ) .
many options exist for the surgical treatment of obturator neuropathy , including transabdominal , laparoscopic , inguinal , and extraperitoneal approaches9 ) . during surgical exploration , critical structures such as the femoral artery
anatomical apprehension of the obturator nerve can be of aid for the accurate planning and appropriate timing of surgery .
the incision can be made at inguinal crease , point approximately one - fifth medially from the asis to the pubic tubercle and the medial inguinal ligament can be a good surface landmark of the obturator nerve . then , the nerve is traced proximally by dissecting to the obturator foramen . most of obturator nerves are located in a triangle bordered by femoral artery , adductor longus muscle and inguinal ligament .
so neurosurgeons should understand the anatomical relationship and the projection of the obturator nerve for saving adjacent structure .
the results of our morphometric study show that the obturator nerve is located beneath the pectineus muscle , at a distance of approximately 1.7 cm and 2.7 cm vertically and horizontally , respectively , from the pubic tubercle . moreover , the shallowest portion of the obturator nerve is distributed approximately one - fifth from the medial inguinal ligament .
the limitations of this study include its small sample size and the use of only two female cadavers .
thus , our report can not be commonly cited to all subjects of both sexes .
in addition , the supine position of cadavers may have affected the results of our study . however
, knowledge concerning obturator neuropathy provided by this study will aid in more accurate diagnoses , active intervention programs , and for the minimization of complications . | objectiveobturator neuropathy is a rare condition .
many neurosurgeons are unfamiliar with the obturator nerve anatomy .
the purpose of this study was to define obturator nerve landmarks around the obturator foramen.methodsfourteen cadavers were studied bilaterally to measure the distances from the nerve root to relevant anatomical landmarks near the obturator nerve , including the anterior superior iliac spine ( asis ) , the pubic tubercle , the inguinal ligament , the femoral artery , and the adductor longus.resultsthe obturator nerve exits the obturator foramen and travels infero - medially between the adductors longus and brevis .
the median distances from the obturator nerve exit zone ( onez ) to the asis and pubic tubercle were 114 mm and 30 mm , respectively .
the median horizontal and vertical distances between the pubic tubercle and the onez were 17 mm and 27 mm , respectively .
the shortest median distance from the onez to the inguinal ligament was 19 mm .
the median inguinal ligament lengths from the asis and the median pubic tubercle to the shortest point were 103 mm and 24 mm , respectively .
the median obturator nerve lengths between the onez and the adductor longus and femoral artery were 41 mm and 28 mm , respectively.conclusionthe obturator nerve exits the foramen 17 mm and 27 mm on the horizontal and sagittal planes , respectively , from the pubic tubercle below the pectineus muscle .
the shallowest area is approximately one - fifth medially from the inguinal ligament .
this study will help improve the accuracy of obturator nerve surgeries to better establish therapeutic plans and decrease complications . |
however , patients without any comorbid anomalies or patients with a mild form of this disease can reach adulthood .
no significant etiological factors have been found in patients , and thus genetic , teratogenic , and mechanical factors have been thought to be responsible .
since it is rarely observed in adulthood , we present a rare case of of pulmonary agenesis in an adult .
a 17-year - old female patient presented to our clinic with a complaint of shortness of breath .
p / a chest radiography revealed a deviation to the left in mediastinal structures , trachea , and heart ; a narrowing between the ribs in the left hemithorax ; a widening between the ribs in the right hemithorax ; and elevation of the right hemidiaphragm .
spirometry findings was consistent with restrictive disease : fvc : 3.01l ( pred % 67.2 ) , fev1 2.59l ( pred % 67 ) , and fev1/fvc : 84.9 ( pred % 102.0 ) .
computed thorax tomography revealed the absence of the left lung parenchyma , deviation of mediastinal structures to the left , a compensatory enlargement of right lung into left hemithorax , and rudimentary bronchus in the left [ figure 2 ] .
the patient underwent thorax ct angiography , abdominal ct , and pulmonary perfusion scintigraphy . in ct angiography ,
furthermore , no parenchymal tissue could be observed in the left lung area [ figure 4 ] .
the image of nonexistent left pulmonary artery and rudimentary left pulmonary vein . left hemithorax ; no parenchyma was observed .
the remaining lung contains twice as much alveoli as normal , but has normal bronchi .
although aplasia does not have the same structures , it has a rudimentary bronchus . in our case , too , the left main bronchus was determined as rudimentary .
as agenesis and aplasia are difficult to distinguish from each other clinically and pathologically , they are often used in the same sense . on the other hand ,
pulmonary hypoplasia is characterized by immature lung tissue or by lung tissue decreased at varying degrees .
pulmonary aplasia ( agenesis ) is thought to result from the negative effects that occur on the 4th week of fetal life .
although its etiology is not fully understood , vitamin a or folic acid deficiency or the use of salicylates may be responsible for it .
its incidence in males and females and the occurrence of the anomaly in the right or left lung are about the same .
hypoplasia and aplasia are often observed together with other malformations ( diaphragma defects , kidney anomalies , extrapulmonary sequestration , muscle , or skeleton system defects ) .
although the most common one is the atrial septal defect , ventricular septal defect , patent ductus arteriosus , or aorta coarctation can also be observed . in our case , too , no pathological findings were found except scoliosis .
although the patients with unilateral lung aplasia ( agenesis ) are believed to die usually in the neonatal period , there are patients who live upto the adulthood , some of whom live without any symptoms .
, secretions not cleared from the rudimentary bronchus and/or comorbid bronchiectasis may partially be responsible for the risk of infection . in our case ,
bronchus was rudimentary and an antibiotic treatment was started to clear the symptoms of a respiratory tract infection . in some cases , a significant decrease in vital capacity and exercise intolerance can be seen .
chronic respiratory failure and eventually respiratory infections can develop in adults . the restrictive pattern detected in our case was thought to be secondary to the aplasic lung disorder . in the diagnosis of this condition ,
methods such as contrast - enhanced ct , bronchography , bronchoscopy , pulmonary angiography , and magnetic resonance imaging are also employed . in our case ,
the diagnosis was made with noninvasive methods such as thorax ct - angiography and ventilation / perfusion scintigraphy . in chest radiography
narrowing between the ribs and elevated hemidiaphragm on this side is also observed . in our case ,
right pulmonary artery was quite long , left pulmonary artery did not exist , and the bronchus was rudimentary .
total atelectasia , diaphragmatic hernia , diaphragmatic eventration , pneumonia , pleural effusion , pulmonary hypoplasia , or pneumonectomy should be considered in the differential diagnosis . | pulmonary agenesis is a rare congenital anomaly , the etiology of which is not clearly known .
other systemic comorbidities such as cardiovascular , gastrointestinal , musculoskeletal , and urogenital system anomalies can be observed in more than half of the patients .
it is usually diagnosed during childhood .
diagnosis in adulthood is very rare .
we present a case of pulmonary agenesis diagnosed in an adult . |
diabetes mellitus ( dm ) is one of the fastest growing public health problems imposing a high burden on dm patients and high financial burden on healthcare systems .
recent estimates of the international diabetes federation ( idf ) predicted that the number of adults with diabetes is expected to rise worldwide from 366 million in 2011 to 552 million by 2030 .
the last colombian national health survey in 2007 revealed a dm prevalence of 3.5% in the colombian population 1869 years of age according to participant self - report .
however , according to the idf there were 2.1 million dm cases in colombia corresponding to a dm prevalence of 7.05% in 2013 for the 2079 year - old population .
it has to be kept in mind that there is a remarkable gap between people diagnosed with dm and those receiving treatment .
it has been shown that only approximately 50% of the people diagnosed with dm receive medical care [ 3 , 4 ] .
however , for the last six years , two official registers have been developed in colombia to track the number of dm and other relevant diseases cases that demand healthcare attention [ 57 ] .
nevertheless , no intent has yet been made to analyze the existing information in order to see whether there are changes in the access to dm treatment within the healthcare system during the past years .
furthermore , it is important for the primary healthcare system to monitor the number of dm patients receiving treatment on a regular basis to evaluate its capacity to detect new dm cases and to follow up whether it is capable to reduce the gap between diagnosed dm cases and those receiving medical care .
the aim of this study was to describe the reported dm prevalence rates of the 2079-year - old population in colombia during 2009 and 2012 reported by the official information sources of the healthcare system .
information on number of patients with dm as primary diagnosis in the population of 2079 years of age was obtained by the integral information system of social protection ( sispro ) and the high cost account ( cac ) , an organization to trace high expenditure diseases .
the sispro is a register developed by the colombian ministry of health and social protection which offers consolidated data from the healthcare services demand in colombia .
all healthcare providers ( hospitals and healthcare centers ) are by law obliged to send information using the icd-10 code of the primary diagnostics of all patients placed in the sispro register ; thus , covers the entire colombian population that demands services within social security health system .
the sispro register is fed by different information sources of healthcare service data after careful validation of each .
data submitted to the sispro register is subject to continuous quality control testing [ 5 , 6 ] .
the data received is cross - checked against other information sources ( such as population census , national health surveys , or other administrative registers ) before being integrated into the sispro register .
if inconsistencies are detected , the data is sent back to the reporting institutions for revision and correction .
in addition , according to the existing regulation several specific projects are carried out to improve primary data sources in order to guarantee a better quality of data received .
the sispro registers the primary diagnostic code of the healthcare visit of both primary care and hospital care visits .
data on healthcare services provided to dm patients of the sispro for the years 20092012 were obtained in october 2013 using the icd10 codes e100e149 , g590 , g632 , h280 , h360 , m142 , n083 , o240o244 , and o249 .
many dm patients contact the primary healthcare system at very advanced stage of their disease presenting already complications of dm .
these patients are usually registered with an icd-10 code related to complications of dm as primary diagnostic code .
therefore , we have included these icd-10 codes in our search to ensure that otherwise invisible cases of dm were included in the analysis .
in addition , the icd-10 differs between insulin - dependent dm ( e10 ) and non - insulin - dependent dm ( e11 ) .
however , since a large proportion of patients with type 2 diabetes are treated with insulin , they are coded as e10 . in addition , the icd-10 codes e12 , e13 , or e14 refer to unspecified dm .
the consequence of this is that the icd-10 codes do not allow a genuine and credible classification of clinical types of dm at least in colombia .
the cac is an agency established by the colombian state in 2008 and managed by the healthcare insurance companies .
the objective of the cac is to monitor high cost illnesses within the social security health system ( sshs ) in order to develop mechanisms for financial management and to promote risk management in relation to high - cost illnesses [ 7 , 8 ] . by law
, the healthcare insurance companies must report all cases of dm , hypertension , and chronic kidney diseases that occur within their healthcare users .
information of all identified cases of dm was received from cac for the years 2009 , 2010 , 2011 , and 2012 [ 912 ] .
it is important to mention that both of the above mentioned registers only provide information about dm cases that have required healthcare attention .
thus , dm cases that have not been in contact with healthcare services do not enter the official registries .
in addition , there is no data available in regard to the type of dm .
information regarding population size and projections was retrieved from the national administrative department of statistics ( dane ) .
dane has also been considered as the official source of information about mortality and some health indicators in colombian [ 14 , 15 ] .
age - standardized reported dm prevalence rates per 100.000 inhabitants for the population of 2079 years of age from 2009 to 2012 were calculated separately for the sispro and the cac registers as a quotient : the numerator was number of cases of dm in people 2079 years of age in the sispro and , respectively , cac register , whereas the number of inhabitants aged 2079 years reported dane population projections were used as the denominator .
the epidat software version 4.0 was used to standardize the reported dm prevalence rates according to age using as reference population estimated for colombia for 2010 according to united nations population .
the estimation of age - standardized reported dm prevalence rates for each of the six regions ( figure 1 ) corresponds to the average age - standardized rates calculated for the provinces in each region .
the age - standardized reported dm prevalence rates are presented per 100.000 people for the age range 2079 years .
they were tested first for linearity using mantel - haenszel test and for statistical significance the chi - square test was used .
the trends in prevalence were subsequently calculated using the linear regression model log(pr ) = a + bt + e , where pr refers to prevalence , t is time , e is the error term , and a and b are the regression coefficients estimated from the data . the change in prevalence per year at the time point t is a constant proportion , 100b percent of the prevalence at t. the estimated average yearly change in percent is exp(b ) 1 , which is approximately b for small changes .
the level of statistical significance was set to 0.05 . finally , for the maps presented in figures 2(a ) , 2(b ) , 2(c ) , and 2(d ) , the annual reported dm prevalence rates of each province were categorized into five categories according to the performance in regard to the reported number of dm patients compared to the highest national reported dm prevalence rates ( 1593/100.000 in 2012 ) in the study period .
the categories were as follows : ( i ) higher than or not more than 19% lower than the highest national value of 2012 ( 1592,6/100.000 ) ; ( ii ) 120% lower than the national value of 2012 ; ( iii ) 2140% lower than the highest national value of 2012 ; ( iv ) 4160% lower than the highest national value of 2012 ; and ( v ) 60% lower than the highest national value of 2012 , respectively .
the trends of reported dm prevalence rates in each region are presented in table 1 .
both registers showed a statistically significantly linear increase in reported dm prevalence rates per 100.000 people during 2009 and 2012 . whereas the reported dm prevalence rates of sispro revealed an increase from 964/100.000 inhabitants ( 2009 ) to 1398/100.000 inhabitants in 2012 ( mean annual increase 141/100.000 ; p value : 0.001 ) , the respective rates in the cac register were 1082/100.000 ( 2009 ) and 1593/100.000 in 2012 ( mean annual increase 165/100.000 ; p value : 0.026 ) .
the cac register reported between 12% and 19% more dm cases/100.000 inhabitants compared to the rate provided by the sispro register .
table 1 shows the age - adjusted reported dm prevalence rates per 100.000 inhabitants 2079 years old in the different regions of colombia according to the sispro and the cac register during 2009 to 2012 .
the reported dm prevalence rates calculated from sispro reported a statistically significant increase in the andes region ( p value : 0.002 ) , the region of orinoco ( p value : 0.039 ) , and the pacific region ( p value : 0.020 ) during the time period , whereas the dm prevalence rates calculated from cac register had a statistical significant linear increase in the age - adjusted reported dm prevalence rates in the regions andes ( p value : 0.022 ) , pacific region ( p value : 0.004 ) , and bogota dc ( p value : 0.035 ) . both registers identified more dm cases/100.000 inhabitants in 2012 compared to 2009 .
however , the annual increase in dm rate ranged from 26 to 153/100.000 people in the sispro register and from 25 to 203/100.000 inhabitants in the cac register , respectively . as during 2009 and 2012
the reported dm prevalence rates of cac were regularly higher than the ones provided by sispro , the changes in the reported dm prevalence rates of the different provinces are only shown for the cac register .
figures 2(a)2(d ) present the development of the dm cases reported in the cac register in the different provinces of colombia during 2009 and 2012 compared to the national reference value of 2012 ( 1593/100.000 ) . whereas 13 provinces reported dm prevalence rates 80% or lower than the national reference value in 2009 , only eight provinces remained that far away from the national reference value in 2012 . mainly , the four provinces in east of the country , choco at the pacific coast , and la guajira in the north - east upper corner of colombia did not show any improvement in detecting dm cases . whereas in 2009 the reported dm prevalence rates of 21 provinces were below 60% of the national reference , the respective number in 2012 was 13 provinces .
the number of provinces reporting at least the national reported dm prevalence rates of 2012 increased from two in 2009 to ten in 2012 .
our study showed that the registries and the information retrieving system of sispro and cac have been improved during 2009 and 2012 resulting into a greater capacity to identify and report dm cases by the healthcare system .
however , some regions and provinces showed a slower or no increase in the reported dm prevalence rates creating a future challenge for the official healthcare information system of colombia .
first , the capacities of the registers to identify the dm cases may have improved due to implementation of different screening or early diagnosis activities within the healthcare providers to detect people with dm .
for instance , many healthcare insurance companies have recently implemented different benchmark system to catch dm patients as early as possible to offer efficient treatment , whereas others may use diabetes risk factors scores combined with laboratory tests . as there is a growing awareness of the burden of dm in colombia ,
many dm screening programs will be developed in colombia in the next years that may lead to a further increase in dm rates 13 detected by the healthcare system .
generally , once individuals are diagnosed , approximately 50% are treated with medication , both in developed and developing countries .
a second reasonable interpretation of our results may be that the diabetes incidence has increased during the past four years in colombia .
both obesity and physical inactivity are two of the most important risk factors for dm . given the increased prevalence of obesity between 2005 and 2010 in the colombian adult population combined with low levels of physical activity
the national survey of the nutritional situation ( ensin ) of 2010 revealed that 35% of women and 34% of men 1864 years of age were overweight .
the corresponding prevalence for obesity was 20% ( women ) and 11.5% ( men ) .
furthermore , only every second 1864-year - old adult reached the current physical activity recommendations of 30 minutes of daily moderate intensity physical activity [ 20 , 21 ] .
thus , the unfavorable development of the major risk factors of dm may be reflected in the increase in the dm rates of people with dm in the healthcare system .
however , we would like to point out that the data present here is not a study on the true prevalence of dm in colombia but rather about the capacity to report dm cases by the healthcare system . according to available evidence ,
the government has put an emphasis on improving these registries in the past years obliging the healthcare providers to improve disease reporting by a series of new legislation .
thus , we think that the increase in the reported dm prevalence rates can rather be explained by the improved capacity of the healthcare system to detect previously unknown dm cases than an increasing prevalence of dm .
it has to be pointed out that the observed increase in reported dm cases in the colombian population may be related to a better and easier access or higher demand to healthcare for dm patients leading to higher dm rates recorded in the reporting system .
furthermore , we hypothesize that the difference observed between the regions in regard to reported dm prevalence rates may be due to the geographical distances from the more developed urban centers . probably , the access to healthcare services is more difficult and the healthcare provider 's information systems may have poorer development compared to the more developed regions of the country .
thus , there is a remarkable gap between the idf estimation and the dm prevalence found by the colombian national health survey in 2007 ( 3.59% ) . besides , considering that there are about 800 000 known dm patients in colombia , it is of great concern that only 50% of them get admitted in the healthcare system .
the data base of sispro only registers the primary diagnostic code leading to an underestimation of dm cases as some patients may have dm as underlying condition to other diseases .
in addition , our analysis is restricted to the dm cases officially reported and does not reflect the true prevalence of dm rather than the dm cases that have been in contact with the healthcare system .
however , the objective of our study was not to estimate the true dm prevalence but to report the dm prevalence rates of the official information sources of the healthcare system . finally , the dm type and the treatment profile that these registered patients are receiving are not described by these sources . in any case , these two registers are the only available sources of information in regard to the number of treated dm patients in colombia as the country does not count with an official register for dm1 or dm2 at the present moment .
the official information sources to track dm patients have improved their ability to detect and record dm patients in colombia .
however , many regions are still lacking behind the national reference showing lower dm detection rates .
the future challenge for the colombian healthcare system is to identify dm patients as early as possible and provide equal access to treatment as early as possible to decrease the burden of dm . | the objective of this study was to describe the reported diabetes mellitus ( dm ) prevalence rates of the 2079-year - old population in colombia from 2009 to 2012 reported by the healthcare system .
information on number of patients treated for dm was obtained by the integral information system of social protection ( sispro ) , the registry of the ministry of health and social protection , and the high cost account ( cac ) , an organization to trace high expenditure diseases . from both sources
age - standardized reported dm prevalence rates per 100.000 inhabitants from 2009 to 2012 were calculated .
whereas the reported dm prevalence rates of sispro revealed an increase from 964/100.000 inhabitants ( 2009 ) to 1398/100.000 inhabitants in 2012 ( mean annual increase 141/100.000 ; p value : 0.001 ) , the respective rates in the cac register were 1082/100.000 ( 2009 ) and 1593/100.000 in 2012 ( mean annual increase 165/100.000 ; p value : 0.026 ) . the number of provinces reporting not less than 19% of the highest national reported dm prevalence rates ( 1593/100.000 ) increased from two in 2009 to ten in 2012 .
apparently , the registries and the information retrieving system have been improved during 2009 and 2012 , resulting in a greater capacity to identify and report dm cases by the healthcare system . |
cytokines are produced by many types of cells including immune cells ( e.g. , macrophages , lymphocytes , and mast cells ) , endothelial cells , fibroblasts , and stromal cells .
current cytokine assays are designed to measure cross - sectional , peripheral cytokine concentrations , regardless of the source of the cytokine(s ) .
many cytokines have an opposing and sometimes complimentary effect to each other ; thereby , modulating the severity of the immune response .
the purpose of this review paper was to shed light on limitations and major methodological issues and how to address them in order to advance the study of cytokines in schizophrenia .
enzyme - linked immunosorbent assay ( elisa ) is the most widely used and the gold standard method used to measure cytokine concentrations .
elisa has a number of limitations : binding affinity of antibodies can vary , the method requires large sample volume , it has a narrow dynamic range , can measure only one protein at a time , and is costly .
multiplex immunoassays can measure multiple cytokines simultaneously , and there are no specific limitations to the multiplex assays other than the two mentioned in the next sentence .
both elisa and multiplex are limited by not being able to distinguish between bioactive and inactive molecules and the matrix and heterophilic ( auto- ) antibody interference .
these antibodies cause false positive and false negative signals by binding to either the capture antibody , detection antibody or to the antigen .
most of the studies included in the meta - analyses , used elisa , but there were a few exceptions . out of the 40 studies ( in supplement ) , 35 studies used elisa , one study each used semimicroassay , radioimmunoassay ( ria ) , bioassay , sandwich , and cytometric bead array .
elisa , ria , and bioassay were used in 83% , 9% , and 8% of the studies , respectively ( supplement ) .
several studies used peripheral blood mononuclear cells ( pbmc ) cultures stimulated with lipopolysaccharide ( lps ) or phytohemagglutinin .
two studies used stimulated t cell culture . in three studies , within the same study , interleukin 2 ( il-2 )
one study did bioassay using the il-6 dependent hybridoma cell line b9 . in none of the studies
were any data presented to compare the similarity of results across different assays , nor were any data presented to suggest which cytokine measurement tests produce the most reliable and consistent results .
another largely unaddressed question regarding the association between cytokines and schizophrenia is whether observed cytokine changes in schizophrenia are part of the causal pathway leading to the disease , or a secondary consequence of treatment , e.g. , weight gain or result from lifestyle concomitants in people with schizophrenia , such as smoking .
the issue of secondary consequence of treatment has been addressed in the miller et al .
the first episode participants were drug nave , suggesting that cytokine abnormalities in schizophrenia are independent of antipsychotic medications .
a further possibility is that cytokines may be elevated as a consequence of the hypothalamic - pituitary - adrenal axis arousal induced by distressing psychiatric symptoms .
careful attention should be paid to the experimental design , sample collection , preparation , and storage ; all these may have an impact on the study results . as previously reported
, future studies should validate the cell sources ( lymphocytes , monocytes , etc . ) of specific cytokines and the complex interactions among cytokines and its association with oxidative stress and other systems .
although peripheral cytokine concentrations are easier to measure and have been mostly used in studies of schizophrenia , more research is needed to examine the association between cerebrospinal fluid and blood concentrations . although the meta - analysis examined the longitudinal changes in cytokines , antipsychotic treatment was not standardized .
future studies are warranted to validate the findings of longitudinal changes in cytokines ; this evidence is scant in the literature .
multiplex assays when combined with gene expression analysis and flow cytometry techniques such as fluorescence - activated cell sorting ( facs ) may be useful to detect abnormalities in specific immune pathways .
pbmc cultures , to evaluate in vitro lps - induced cytokine production , may be a better technology than measuring cytokines in the serum .
cytokines in serum may be vulnerable to differential concentrations between the site of cytokine release and blood concentrations and the relatively short half - life of cytokines .
significant increases in peroxisome proliferator - activated receptor gamma , sterol regulatory element - binding transcription factor 1 , il-6 and tumor necrosis factor alpha , and decreases in peroxisome proliferator - activated receptor alpha and c / enhancer - binding protein alpha and mrna levels using stimulated pbmc were found in 31 participants with schizophrenia compared to 31 controls .
investigators should decide , after consulting with a psychoneuroimmunologist , which tests to be done .
in sum , there is considerable evidence for a role of inflammation in the pathophysiology of schizophrenia . the use of standardized methodology for cytokine measurement will enhance the replicability of results across studies .
this will advance the field for potential integration of pathophysiology with novel therapeutic discovery targeting inflammatory mechanisms .
| there is mounting evidence that inflammation is a major factor in the pathophysiology of schizophrenia .
inflammatory status is commonly ascertained by measuring peripheral cytokine concentrations .
an issue concerning research on inflammation and schizophrenia relates to assay methodology .
enzyme - linked immunosorbent assay ( elisa ) is the most widely used and the gold standard method used to measure cytokine concentrations .
elisa has a number of limitations .
both elisa and multiplex are limited by not being able to distinguish between bioactive and inactive molecules and the matrix and heterophilic ( auto- ) antibody interference .
multiplex assays when combined with gene expression analysis and flow cytometry techniques such as fluorescence - activated cell sorting may be useful to detect abnormalities in specific immune pathways .
peripheral blood mononuclear cells cultures , to evaluate in vitro lipopolysaccharide - induced cytokine production , may be a better technology than measuring cytokines in the serum .
the purpose of this paper is to shed light on major methodological issues that need to be addressed in order to advance the study of cytokines in schizophrenia .
we make a few recommendations on how to address these issues . |
despite the diverse etiologies of renal parenchymal calcification ( nephrocalcinosis ) , nephrocalcinosis combined with electrolyte imbalance of hypokalemia can narrow down the differential diagnosis into hereditary tubulopathy such as bartter 's syndrome , chronic laxative or diuretic abuse , type 1 renal tubular acidosis ( rta ) , and recently recognized acute phosphate poisoning such as oral sodium phosphate bowel preparation ( ospbp ) ( 1 , 2 ) . since the first introduction of an unusual form of cortical nephrocalcinosis due to " acute phosphate nephropathy " by desmeules et al .
( 2 ) in 2003 , osp - bp associated acute or chronic renal injury has been documented in several other reports ( 1 , 3 ) .
also , transient hypocalcemia and hypokalemia after osp - bp was noted as electrolyte imbalances in more than 50% of elderly patients ( 4 ) .
in contrast , the prominent clinical features of type 1 rta are characterized by medullary nephrocalcinosis / urolithiasis and bone involvement due to sustained hypokalemia and metabolic acidosis . in the present case ,
the unique clinical experience of acute phosphate poisoning after osp - bp leading to the disclosure of previously unrecognized chronic type 1 rta due to sjgren 's syndrome is introduced .
a 30-yr - old woman presented to the emergency department ( ed ) in extreme mental agitation with the recent onset of carpal spasm , a tingling sensation in the lower extremities , and circumoral numbness about 2 hr after the second dose of oral sodium phosphate ( osp ) on march 14 , 2012 , which she took for the bowel preparation of a screening colonoscopy .
she had watery diarrhea twice with abdominal discomfort before presentation to ed . in ed , vital signs were stable but with a respiratory rate of 24/min , and other physical examinations were unremarkable except for the presenting neuromusclar manifestations of hypocalcemia including the positive chvostek 's sign .
as shown in table 1 , laboratory findings with normal kidney function in ed were hyperphosphatemia ( 9.5 mg / dl ; normal , 2.5 - 4.5 mg / dl ) , hypocalcemia ( total calcium of 6.5 mg / dl and ionized calcium of 1.90 mm / l ; normal , 2.26 - 2.64 mm / l ) , hypokalemia ( 3.3 meq / l ) , and respiratory alkalosis ( alkaline arterial ph , 7.53 ; bicarbonate , 21.1 meq / l ; pco2 , 24.8 mmhg ) . after the intravenous injection of 1 gram of calcium gluconate in ed , carpal spasm resolved rapidly , and hyperphosphatemic hypocalcemia was no longer noticed . on subsequent hospital days ( day 2 and 3 ) , however , sustained hypokalemia ( 3.3 to 3.4 meq / l ) with inappropriately high transtubular potassium gradient ( 7.0 ) suggesting renal potassium wasting despite dietary potassium supplementation , metabolic acidosis with normal blood anion gap ( 9 to 11 meq / l ; reference , 10 2
meq / l ) , and alkaline urine ph ( > 6.0 ) with positive urine anion gap ( > 16
meq / l ) suggested type 1 rta , which was ascertained by other positive evidences for the impaired urinary acidification of distal tubules by loopdiuretic ( torsemide ) test ( urine ph > 6.5 ) and urine to blood pco2 gradient ( > 20 mmhg ) upon intravenous alkali load with sodium bicarbonate . on further questioning in careful history taking , she admitted chronic gritty sensation of eyes using artificial eye drops frequently and dry mouth with polydipsia and polyuria more than 5 yr , which suggested an impaired exocrine gland secretion mostly due to sjgren 's syndrome . also , keratoconjunctivitis sicca was confirmed by schirmer 's test .
furthermore , several autoantibodies for sjgren 's syndrome were positive with high titers ; antinuclear antibodies , antibodies to the ribonucleoprotein antigen la ( ss - b ) and ro ( ss - a ) , and rheumatic factor . in imaging tests ,
1 ) , and the salivary scintigraphy showed the evidence of salivary gland dysfunction in both submandibular and parotid glands ( fig .
subsequently , oral intake of potassium citrate ( urocitra - k , 1,080 mg / tablet ) was initiated , of which dose was adjusted for the adequate alkali supplementation as well as for the correction of hypokalemia . since then , hypokalemia and metabolic acidosis were no longer noticed .
carpal spasm in this patient is most likely caused by hypocalcemia because of the transient hyperphosphatemia leading to the precipitation of calcium - phosphate complexes with acute phosphate poisoning after the use of osp - bp .
the supportive evidence is the presence of hyperphosphatemic hypocalcemia on presentation with the complete resolution of the symptoms and hyperphosphatemia within 2 hr of admission ( table 1 ) .
also , the acute respiratory alkalosis ( ph 7.53 , pco2 24.8 mmhg ) as a result of the extreme mental agitation caused by the presenting neurological symptoms probably further contributed to a reduction in free ionized calcium concentration ( 5 ) . because of the lower cost and the better tolerance , osp remains popular in the bowel preparation for colonoscopy in clinical practice .
however , the bowel preparation using osp is often associated with mild to severe electrolyte imbalances , such as hyperphosphatemia , hypocalcemia , hypernatremia , hyponatremia , hypokalemia , and high anion - gap metabolic acidosis . even acute and subsequent chronic renal injury due to
deposition of calcium - phosphate crystals in distal renal tubules after osp can be only proven by renal biopsy since the intrarenal deposition in acute hyperphosphatemia is usually not sufficient to be detectable in radiological imaging ( 1 - 3 , 6 ) .
furthermore , nephrocalcinosis associated with acute phosphate nephropathy is more predominantly localized in the renal cortex than medulla .
1 ) is seen usually with sustained hypercalcemia and/or hypercalciuria related to malignancy , hyperparathyroidism , furosemide or laxative abuse , sarcoidosis , type 1 rta , etc ( 3 ) . following the admission , as shown in table 1 and fig . 1 , sustained hypokalemia with renal potassium loss ( high transtubular potassium gradient ( ttkg ) 7.0 ) , normal anion - gap metabolic acidosis , the persistent urine ph > 6.0 in the presence of metabolic acidosis , and medullary nephrocalcinosis suggested the impaired distal urinary acidification due to type 1 rta ( 7 ) .
the classical test using oral ammonium - chloride loading to assess the impaired distal urinary acidification was not performed because of the substantial pre - existing systemic acidemia . instead
, the oral administration of torsemide rather than furosemide , recently known to be more specific in assessing urinary acidification of the distal nephron in a study , showed sustained alkaline urine ph more than 6.5 ( 8) .
furthermore , the sodium bicarbonate ( nahco3 ) loading test also revealed the urine - to - blood pco2 gradient that was persistently less than 11.2 mmhg when the urine ph ( 7.62 ) exceeded the corresponding blood ph ( 7.39 ) ( 9 ) . hence , there is little doubt for the certainty of the diagnosis of type 1 rta in this patient .
the renal involvement of the disease is often associated with rta ( 33% ) , predominantly type 1 rta and less frequently type 2 rta ( 10 ) . in an immunocytochemical study ,
the absence or decrease of h - atpase in the intercalated cells of cortical collecting tubules of distal nephron has been shown , and appears to play an important role in the pathogenesis of type 1 rta in patients with sjgren 's syndrome ( 11 ) .
chronic metabolic acidosis results in negative calcium balance due to the renal wasting of calcium ( 12 ) .
although serum calcium is usually maintained within normal limits because of the compensating secondary hyperparathyroidism , the ionized calcium concentration in the serum must be lower than usual in the presence of chronic metabolic acidosis in order to induce secondary hyperparathyroidism .
thus , any additional factor that reduces serum calcium such as acute hyperphosphatemia as in this case would have a greater hypocalcemic effect in an untreated patient with type 1 rta .
the major underlying mechanism of nephrocalcinosis in type 1 rta is metabolic acidosis : chronic metabolic acidosis stimulates bone resorption and impairs renal tubular reabsorption of calcium , both of which contribute to hypercalciuria ; nephrocalcinosis is further aggravated by a low urinary citrate excretion , which in the case of type 1 rta is due to metabolic acidosis and hypokalemia ; metabolic acidosis leads to hypokalemia because of the increased delivery of sodium to the cortical collecting duct and the stimulation of aldosterone ( 5 , 13 , 14 ) .
hence , the mainstay of the treatment for nephrocalcinosis is to prevent its progression by correcting the biochemical abnormalities associated with type 1 rta .
the presence of hypokalemia in type 1 rta by inducing low intracellular ph of the proximal tubular cells contributes to decreased excretion of urinary citrate ( 14 ) .
in fact , the increase in citrate excretion following the administration of potassium citrate is the result of correction of metabolic acidosis , not the citrate administration per se .
although potassium bicarbonate for the therapy of type 1 rta can be equally effective , it causes more side effects , such as gas formation of co2 in the stomach as bicarbonate reacts with hcl .
therefore , potassium citrate by either as a drug or from natural food sources including beverages would be an ideal substance for adequate alkali supplementation ( 1 - 2 mm / kg / d in adults ) and for the correction of hypokalemia and hypocitraturia as well . in summary , the precipitation of calcium - phosphate complexes by acute hyperphosphatemia after osp - bp caused acute hypocalcemia , which was further aggravated by acute respiratory alkalosis , leading to the manifestation of hypocalcemic tetany .
subsequently , the sustained hypokalemia , nephrocalcinosis in medulla , and biochemical findings of impaired ability of urinary acidification unveiled the secondary type 1 rta due to sjgren 's syndrome . | although renal calcium crystal deposits ( nephrocalcinosis ) may occur in acute phosphate poisoning as well as type 1 renal tubular acidosis ( rta ) , hyperphosphatemic hypocalcemia is common in the former while normocalcemic hypokalemia is typical in the latter .
here , as a unique coexistence of these two seperated clinical entities , we report a 30-yr - old woman presenting with carpal spasm related to hypocalcemia ( ionized calcium of 1.90
mm / l ) due to acute phosphate poisoning after oral sodium phosphate bowel preparation , which resolved rapidly after calcium gluconate intravenously . subsequently ,
type 1 rta due to sjgren 's syndrome was unveiled by sustained hypokalemia ( 3.3 to 3.4
meq / l ) , persistent alkaline urine ph ( > 6.0 ) despite metabolic acidosis , and medullary nephrocalcinosis . through this case report
, the differential points of nephrocalcinosis and electrolyte imbalances between them are discussed , and focused more on diagnostic tests and managements of type 1 rta . |
infants born in south asia tend to have lower mean birth weights than infants born elsewhere in the world , including those born in other low - income countries.1 this is , in part , thought to reflect poor maternal nutrition throughout their life course , including during pregnancy .
however , infants born in high - income countries , such as the uk and the usa to mothers of south asian origin , are also considerably lighter than babies born to white mothers2
3 lower birth weight is related to adverse perinatal outcomes4
5 and has also been associated with a number of common chronic diseases of adulthood , including cardiovascular disease and type 2 diabetes.68 it has therefore been suggested that the risk factors for lower birth weight among south asian infants should be identified and interventions aimed at increasing their birth weight be developed.3
9 however , there is some evidence that , relative to their lower birth weight , south asian infants are more adipose at birth than european infants ( ie , that for a given birth weight they carry relatively more fat mass).1013 this would be consistent with findings in infants ( 612 weeks ) , children and adults suggesting that for a given body mass those of south asian origin have more total fat and are more prone to centrally distributed fat than european origin adults and children.1417 if south asian - origin infants do have more fat at birth than european infants , then any public health interventions to increase their birth weight could inadvertently increase fat mass and potentially worsen their later cardiometabolic health , thus increasing the already existent ethnic inequalities in diabetes and coronary heart disease.18 evidence to date for the existence of such differences at birth is scant . to our knowledge ,
only four previous studies have compared markers of fat mass or distribution between south asian and white european origin infants at birth .
all suggested that south asian infants tended to have greater central adiposity and fat mass for a given weight than the european infants.1013 however , these studies had relatively small sample sizes ( 224 , 969 , 1040 and 560 , respectively ) and three of them compared south asian infants born in india to european infants born in the uk or the netherlands.1013 it is possible that the differences in these studies reflect geographical - related rather than ethnic - related mechanisms . to our knowledge , just one small study has compared the uk - born south asian - origin infants ( n=165 ) and british infants ( n=343).13 one method that has been used to try and understand mechanisms underlying birth weight differences has been to look across generations .
the assumption here is that if poor maternal nutrition is an important determinant of the birth weight differences , then when mothers ( as well as infants ) have been born in a more affluent country and potentially exposed to better nutrition , the difference should be smaller .
furthermore , the narrowing of the difference would be expected to increase across generations as populations become more assimilated and intergenerational effects on poor growth are less prominent , though we acknowledge that some immigrant populations may not assimilate and may maintain similar diets to those prior to immigration .
of the five previous studies that have looked at birth weight differences in the uk between european and south asian origin infants across generations,3
9
1921 four found no clear evidence of a narrowing of the difference with subsequent generations,3
9
19
20 and one small study ( n=331 ) found some increase in birth weight in second generation compared with the first.21 in most of these studies , generation has been defined according to maternal place of birth .
this approach assumes that paternal place of birth does not contribute to the ethnic differences in birth weight , but both paternal and maternal birth weight and adult height are associated with offspring birth weight22 and it is possible that both mothers and fathers nutritional status and other environmental exposures could influence the offspring 's birth size .
fathers could influence infant birth weight indirectly via fathers influencing their partners diet and hence pregnancy nutritional status ( ie , if fathers are uk born , the mother may be more or less likely to have changed her diet to that of the uk population than if the father is not uk born ) .
it has also been suggested that fathers can influence offspring growth via parent of origin / epigenetic effects,23 , though we are as yet unaware of any direct evidence to support this . to our knowledge ,
no previous study has compared differences in indicators of birth fatness ( skinfolds and cord leptin ) between different generations of the uk born south asian infants .
the aim of this study was to compare the birth weight , skinfold thickness and cord leptin between pakistani and white british infants and to investigate explanatory factors , including the place of birth of parents and grandparents .
the skinfold thicknesses are used as indicators of fatness24 with subscapular skinfolds indicating centrally distributed fat25 and allow comparisons with previous studies that have also used skinfold measurements to compare adiposity between south asian and european origin infants.1013 in addition , we have compared cord leptin , which provides a more accurate reflection of total infant fat mass26 between the two groups .
the born in bradford ( bib ) study is a prospective birth cohort study that recruited women during pregnancy and has followed them , their infants and their partners into the child 's infancy . to be eligible for the study , women had to attend booking clinic between march 2007 and december 2010 and be booked to give birth in bradford .
full details of the study methodology have been previously reported.27 women were recruited at their oral glucose tolerance test ( ogtt ) appointment ; all women booked for delivery in bradford are offered a 75 g ogtt at around 2628 weeks gestation . women who attended this appointment completed an interviewer - administered questionnaire and had their height and weight measured .
interviews were conducted in a range of south asian languages ( including mirpuri , bengali , punjabi ) . for these analyses ,
infants born at less than 37 weeks gestation , stillbirths , multiple births and infants born to parents of ethnic origin other than white british or pakistani were all excluded ( figure 1 ) .
birth weight was abstracted from the medical records and in all participants was recorded immediately following birth .
subscapular and triceps skinfold measurements were collected by trained research administrators and most were measured within the first 24 h following birth and all within 72 h.28 measurements were obtained using harpenden calipers ( holtain ltd ) on the left side of the body .
we have previously shown good levels of within and between assessor reliability for all the birth size measurements.25 cord blood samples were obtained at delivery by the attending midwife on a consecutive subsample ( n=1387 ) .
samples were refrigerated at 4c in edta tubes until collected by laboratory staff within 12 h. samples were spun , frozen and stored at 80c .
once all samples had been collected , they were transferred to the biochemistry department of glasgow royal infirmary for analysis .
leptin was measured by a highly sensitive in - house elisa with better sensitivity at lower levels than commercial assays as previously described.29 ethnicity was self - reported at interview , with participants given response options based on the uk office of national statistics guidance.30 pakistani - origin women completed a detailed ancestry interview which included the details of the place of birth of themselves , their partner and all four grandparents .
generational status of pakistani infants was based on these data . using information on both parents and all four grandparents produced 66 possible unique categories .
we examined the numbers , mean birth weight and sd in all categories and then combined the groups based on sample size and similarities in mean birth weight and migration history .
this resulted in 17 categories within which 90% fell into one of the four main categories : both parents uk born and all grandparents south asian bornmother uk born , father and all grandparents south asian bornfather uk born , mother and all grandparents south asian bornboth parents south asian born and all grandparents south asian born both parents uk born and all grandparents south asian born mother uk born , father and all grandparents south asian born father uk born , mother and all grandparents south asian born both parents south asian born and all grandparents south asian born the remaining 10% fell across the other 13 categories , each of which included relatively few participants and so these groups were combined to form one other category .
as all grandparents were south asian born ( with the exception of the other group ) , and to simplify interpretation , the generation groups were labelled according to the place of birth of parents only for the remainder of the paper .
few characteristics could plausibly influence a woman 's ethnicity and therefore confound the association of this with outcomes.31
32 however , we were interested in characteristics that are known to be influenced by ethnicity and that might be on a causal pathway explaining birth size differences .
for example , women of white british origin are more likely to smoke during pregnancy than those of pakistani origin and taking account of this may in fact increase the ethnic difference in birth weight . while such characteristics can not be on a mediating causal pathway ( since accounting for their ethnic differences would likely increase rather than decrease birth weight differences ) , we felt it important to explore the effect of adjusting for them on ethnic differences in birth size . a priori , we considered maternal early pregnancy body mass index ( bmi ) , height , age , parity , smoking , alcohol consumption , socioeconomic position ( maternal education , housing tenure , whether they received benefits ) , living with a partner , fasting and postload glucose , fasting insulin , hypertensive disorders of pregnancy , infant sex and gestational age to be characteristics that might explain or mask ethnic differences in outcomes .
online supplementary table s1 briefly summarises our a priori assumptions of directions of association between ethnicity and each of the characteristics and between each of these and birth weight and hence the potential impact that they might have on birth weight differences between pakistani and white british infants .
maternal weight ( seca digital scales ) and height ( leicester height measure ) were measured in light clothing and unshod.27 fasting and postload glucose and fasting insulin were obtained from the ogtt samples which were assayed immediately after sampling at the biochemistry department of bradford royal infirmary using the glucose oxidase method on siemen 's advia 2400 chemistry autoanalysers and siemen 's advia centaur assay .
information for all other covariables was obtained from medical records or the mother 's questionnaire.27 all analyses were performed using stata ( v.11 ) .
multivariable linear regression was used to examine the associations between ethnicity and birth weight , skinfolds and cord leptin levels ( pakistani infants compared with white british infants ) and to examine these associations by generation ( each pakistani generation group compared with white british infants ) .
we adjusted separately for each covariable to see how these influenced the associations and then adjusted ( 1 ) jointly for those that we thought a priori ( see online supplementary table s1 ) would plausibly be on the causal pathway for lower birth size in pakistani origin infants ; ( 2 ) jointly for those that did reduce the difference when adjusted for individually ; and ( 3 ) for all covariables simultaneously .
we generated sex and gestational age internal standardised z - scores for birth weight and skinfold thicknesses using six gestational age categories ( 37 ; 38 ; 39 ; 40 ; 41 ; 4242 ) so that these were all assessed on the same scale and could be directly compared .
we then regressed these scores on ethnicity to calculate the mean difference in z - score between white british and pakistani infants .
further , we adjusted for birth weight in the regression analyses of skinfolds ( z - scores ) and cord leptin .
cord leptin concentration had a positively skewed distribution and was natural log transformed to achieve approximate normality of the residuals in the regression models .
the resultant coefficients were back transformed to give ratios of geometric means which have a null value of 1 .
owing to the smaller leptin sample size , we only included covariables in the final model that altered the coefficient when each was examined individually .
for the main analyses , we included only participants with complete data on exposure , each outcome and all covariables .
this resulted in 8704 participants for birth weight analyses ; 6041 for subscapular skinfolds ; 6058 for triceps skinfolds ; and 1186 for cord leptin analyses .
in order to examine whether missing covariable data might have biased our results , we repeated unadjusted analyses and analyses adjusting just for one covariable ( taking each in turn ) in maximal samples ( ie , not restricting to those with complete data on all covariables ) and compared results with the equivalent results in those with complete data on all covariables ( see online supplementary tables s3s6 ) .
the born in bradford ( bib ) study is a prospective birth cohort study that recruited women during pregnancy and has followed them , their infants and their partners into the child 's infancy . to be eligible for the study , women had to attend booking clinic between march 2007 and december 2010 and be booked to give birth in bradford .
full details of the study methodology have been previously reported.27 women were recruited at their oral glucose tolerance test ( ogtt ) appointment ; all women booked for delivery in bradford are offered a 75 g ogtt at around 2628 weeks gestation . women who attended this appointment completed an interviewer - administered questionnaire and had their height and weight measured .
interviews were conducted in a range of south asian languages ( including mirpuri , bengali , punjabi ) . for these analyses ,
infants born at less than 37 weeks gestation , stillbirths , multiple births and infants born to parents of ethnic origin other than white british or pakistani were all excluded ( figure 1 ) .
birth weight was abstracted from the medical records and in all participants was recorded immediately following birth .
subscapular and triceps skinfold measurements were collected by trained research administrators and most were measured within the first 24 h following birth and all within 72 h.28 measurements were obtained using harpenden calipers ( holtain ltd ) on the left side of the body .
we have previously shown good levels of within and between assessor reliability for all the birth size measurements.25 cord blood samples were obtained at delivery by the attending midwife on a consecutive subsample ( n=1387 ) .
samples were refrigerated at 4c in edta tubes until collected by laboratory staff within 12 h. samples were spun , frozen and stored at 80c .
once all samples had been collected , they were transferred to the biochemistry department of glasgow royal infirmary for analysis .
leptin was measured by a highly sensitive in - house elisa with better sensitivity at lower levels than commercial assays as previously described.29
ethnicity was self - reported at interview , with participants given response options based on the uk office of national statistics guidance.30 pakistani - origin women completed a detailed ancestry interview which included the details of the place of birth of themselves , their partner and all four grandparents .
generational status of pakistani infants was based on these data . using information on both parents and all four grandparents produced 66 possible unique categories .
we examined the numbers , mean birth weight and sd in all categories and then combined the groups based on sample size and similarities in mean birth weight and migration history .
this resulted in 17 categories within which 90% fell into one of the four main categories : both parents uk born and all grandparents south asian bornmother uk born , father and all grandparents south asian bornfather uk born , mother and all grandparents south asian bornboth parents south asian born and all grandparents south asian born both parents uk born and all grandparents south asian born mother uk born , father and all grandparents south asian born father uk born , mother and all grandparents south asian born both parents south asian born and all grandparents south asian born the remaining 10% fell across the other 13 categories , each of which included relatively few participants and so these groups were combined to form one other category .
other group ) , and to simplify interpretation , the generation groups were labelled according to the place of birth of parents only for the remainder of the paper .
few characteristics could plausibly influence a woman 's ethnicity and therefore confound the association of this with outcomes.31
32 however , we were interested in characteristics that are known to be influenced by ethnicity and that might be on a causal pathway explaining birth size differences .
for example , women of white british origin are more likely to smoke during pregnancy than those of pakistani origin and taking account of this may in fact increase the ethnic difference in birth weight .
while such characteristics can not be on a mediating causal pathway ( since accounting for their ethnic differences would likely increase rather than decrease birth weight differences ) , we felt it important to explore the effect of adjusting for them on ethnic differences in birth size . a priori , we considered maternal early pregnancy body mass index ( bmi ) , height , age , parity , smoking , alcohol consumption , socioeconomic position ( maternal education , housing tenure , whether they received benefits ) , living with a partner , fasting and postload glucose , fasting insulin , hypertensive disorders of pregnancy , infant sex and gestational age to be characteristics that might explain or mask ethnic differences in outcomes .
online supplementary table s1 briefly summarises our a priori assumptions of directions of association between ethnicity and each of the characteristics and between each of these and birth weight and hence the potential impact that they might have on birth weight differences between pakistani and white british infants .
maternal weight ( seca digital scales ) and height ( leicester height measure ) were measured in light clothing and unshod.27 fasting and postload glucose and fasting insulin were obtained from the ogtt samples which were assayed immediately after sampling at the biochemistry department of bradford royal infirmary using the glucose oxidase method on siemen 's advia 2400 chemistry autoanalysers and siemen 's advia centaur assay .
multivariable linear regression was used to examine the associations between ethnicity and birth weight , skinfolds and cord leptin levels ( pakistani infants compared with white british infants ) and to examine these associations by generation ( each pakistani generation group compared with white british infants ) .
we adjusted separately for each covariable to see how these influenced the associations and then adjusted ( 1 ) jointly for those that we thought a priori ( see online supplementary table s1 ) would plausibly be on the causal pathway for lower birth size in pakistani origin infants ; ( 2 ) jointly for those that did reduce the difference when adjusted for individually ; and ( 3 ) for all covariables simultaneously .
we generated sex and gestational age internal standardised z - scores for birth weight and skinfold thicknesses using six gestational age categories ( 37 ; 38 ; 39 ; 40 ; 41 ; 4242 ) so that these were all assessed on the same scale and could be directly compared .
we then regressed these scores on ethnicity to calculate the mean difference in z - score between white british and pakistani infants .
further , we adjusted for birth weight in the regression analyses of skinfolds ( z - scores ) and cord leptin .
cord leptin concentration had a positively skewed distribution and was natural log transformed to achieve approximate normality of the residuals in the regression models .
the resultant coefficients were back transformed to give ratios of geometric means which have a null value of 1 .
owing to the smaller leptin sample size , we only included covariables in the final model that altered the coefficient when each was examined individually .
for the main analyses , we included only participants with complete data on exposure , each outcome and all covariables .
this resulted in 8704 participants for birth weight analyses ; 6041 for subscapular skinfolds ; 6058 for triceps skinfolds ; and 1186 for cord leptin analyses .
in order to examine whether missing covariable data might have biased our results , we repeated unadjusted analyses and analyses adjusting just for one covariable ( taking each in turn ) in maximal samples ( ie , not restricting to those with complete data on all covariables ) and compared results with the equivalent results in those with complete data on all covariables ( see online supplementary tables s3s6 ) .
pakistani - origin mothers were on average older , shorter and had a lower bmi than white british origin mothers ( see online supplementary table s2 ) .
few pakistani mothers reported smoking during pregnancy and none reported having consumed alcohol while pregnant .
there were fewer uk - born pakistani mothers in this sample than south asian born and the largest pakistani generation group was that where both parents were born in south asia .
some characteristics varied across generation groups according to the maternal birthplace including employment and smoking during pregnancy which were both more common among uk - born pakistani mothers .
other characteristics differed by the partner 's place of birth irrespective of whether the mother was uk or south asian born , including maternal bmi which was higher among pakistani mothers with a south asian - born partner compared with those with a uk - born partner .
birth weight was lower among pakistani infants than white british infants ( figure 2 and see online supplementary table s3 ; unadjusted mean difference 235 95% ci 256 to 214 ) .
maternal height , maternal bmi and gestational age all explained some of the differences , but important differences remained even with adjustment simultaneously for these three characteristics ( see online supplementary table s3 ) .
smoking , maternal fasting glucose , parity and living with a partner all masked some of the difference .
the combined effect of adjusting for all covariables simultaneously was that the mean birth weight difference was similar to the unadjusted difference ( figure 2 and see online supplementary table s3 ) . across generation groups ,
the adjusted mean difference in birth weight was least where both parents were uk born ( 217 95% ci 261 to 173 ) and greatest when just the mother was uk born ( 253 95% ci 285 to 222 ) , but there was no strong statistical evidence that the association of ethnicity with birth weight varied by generational group ( all p values 0.1 ) .
unadjusted and adjusted mean difference in birth weight ( 95% ci ) between pakistani and white british infants .
adjusted for smoking ; alcohol ; maternal age ; maternal hypertension ; maternal fasting glucose ; maternal height ; maternal body mass index ; parity ; gestation ; sex ; socioeconomic position ( maternal education , housing tenure , receipt of means tested benefits ) ; and living with partner .
fasting glucose , postload glucose and fasting insulin were highly correlated thus postload glucose and fasting insulin were excluded from the fully adjusted model as they had the least effect and no impact on associations over that seen for fasting glucose .
subscapular and triceps skinfold thickness were each smaller among pakistani infants compared with white british infants ( figures 3 and 4 , see online supplementary tables s4 and s5 ) .
the adjusted mean difference in subscapular and triceps skinfold was greatest when mothers were south asian born and fathers were uk born ( 0.36 95% ci 0.47 to 0.26 and 0.32 95% ci 0.42 to 0.21 , respectively ) and least for subscapular skinfold when both parents were uk born ( 0.22 95% ci 0.36 to 0.08 ) and for triceps skinfold where mothers were uk born and fathers south asian born ( 0.22 95% ci 0.32 to 0.12 ) .
as with birth weight , differences between groups were generally small and again there was no strong statistical evidence of differences between generational groups ( all p values 0.1 and 0.3 , respectively , for subscapular and triceps differences ) .
adjusted for smoking ; alcohol ; maternal age ; maternal hypertension ; maternal fasting glucose ; maternal height ; maternal body mass index ; parity ; gestation ; sex ; socioeconomic position ( maternal education , housing tenure , receipt of means tested benefits ) ; and living with partner .
fasting glucose , postload glucose and fasting insulin were highly correlated thus postload glucose and fasting insulin were excluded from the fully adjusted model as they had the least effect and no impact on associations over that seen for fasting glucose .
unadjusted and adjusted mean difference in triceps skinfold between pakistani and white british infants . adjusted for smoking ; alcohol ; maternal age ; maternal hypertension ; maternal fasting glucose ; maternal height ; maternal body mass index ; parity ; gestation ; sex ; socioeconomic position ( maternal education , housing tenure , receipt of means tested benefits ) ; and living with partner .
fasting glucose , postload glucose and fasting insulin were highly correlated thus postload glucose and fasting insulin were excluded from the fully adjusted model as they had the least effect and no impact on associations over that seen for fasting glucose .
mean z - score differences were lower for subscapular and triceps skinfold thickness than birth weight ( table 1 ) ; once the differences in birth weight were adjusted for , the z - score differences in skinfold thickness were markedly reduced or became weakly positive ( table 2 ) .
cord blood leptin levels were similar among pakistani and white british infants without the adjustment for birth weight ( table 3 ) ; but , with adjustment for birth weight , pakistani infants had a cord leptin value on average 30% higher ( ratio of geometric means 1.30 95% ci 1.17 to 1.44 ) than white british infants .
adjusted mean z - score differences in pakistani relative to white british infants * adjusted for smoking ; alcohol ; maternal age ; maternal hypertension ; maternal fasting glucose ; maternal height ; maternal bmi ; parity ; gestation ; sex ; socioeconomic position ( maternal education , housing tenure , receipt of means tested benefits ) ; and living with partner .
fasting glucose , postload glucose and fasting insulin were highly correlated thus postload glucose and fasting insulin were excluded from the fully adjusted model as they had the least effect and no impact on associations over that seen for fasting glucose .
bmi , body mass index . adjusted mean z - score differences in pakistani relative to white british infants with adjustment for birth weight * adjusted for smoking ; alcohol ; maternal age ; maternal hypertension ; maternal fasting glucose ; maternal height ; maternal bmi ; parity ; gestation ; sex ; socioeconomic position ( maternal education , housing tenure , receipt of means tested benefits ) ; living with partner ; and birth weight .
fasting glucose , postload glucose and fasting insulin were highly correlated thus postload glucose and fasting insulin were excluded from the fully adjusted model as they had the least effect and no impact on associations over that seen for fasting glucose .
bmi , body mass index . adjusted differences in cord leptin between pakistani and white british infants values are ratios of geometric means ( 95% ci ) . *
to maintain statistical power , only those covariables that had an effect were included in the final model : smoking ; maternal fasting glucose ; maternal postload glucose ; maternal fasting insulin ; maternal bmi ; and living with a partner .
these results suggest a striking difference in birth weight between pakistani and white british infants which persists even after the adjustment for a wide range of potential mediating characteristics and is maintained over successive generations .
we found evidence of greater relative adiposity in pakistani infants compared with those of white british origin .
notably , once birth weight had been taken into account , cord leptin , used as a marker of total fat mass , was higher in pakistani origin compared with white british - origin infants suggesting that infants of pakistani origin are lighter , but relatively fatter , than those of white british origin .
this is by far the largest study to date to examine these associations and highlights the importance of considering body composition , and not just weight , when considering ethnic differences in birth size .
our finding that birth weight differences between pakistani and white british origin infants do not vary notably by pakistani generation is consistent with most previous studies9
19
20 however , we have added to this previous literature by showing that this is also the case when generation is defined by paternal ( and not only maternal ) place of birth .
we found marked differences in pregnancy , behaviour and socioeconomic characteristics between pakistani and white british mothers that had varying effects on the associations between ethnicity , generation and birth size .
smoking , maternal fasting glucose , parity and living with a partner all masked some of the differences , and also gestational length , maternal height and bmi individually explained some . from these results , there is no clear way in which the ethnic differences in birth size could be reduced .
levels of smoking are modifiable , but reducing smoking in white british women , while desirable , would only increase ethnic differences .
effective treatment of hyperglycaemia in pregnant women would be beneficial , but based on our results would likely increase ethnic differences in birth weight , although given the relationship of greater maternal glycaemia with greater adiposity ( see online supplementary table s6 ) , could reduce the greater relative adiposity in pakistani - origin infants .
characteristics such as parity , marital status , gestational length and maternal height are not clearly modifiable during pregnancy .
finally , interventions to increase bmi in pakistani mothers would potentially be disadvantageous for both the mother 's and her offspring 's health , as they would increase the mother 's risk of gestational diabetes and later adverse cardiometabolic health and potentially the risk of greater relative adiposity in offspring .
our findings add importantly to the four previous studies1013 exploring whether the ethnic difference in relative body fat found in studies comparing south asian and white european origin adults and schoolchildren,14
15 is present at birth .
our study is considerably larger than these previous studies and compares pakistani infants to white british infants who were all born in the same uk maternity unit , and thus controls for the possible differences that may occur between births in a south asian country and those in the uk . while generally consistent with previous studies , we found no clear evidence of greater relative central adiposity in pakistani infants compared with white british infants which some previous studies have reported based largely on comparing subscapular to triceps differences between ethnic groups.1013 these studies have been smaller than ours and largely compared south asian - born infants to uk - born european infants , and it is possible that central adiposity is greater in south asian infants born in south asia , but not in those born in the uk .
this has some support from a study of uk schoolchildren which found , consistent with our results , greater markers of general adiposity , but a lower waist circumference in uk south asian compared with white european children.33 the key strengths of this study are its large sample size , detailed information on place of birth of mothers , fathers and all four grandparents , the ability to adjust for a much wider range of possible explanatory or masking variables than any previous study and the availability of cord leptin to assess relative differences in fat mass .
however , patterns of missing data were similar by ethnic group and unadjusted associations in each group did not differ markedly between those with some data and those with complete data on all covariables .
it is possible that bmi is a less accurate measure of adiposity among south asian mothers than white british mothers ; however , the plausible mechanism by which maternal bmi might influence offspring fatness would be via glucose intolerance , and here we have used a gold standard ( ogtt ) measure of this and have shown that adjustment for fasting glucose , postload glucose or fasting insulin do not substantively explain the ethnic differences reported here .
cord leptin measurements were only available for a subgroup , but these were a consecutive sample and we can see no reason why collecting these over a specific time period would result in a select group ; similar proportions from each ethnic group had cord leptin data .
we had hoped to be able to explore the generational effects across a larger range of generational groups , but for the vast majority of participants in the study all four grandparents were born in south asia and so we were unable to examine whether place of birth of grandparents influenced the differences .
nonetheless , this pattern of migration to bradford is in itself interesting and relevant to future health needs in the city . while our findings for birth weight are largely consistent with a number of other studies that have explored broader south asian groups and/or looked at different groups ( eg , pakistani , indian and bangladeshi origin ) and found in general all have lower birth weight than white british infants,3
9
13
19
20 we can not be certain that the results are generalisable to other south asian groups .
there are participants of indian and bangladeshi origin in bib , but the numbers were too small for meaningful analyses , particularly across generations or for cord leptin concentration , therefore they were excluded from this paper .
these results suggest a striking difference in birth weight between pakistani and white british infants which persists even after the adjustment for a wide range of potential mediating characteristics and is maintained over successive generations .
we found evidence of greater relative adiposity in pakistani infants compared with those of white british origin .
notably , once birth weight had been taken into account , cord leptin , used as a marker of total fat mass , was higher in pakistani origin compared with white british - origin infants suggesting that infants of pakistani origin are lighter , but relatively fatter , than those of white british origin .
this is by far the largest study to date to examine these associations and highlights the importance of considering body composition , and not just weight , when considering ethnic differences in birth size .
our finding that birth weight differences between pakistani and white british origin infants do not vary notably by pakistani generation is consistent with most previous studies9
19
20 however , we have added to this previous literature by showing that this is also the case when generation is defined by paternal ( and not only maternal ) place of birth .
we found marked differences in pregnancy , behaviour and socioeconomic characteristics between pakistani and white british mothers that had varying effects on the associations between ethnicity , generation and birth size .
smoking , maternal fasting glucose , parity and living with a partner all masked some of the differences , and also gestational length , maternal height and bmi individually explained some . from these results , there is no clear way in which the ethnic differences in birth size could be reduced .
levels of smoking are modifiable , but reducing smoking in white british women , while desirable , would only increase ethnic differences .
effective treatment of hyperglycaemia in pregnant women would be beneficial , but based on our results would likely increase ethnic differences in birth weight , although given the relationship of greater maternal glycaemia with greater adiposity ( see online supplementary table s6 ) , could reduce the greater relative adiposity in pakistani - origin infants .
characteristics such as parity , marital status , gestational length and maternal height are not clearly modifiable during pregnancy .
finally , interventions to increase bmi in pakistani mothers would potentially be disadvantageous for both the mother 's and her offspring 's health , as they would increase the mother 's risk of gestational diabetes and later adverse cardiometabolic health and potentially the risk of greater relative adiposity in offspring .
our findings add importantly to the four previous studies1013 exploring whether the ethnic difference in relative body fat found in studies comparing south asian and white european origin adults and schoolchildren,14
15 is present at birth .
our study is considerably larger than these previous studies and compares pakistani infants to white british infants who were all born in the same uk maternity unit , and thus controls for the possible differences that may occur between births in a south asian country and those in the uk . while generally consistent with previous studies , we found no clear evidence of greater relative central adiposity in pakistani infants compared with white british infants which some previous studies have reported based largely on comparing subscapular to triceps differences between ethnic groups.1013 these studies have been smaller than ours and largely compared south asian - born infants to uk - born european infants , and it is possible that central adiposity is greater in south asian infants born in south asia , but not in those born in the uk .
this has some support from a study of uk schoolchildren which found , consistent with our results , greater markers of general adiposity , but a lower waist circumference in uk south asian compared with white european children.33
the key strengths of this study are its large sample size , detailed information on place of birth of mothers , fathers and all four grandparents , the ability to adjust for a much wider range of possible explanatory or masking variables than any previous study and the availability of cord leptin to assess relative differences in fat mass .
however , patterns of missing data were similar by ethnic group and unadjusted associations in each group did not differ markedly between those with some data and those with complete data on all covariables .
it is possible that bmi is a less accurate measure of adiposity among south asian mothers than white british mothers ; however , the plausible mechanism by which maternal bmi might influence offspring fatness would be via glucose intolerance , and here we have used a gold standard ( ogtt ) measure of this and have shown that adjustment for fasting glucose , postload glucose or fasting insulin do not substantively explain the ethnic differences reported here .
cord leptin measurements were only available for a subgroup , but these were a consecutive sample and we can see no reason why collecting these over a specific time period would result in a select group ; similar proportions from each ethnic group had cord leptin data . we had hoped to be able to explore the generational effects across a larger range of generational groups , but for the vast majority of participants in the study all four grandparents were born in south asia and so we were unable to examine whether place of birth of grandparents influenced the differences . nonetheless , this pattern of migration to bradford is in itself interesting and relevant to future health needs in the city . while our findings for birth weight are largely consistent with a number of other studies that have explored broader south asian groups and/or looked at different groups ( eg , pakistani , indian and bangladeshi origin ) and found in general all have lower birth weight than white british infants,3
9
13
19
20 we can not be certain that the results are generalisable to other south asian groups .
there are participants of indian and bangladeshi origin in bib , but the numbers were too small for meaningful analyses , particularly across generations or for cord leptin concentration , therefore they were excluded from this paper .
to conclude , our results suggest that despite being markedly lighter , pakistani infants have greater fat mass ( as assessed by cord leptin ) for a given birth weight than white british - origin infants .
further studies are needed to explore whether efforts to increase birth weight in pakistani infants and hence to reduce the ethnic difference could inadvertently result in even greater relative adiposity which in turn may worsen long - term health outcomes for pakistani infants .
what is already known on this subjectinfants born in south asia are on average smaller and lighter at birth than infants born elsewhere in the world , and this difference persists among the south asian - origin infants born in high - income countries including the uk.previous studies have found no clear evidence that mean birth weight is increasing in subsequent generations of the uk south asians.south asian children and adults have greater fat mass for a given weight or body mass index , but whether this is the case at birth is unclear .
what this study addsfor a given birth weight , pakistani - origin infants have greater adiposity than white british - origin infants born at the same uk maternity hospital.ethnic differences in adiposity do not vary notably depending on whether both mothers and fathers of pakistani - origin infants are born in the uk or south asia.efforts to increase birth weight in south asian - origin infants could inadvertently result in even greater relative adiposity .
infants born in south asia are on average smaller and lighter at birth than infants born elsewhere in the world , and this difference persists among the south asian - origin infants born in high - income countries including the uk .
previous studies have found no clear evidence that mean birth weight is increasing in subsequent generations of the uk south asians .
south asian children and adults have greater fat mass for a given weight or body mass index , but whether this is the case at birth is unclear . for a given birth weight , pakistani - origin infants have greater adiposity than white british - origin infants born at the same uk maternity hospital .
ethnic differences in adiposity do not vary notably depending on whether both mothers and fathers of pakistani - origin infants are born in the uk or south asia .
efforts to increase birth weight in south asian - origin infants could inadvertently result in even greater relative adiposity .
| backgroundprevious studies have shown markedly lower birth weight among infants of south asian origin compared with those of white european origin .
whether such differences mask greater adiposity in south asian infants and whether they persist across generations in contemporary uk populations is unclear .
our aim was to compare birth weight , skinfold thickness and cord leptin between pakistani and white british infants and to investigate the explanatory factors , including parental and grandparental birthplace.methodswe examined the differences in birth weight and skinfold thickness between 4649 pakistani and 4055 white british infants born at term in the same uk maternity unit and compared cord leptin in a subgroup of 775 pakistani and 612 white british infants.resultspakistani infants were lighter ( adjusted mean difference 234 g 95% ci 258 to 210 ) and were smaller in both subscapular and triceps skinfold measurements .
the differences for subscapular and triceps skinfold thickness ( mean z - score difference 0.27 95% ci 0.34 to 0.20 and 0.23 95% ci 0.30 to 0.16 , respectively ) were smaller than the difference in birth weight ( mean z - score difference 0.52 95% ci 0.58 to 0.47 ) and attenuated to the null with adjustment for birth weight ( 0.03 95% ci 0.03 to 0.09 and 0.01 95% ci 0.08 to 0.05 , respectively ) .
cord leptin concentration ( indicator of fat mass ) was similar in pakistani and white british infants without adjustment for birth weight , but with adjustment became 30% higher ( 95% ci 17% to 44% ) among pakistani infants compared with white british infants .
the magnitudes of difference did not differ by generation.conclusionsdespite being markedly lighter , pakistani infants had similar skinfold thicknesses and greater total fat mass , as indicated by cord leptin , for a given birth weight than white british infants .
any efforts to reduce ethnic inequalities in birth weight need to consider differences in adiposity and the possibility that increasing birth weight in south asian infants might inadvertently worsen health by increasing relative adiposity . |
erythropoietin ( epo ) or hematopoietin , a member of the type1cytokine superfamily , is a glycoprotein hormone , which is responsible for the regulation of erythropoiesis through inhibiting of apoptosis , proliferation and differentiation of erythroid precursor cells .
discovery of epo and epo receptor in neural cells indicated that , in addition to erythropoiesis , epo has protective effects in the brain ( 1 , 2 ) . studies over the past years revealed that epo can protect neurons from injury and has an important role in the survival and proliferation in neural progenitor cells ( 3 , 4 ) .
administration of recombinant human epo in a rabbit model of subarachnoid hemorrhage induced acute cerebral ischemia , considerably decreased acute ischemic neuronal damage and increased the epo concentration in the cerebrospinal fluid ( 5 ) .
it has been shown that epo can induce a wide range of cellular responses to protect and repair brain injury in different stress conditions like hypoxia and excitotoxicity ( 4 , 6 ) .
preventive effects of epo against oxidative damage through increasing antioxidant enzymes such as superoxide dismutase and glutathione peroxidase have also been reported ( 7 ) .
epo could reduce inflammation by inhibition of inflammatory mediators including tnf- , interleukin-6 ( il-6 ) , il-1beta , il- 1alpha and interferon- ( 8) . moreover epo is involved in the recovery of traumatic brain and spinal cord injuries by inhibition of apoptosis and anti - oxidant properties , induction of neurogenesis and angiogenesis . according to documents
a great potential of epo in the recovery of stroke , multiple sclerosis , alzheimer , huntington , parkinson , traumatic brain and spinal cord injuries , has been shown(4 , 9 ) .
affinity chromatography technique has been widely used to isolate specific target proteins from a complex proteome . in order to isolate bound protein targets ,
the eluted proteins can then separate by gel electrophoresis and analyzed by mass spectrometry ( 1 ) .
drug target deconvolution is a process in which the biological role of a drug , a small molecule , is characterized through the identification of the proteins that interact with the drug and , so that , initiate the biological effect .
then , the biological relevant targetsareidentified froma mixture of proteins identified in such an approach . beside the medically desired action of the drug , the identification of other proteins that could interact with the drug , could help to identify the side effects and toxicity at a very early stage of drug development ( 10 ) .
in this project we hypothesized that some of therapeutic effects are through the direct interaction between erythropoeitin and proteins .
the aim of this study we investigated erythropoietin interacting proteins using affinity chromatography based target deconvolution .
animal study was approved by mums ( mashhad university of medical sciences ) ethics committee .
animals were housed at temperature of 252c on a 12-hr light / dark cycle with free access to food and water .
twelve male balb / c mice ( 6 weeks old , 2030 g ) were sacrificed by decapitation .
brain tissues ( 200 mg ) were homogenized in 1 ml of lysis buffer , containing 50 mm tris ph 7.4 , 2 mm egta , 2 mm edta , 2 mm na3vo4 , 1% triton x-100 and 10 mm 2-mercaptoethanol , 1 mm phenylmethylsulfonyl fluoride ( pmsf ) , and complete protease inhibitor cocktail ( sigma p8340 , usa ) were added just before homogenization of tissues using a polytron homogenizer ( ika r t10 , germany ) in ice .
supernatants were collected and in order to interact between epo and its target in brain , about 5 g epo added to brain homogenates ( 500 ul of 1 mg / ml ) and incubate at 4c for 30 min .
epo protein targets were isolated using ip . according to pierce crosslink immune - precipitation kit protocol ( thermo scientific , cat number=26147 ) .
briefly , 5 g epo antibody was covalently cross linked to protein a / g resin using disuccinimidylsuberate ( dss ) .
500 l of 1 mg / ml tissue lysates were added to control resin , to eliminate proteins with unspecific interactions with beads . after a brief centrifugation
, supernatant was added to epo antibody - cross linked resin ( ip step ) .
after few washes , epo binding proteins were eluted using elution buffer provided in kit ( ph 2.8 ) .
concentrated elute was dialyzed against deionized water using a membrane with 2000 da cutoff ( spectra , usa ) for 3 days at 28c and then freeze dried .
freeze dried elutes , from ip , step were dissolved in 20 l of 2x sds sample buffer , samples were incubated in boiling water for 5 min and then subjected to sds - page electrophoresis ( biorad ) .
gels were silver stained and protein bands were excised and collected in microtubes containing 1% acetic acid .
freeze - dryed elutes were dissolved in rehydration buffer containing 6 m urea , 2 m thiourea , 2% 3-[(3 cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( chaps ) , 50 mm dithiothreitol ( dtt ) , 20% bio - lyte ( biorad ) .
125 l of lysate was loaded to non - linear immobilized ph gradient ( ipg ) strips ( ph range : 310 , 7 cm , biorad ) toseparate epo target proteins according to their isoelectric point . following active rehydration at 50v for 12 hrs
, isoelectric focusing was performed using protean ief cell ( biorad ) at 20c in 4 steps as follow : 250 v for 15min ; 4000 v for 1 hrs ; 35000 v for 1 hrs .
ipgs were incubated in equilibration buffer [ 375 mmtris ( ph 8.8 ) , 6 m urea , 2.5% sds and 30% glycerol ] for 12 min .
ipgs were placed on top of 12% sds - page and sealed with agarose solution [ 25 mmtris ( ph 8.8 ) , 84 mm glycin , 0.5% agarose , 0.1% sds and amount of tracking dye bromophenol blue ] . electrophoresis was performed for 80 min at 120 v. after electrophoresis , gels were silver stained .
protein spots were cut off utilizing sterile scalpel from gels and conveyed to a micro tube containing 1% acid acetic and posted to the center for genomic sciences at the university of hong kong for identification and characterization by maldi - tof / tof .
mass data were acquired in the genome research centre , university of hong kong with 4800 maldi - tof / tof analyzer ( abi ) in positive ion reflector mode .
data were investigated in both ncbinr and swissprot databases . for analyzing mass data mascot ( version 2.1.0 , matrix science , london , uk ) was applied .
mascot parameters were set as follow : taxonomy : rat , fixed modification : carbamidomethyl ( cysteine ) , variable modification : oxidation ( methionine ) , ms / ms fragment tolerance:0.2 da , precursor tolerance : 75 ppm , peptide charge : + 1 , monoisotopic .
proteins with a score of > 50 and confidence interval ( ci ) > 95% were accepted .
categorization of proteins was performed according to biological function and process using panther classification system ( http://www.pantherdb.org ) .
animal study was approved by mums ( mashhad university of medical sciences ) ethics committee .
animals were housed at temperature of 252c on a 12-hr light / dark cycle with free access to food and water .
twelve male balb / c mice ( 6 weeks old , 2030 g ) were sacrificed by decapitation .
brain tissues ( 200 mg ) were homogenized in 1 ml of lysis buffer , containing 50 mm tris ph 7.4 , 2 mm egta , 2 mm edta , 2 mm na3vo4 , 1% triton x-100 and 10 mm 2-mercaptoethanol , 1 mm phenylmethylsulfonyl fluoride ( pmsf ) , and complete protease inhibitor cocktail ( sigma p8340 , usa ) were added just before homogenization of tissues using a polytron homogenizer ( ika r t10 , germany ) in ice .
supernatants were collected and in order to interact between epo and its target in brain , about 5 g epo added to brain homogenates ( 500 ul of 1 mg / ml ) and incubate at 4c for 30 min .
epo protein targets were isolated using ip . according to pierce crosslink immune - precipitation kit protocol ( thermo scientific , cat number=26147 ) .
briefly , 5 g epo antibody was covalently cross linked to protein a / g resin using disuccinimidylsuberate ( dss ) .
500 l of 1 mg / ml tissue lysates were added to control resin , to eliminate proteins with unspecific interactions with beads . after a brief centrifugation
, supernatant was added to epo antibody - cross linked resin ( ip step ) .
after few washes , epo binding proteins were eluted using elution buffer provided in kit ( ph 2.8 ) .
concentrated elute was dialyzed against deionized water using a membrane with 2000 da cutoff ( spectra , usa ) for 3 days at 28c and then freeze dried .
freeze dried elutes , from ip , step were dissolved in 20 l of 2x sds sample buffer , samples were incubated in boiling water for 5 min and then subjected to sds - page electrophoresis ( biorad ) .
gels were silver stained and protein bands were excised and collected in microtubes containing 1% acetic acid .
freeze - dryed elutes were dissolved in rehydration buffer containing 6 m urea , 2 m thiourea , 2% 3-[(3 cholamidopropyl ) dimethylammonio]-1-propanesulfonate ( chaps ) , 50 mm dithiothreitol ( dtt ) , 20% bio - lyte ( biorad ) .
125 l of lysate was loaded to non - linear immobilized ph gradient ( ipg ) strips ( ph range : 310 , 7 cm , biorad ) toseparate epo target proteins according to their isoelectric point . following active rehydration at 50v for 12 hrs
, isoelectric focusing was performed using protean ief cell ( biorad ) at 20c in 4 steps as follow : 250 v for 15min ; 4000 v for 1 hrs ; 35000 v for 1 hrs .
ipgs were incubated in equilibration buffer [ 375 mmtris ( ph 8.8 ) , 6 m urea , 2.5% sds and 30% glycerol ] for 12 min .
ipgs were placed on top of 12% sds - page and sealed with agarose solution [ 25 mmtris ( ph 8.8 ) , 84 mm glycin , 0.5% agarose , 0.1% sds and amount of tracking dye bromophenol blue ] .
electrophoresis was performed for 80 min at 120 v. after electrophoresis , gels were silver stained .
protein spots were cut off utilizing sterile scalpel from gels and conveyed to a micro tube containing 1% acid acetic and posted to the center for genomic sciences at the university of hong kong for identification and characterization by maldi - tof / tof .
mass data were acquired in the genome research centre , university of hong kong with 4800 maldi - tof / tof analyzer ( abi ) in positive ion reflector mode .
data were investigated in both ncbinr and swissprot databases . for analyzing mass data mascot ( version 2.1.0 , matrix science , london , uk ) was applied .
mascot parameters were set as follow : taxonomy : rat , fixed modification : carbamidomethyl ( cysteine ) , variable modification : oxidation ( methionine ) , ms / ms fragment tolerance:0.2 da , precursor tolerance : 75 ppm , peptide charge : + 1 , monoisotopic . proteins with a score of > 50 and confidence interval ( ci ) > 95% were accepted .
categorization of proteins was performed according to biological function and process using panther classification system ( http://www.pantherdb.org ) .
in order to find cellular targets of epo in the mice brain tissues , ip was performed . to eliminate unspecific binding of non - target proteins
, tissue extracts were incubated with control agarose beads for 30 min , after a brief centrifugation , supernatant was incubated with epo antibody - cross linked resin .
target proteins were eluted and subjected to 1d ( one - dimensional gel electrophoresis ) and 2d ( two - dimensional gel electrophoresis ) and ms analysis respectively .
several bands and spots 1d and 2d gels were detected as targets of epo , respectively .
bands with enough protein for detection by mass spectroscopy were excised , in gel digested and identified ( figure 1 ) .
a ) image of cellular targets of epo in mouse brain separated by sds - page in a 12 % gel .
b)image of cellular targets of epo in mouse brain separated by ief in a 7 cm ipg strip containing nonlinear ph gradient 310 followed by two - dimensional gel electrophoresis .
protein detection was by silver - staining eight proteins were identified among the detected spots and bands including tubulin beta , actin cytoplasmic 2 , t - complex protein 1,tpr and ankyrin repeat - containing protein 1 , centromere - associated protein e , kinesin - like protein kif7 , growth arrest - specific protein 2 and pleckstrin homology - like domain family b member 2 .
the information of identified proteins including protein name / gene names , sample name , swiss - prot accession number , confidence interval ( c.i.% ) , protein score , sequence coverage ( % ) , theoretical mw ( da)/pi , unique peptides detected , sequence of identified peptide with the highest ion score were listed in table 1 .
identified proteins by maldi / tof / tof in mouse brain gene names of epo target proteins were submitted to panther database and categorized based on the biological process ( figure 2 ) .
gene names of epo target proteins were submitted to panther database and classified according on biological process to seven group .
most of identified proteins were involved in cellular component and cellular process proteins were divided to seven classes including , cellular component organization , cellular process , immune system process , developmental process , localization , metabolic process and response to stimulus .
in the present study , affinity chromatography based target deconvolution was chosen to find potential targets of epo in the mice brain . utilizing a proteomics approach and ip , we have identified 8proteins which can be considered as potential epo target proteins in the mice brain tissue .
neuroprotective effects of epo have been reported in several studies ( 1 , 2 ) . different mechanisms inclu - ding antiapoptotic , increased no level in the brain vessels , anti - inflammation , maintenance of neuronal structure and survival , regulation of neuronal proliferation and differentiation have been attributed to epo neuroprotective(1 ) .
this study revealed some novel epo target proteins for the first time in mice brain tissue .
these proteins were divided into seven groups based on biological process including cellular component organization , cellular process , immune system process , developmental process , localization , metabolic process and response to stimulus .
it is well known that epo could prevent neuronal apoptosis via several pathways , which include the activation of pkb ( protein kinase - b ) and the subsequent inhibition of caspase-8 , -1 , and -3 , ( 11)furthermore epo could increase the activity of akt1(12 ) .
gas2 , a component of the microfilament system , belongs to the class of gas genes whose expression is induced at growth arrest .
gas2 is a target for caspase-7 and caspase-3 and can induce apoptosis ( 13 ) .
it was identified that gas- 2 can be expressed in frontal cortex in cocaine exposed mice .
in addition gas2 , can be acted as a inducer of microfilaments and microtubules modify - cations in the growth arrested cells ( 15 ) . due to the direct role of gas2 protein in several apoptosis pathways , it can be hypothesized that epo , as an inhibitor of apoptosis , can act through gas2 and caspase pathways .
pleckstrins are part of ll5 proteins which are presented as a branch in akt / protein kinase - b .
one study showed that the activation of pleckstrin branch in akt / pkb resulted in anti - apoptotic effects in ischemic astrocytes of rat brain hippocampus ( 16 ) .
in addition ll5 can regulate intra - cellular and membrane skeleton in response to extracellular signaling ( 17 ) .
considering the role of pleckstrin in apoptosis and cell survival , organization and maintenance of cell structure and its presence as a part of akt / protein kinase - b , effective interaction of this protein and epo in our study is verified . according to the documents , actin cytoplasmic
it is recognized that actin acts as a substrate for tissue transglutaminase which can be considered as a regulator of apoptosis biochemical events through transglutaminase dependent cross - linking .
it is also reported that fragmentation of actins may be associated with induction of apoptosis .
higher actin content is associated with decrease of ros formation and increase in cell survival .
studies showed that actin inhibitors ( eg , jasplakinolide ) could induce ros production ( 18 ) .
studies on t - cells dependent il-2 , indicated that jasplakinolide diminished the functionality of actin resulting t - cell apoptosis via increased activity of caspase- 3(19 ) .
it was identified that actin regulatory proteins known as rsp5p , end3p , sla1p ubiquitin ligase ( ubiquitin ligase rsp5p ) are primary regulators of oxidative stress associated with actin ( 20 ) .
function removal of end3p and sla1p led to poor cytoskeleton stability which causes loss of mitochondrial membrane potential and increased oxidative stress ( 21 ) .
actin is one of the main components of structural cytoskeleton in brain and spinal cord neurons .
many regulatory of actin dependent proteins which are presented in neurons are expressed during axonal growth and dendrite formation .
the amount of drebrin is decreased in the frontal and temporal cortex in alzheimer s disease . in neurodegenerative diseases
actin fragmentation could induce changes in cell morphology , resulting in dysfunction of neuronal receptors signaling ( 22 ) .
it has been indicated that another protein which has a role in actins regulation through attachment to it , is apolipoprotein - e ( apo e ) .
regarding the beneficial effects of epo on alzheimer disease , it can be concluded that actin could be considered as one of the main proteins which can interact with epo .
effects of epo on protein species involved in cell cycle regulation , development and differentiation .
it has been established that epo / epor systemplay a role both in neurodevelopment and brain homoeostasis .
according to the results tpr and ankyrin repeat - containing protein 1 is another epo target proteins .
this protein has six ankyrin repetitive sequences and four tpr ( tetra - tricopeptide repeat ) repetitive sequences , which is distributed in the brain hippocampus and hypothalamus .
ankyrin repetitive sequences have been found in many proteins which are involved in cell signaling , maintenance of cytoskeletal structure , cell cycle regulation , inflammatory responses , growth , differentiation and other transport phenomena ( 24 ) .
tpr sequences is also involved in various functions including cell cycle regulation , protein transport and folding proteins ( 25 ) . according to the important role of this protein in the cell cycle regulation , growth , differentiation and inflammatory response ,
it may be considered as one of the functional pathway of epo in the nervous system .
kinesin - like protein ( kif7 ) is also involved in neuronal proliferation , development and microtubule - based transport systems ( 26 ) .
this protein is also considered as a regulator in sonic hedgehog pathway ( shh pathway ) during cell development ( 27 ) .
it has been reported that mutation in the kif7gene leads to joubertsyndrome which is a brain abnormality .
centromere - associated protein - e ( cenp - e ) is a critical mitotic kinesin which is essential for efficient , stable microtubule capture at kinetochores .
it can bind to a kinetochore - associated kinase implicated in the mitotic checkpoint , which is the major cell cycle control pathway .
one study revealed that cenp - e was over expressed in glioblastoma and astrocytic tumors ( 29 ) .
another epo target protein is t - complex protein 1 which acts as a chaperone that is associated with atp hydrolysis .
it has important roles in actin and tubulin folding , growth and development of nerve cells ( 30 ) .
t - complex protein 1 was identified in rat hippocampus and human cortical neurons ( 31 , 32 ) tubulin , specifically expressed in neurons , is involved in microtubule formation . in mitosis , microtubules have a special role in appropriate chromosomal division in cells ( 33 ) .
mesenchymial stem cells ( mscs ) may improve symptoms in neurodegenerative diseases . under normal concentration of oxygen ( normoxic ) , human mscs
epo administration in normoxic conditions results in expression of tubulin beta-3 and induces cells with neuronal morphology ( 34 ) .
the relevance of tubulin protein and huntington s disease is also identified ( 35 ) .
it was reported that co- administration of epo and paclitaxel can inhibit neuronal axonal damage due to the lower level of detyrosinated tubulin ( 36 ) .
detyrosinated tubulin is a marker for microtubule stabilization . stabilized microtubules disrupt mitotic processes and arrest the cell division .
considering the role of tubulin in neural cell death , neuronal structure and mitosis , the interaction of epo and beta tubulin in neurons could be concluded .
it is well known that epo could prevent neuronal apoptosis via several pathways , which include the activation of pkb ( protein kinase - b ) and the subsequent inhibition of caspase-8 , -1 , and -3 , ( 11)furthermore epo could increase the activity of akt1(12 ) .
gas2 , a component of the microfilament system , belongs to the class of gas genes whose expression is induced at growth arrest .
gas2 is a target for caspase-7 and caspase-3 and can induce apoptosis ( 13 ) .
it was identified that gas- 2 can be expressed in frontal cortex in cocaine exposed mice .
in addition gas2 , can be acted as a inducer of microfilaments and microtubules modify - cations in the growth arrested cells ( 15 ) . due to the direct role of gas2 protein in several apoptosis pathways , it can be hypothesized that epo , as an inhibitor of apoptosis , can act through gas2 and caspase pathways .
pleckstrins are part of ll5 proteins which are presented as a branch in akt / protein kinase - b .
one study showed that the activation of pleckstrin branch in akt / pkb resulted in anti - apoptotic effects in ischemic astrocytes of rat brain hippocampus ( 16 ) .
in addition ll5 can regulate intra - cellular and membrane skeleton in response to extracellular signaling ( 17 ) .
considering the role of pleckstrin in apoptosis and cell survival , organization and maintenance of cell structure and its presence as a part of akt / protein kinase - b , effective interaction of this protein and epo in our study is verified .
it is recognized that actin acts as a substrate for tissue transglutaminase which can be considered as a regulator of apoptosis biochemical events through transglutaminase dependent cross - linking .
it is also reported that fragmentation of actins may be associated with induction of apoptosis .
higher actin content is associated with decrease of ros formation and increase in cell survival .
studies showed that actin inhibitors ( eg , jasplakinolide ) could induce ros production ( 18 ) .
studies on t - cells dependent il-2 , indicated that jasplakinolide diminished the functionality of actin resulting t - cell apoptosis via increased activity of caspase- 3(19 ) .
it was identified that actin regulatory proteins known as rsp5p , end3p , sla1p ubiquitin ligase ( ubiquitin ligase rsp5p ) are primary regulators of oxidative stress associated with actin ( 20 ) .
function removal of end3p and sla1p led to poor cytoskeleton stability which causes loss of mitochondrial membrane potential and increased oxidative stress ( 21 ) .
actin is one of the main components of structural cytoskeleton in brain and spinal cord neurons .
many regulatory of actin dependent proteins which are presented in neurons are expressed during axonal growth and dendrite formation .
the amount of drebrin is decreased in the frontal and temporal cortex in alzheimer s disease . in neurodegenerative diseases
actin fragmentation could induce changes in cell morphology , resulting in dysfunction of neuronal receptors signaling ( 22 ) .
it has been indicated that another protein which has a role in actins regulation through attachment to it , is apolipoprotein - e ( apo e ) .
regarding the beneficial effects of epo on alzheimer disease , it can be concluded that actin could be considered as one of the main proteins which can interact with epo .
effects of epo on protein species involved in cell cycle regulation , development and differentiation .
it has been established that epo / epor systemplay a role both in neurodevelopment and brain homoeostasis .
according to the results tpr and ankyrin repeat - containing protein 1 is another epo target proteins .
this protein has six ankyrin repetitive sequences and four tpr ( tetra - tricopeptide repeat ) repetitive sequences , which is distributed in the brain hippocampus and hypothalamus .
ankyrin repetitive sequences have been found in many proteins which are involved in cell signaling , maintenance of cytoskeletal structure , cell cycle regulation , inflammatory responses , growth , differentiation and other transport phenomena ( 24 ) .
tpr sequences is also involved in various functions including cell cycle regulation , protein transport and folding proteins ( 25 ) . according to the important role of this protein in the cell cycle regulation , growth , differentiation and inflammatory response ,
it may be considered as one of the functional pathway of epo in the nervous system .
kinesin - like protein ( kif7 ) is also involved in neuronal proliferation , development and microtubule - based transport systems ( 26 ) .
this protein is also considered as a regulator in sonic hedgehog pathway ( shh pathway ) during cell development ( 27 ) .
it has been reported that mutation in the kif7gene leads to joubertsyndrome which is a brain abnormality .
centromere - associated protein - e ( cenp - e ) is a critical mitotic kinesin which is essential for efficient , stable microtubule capture at kinetochores .
it can bind to a kinetochore - associated kinase implicated in the mitotic checkpoint , which is the major cell cycle control pathway .
one study revealed that cenp - e was over expressed in glioblastoma and astrocytic tumors ( 29 ) .
another epo target protein is t - complex protein 1 which acts as a chaperone that is associated with atp hydrolysis .
it has important roles in actin and tubulin folding , growth and development of nerve cells ( 30 ) .
t - complex protein 1 was identified in rat hippocampus and human cortical neurons ( 31 , 32 ) tubulin , specifically expressed in neurons , is involved in microtubule formation . in mitosis , microtubules have a special role in appropriate chromosomal division in cells ( 33 ) .
mesenchymial stem cells ( mscs ) may improve symptoms in neurodegenerative diseases . under normal concentration of oxygen ( normoxic ) , human mscs
epo administration in normoxic conditions results in expression of tubulin beta-3 and induces cells with neuronal morphology ( 34 ) .
the relevance of tubulin protein and huntington s disease is also identified ( 35 ) .
it was reported that co- administration of epo and paclitaxel can inhibit neuronal axonal damage due to the lower level of detyrosinated tubulin ( 36 ) .
considering the role of tubulin in neural cell death , neuronal structure and mitosis , the interaction of epo and beta tubulin in neurons could be concluded .
epo is a promising therapeutic drug for the treatment of neurological diseases , in this study we successfully used ip and proteomics techniques for elucidation of the target proteins involved in epo - induced neuroprotection for potentially powerful therapeutical tool .
identified proteins may help us to have a better understanding about the mechanism of protective effects of epo in the brain .
| objective(s):erythropoietin ( epo ) , is a 34kda glycoprotein hormone , which belongs to type 1 cytokine superfamily .
epo involves in erythrocyte maturation through inhibition of apoptosis in erythroid cells . besides its main function
, protective effects of epo in heart and brain tissues have been reported .
epo has a critical role in development , growth , and homeostasis of brain .
furthermore epo has great potential in the recovery of different brain diseases which are still under studying . in this research ,
epo binding pattern to brain proteins in animal model was studied.materials and methods : epo antibody was covalently crosslinked to protein a / g agarose . in order to interact between epo and its target in brain ,
about 5 g epo added to brain homogenates(500ul of 1 mg / ml ) and incubate at 4 c for 30 min .
brain tissue lysate were added to agarose beads , after isolation of target proteins(epo - protein ) both one and two - dimensional gel electrophoresis were performed .
proteins were identified utilizing maldi - tof / tof and mascot software.results:this research showed that epo could physically interact with eightproteins including tubulin beta , actin cytoplasmic 2 , t - complex protein 1 , tpr and ankyrin repeat - containing protein 1 , centromere - associated protein e , kinesin - like protein kif7 , growth arrest - specific protein 2 and pleckstrin homology - like domain family b member 2.conclusion:since epo is a promising therapeutic drug for the treatment of neurological diseases , identified proteins may help us to have a better understanding about the mechanism of protective effects of epo in the brain .
our data needs to be validated by complementary bioassays . |
malaria , a disease caused by a protozoan pathogen of the genus plasmodium , remains one of the most devastating human diseases , causing as many as 250 million new cases and nearly one million deaths annually worldwide .
this disease is especially worrisome in africa , where one out of every five infant deaths is related to its effects .
although malaria can be caused by mainly four species of the genus , plasmodium falciparum is responsible for the most severe and deadly form of the disease and is to blame for 90% of malaria - related deaths occurring in african children below the age of five . at present , the available drugs used to fight the disease are ( a ) aryl aminoalcohol compounds , such as quinine , ( b ) antifolates - dihydrofolate reductase inhibitors , such as pyrimethamine [ 4 , 5 ] , or ( c ) artemisinin derivatives . therapeutic treatment has , however , encountered serious obstacles , as this parasite has developed at least partial resistance to nearly every antimalarial regimen introduced to date , including the first - line drugs chloroquine and sulfadoxine - pyrimethamine . as
single - drug treatments are no longer adequate , combinations of two or more drugs will probably offer improved efficacy and reduced risk of emergence of drug - resistant parasites .
current examples of drug combinations are artemisinin - amodiaquine and artemether - lumefantrine ( coartem ) .
the lack of an effective prophylactic vaccine and the generation and dissemination of drug - resistant strains clearly indicate the need for the development of new therapeutic approaches and identification of novel targets .
p. falciparum , a malarial parasite of the phylum apicomplexa , has been found to contain an apicoplast , an organelle that originally arose from a cyanobacterium through a secondary endosymbiotic process , and , thus , possesses four membranes . during these endosymbiotic processes , resultant of millions of years of adaptive selection ,
the endosymbiont and host were integrated in a relationship involving gene losses and gene transfers , resulting in a reduction of the apicoplast genome to a bare minimum .
the majority of the apicoplast proteins are encoded by genes in the nuclear genome of the host , and the proteins that reside in the apicoplast and are encoded by the nucleus are posttranslationally targeted to the apicoplast organelle via the secretory pathway , owing to the existence of a bipartite n - terminal leader sequence .
in fact , the apicoplast is indispensable for the malarial parasite , since several vital metabolic processes for the parasite occur in this organelle . among these processes
fatty acid biosynthesis is critical for the parasite development as fatty acids are the major components of cell membranes .
moreover , fatty acids are important source of energy , play a key role in signal transduction as well as in protein acylation , and are needed for the growth , differentiation , and homeostasis in p. falciparum .
it is also known that lipid biosynthesis is highly increased during the erythrocytic phases , when the parasite grows and divides extremely fast .
as the parasite invades its host , it attempts to protect itself by creating a so - called parasitophorous vacuole , in part as a protection from the host 's immune system . in this process , the parasite needs to produce its own fatty acids de novo so as to form and expand its membrane .
the enzymes involved in the biosynthesis of fatty acids are organized in two distinct ways within living systems .
fungi , mammals , and some mycobacteria accomplish fatty acid synthesis through the action of multifunctional proteins , each reaction being catalysed by a distinct region ( domain ) of these single - polypeptide proteins .
these enzymes are classified as type i fatty acid synthases ( fas i ) and are also described as the associative type of fatty acid synthases , as successive steps in the fatty acid synthetic reaction occur at specific domains [ 15 , 16 ] .
in contrast , plants and many bacteria use the type ii or dissociated fatty acid synthases ( fas ii ) , whose enzymes are best characterized in escherichia coli .
each step of the fas ii system is catalysed by a specific enzyme encoded by a separate gene . since the fas ii is absent in humans , it represents a great opportunity for therapeutic applications .
the fatty acid chain elongation module of fas ii consists of the cycling of the growing fatty acyl moiety through four key enzymes .
elongation is initiated by -ketoacyl - acp synthase i / ii ( fabb / fabf ) enzyme , which catalyses the condensation of the growing fatty acyl acp with malonyl - acp to yield a -ketoacyl - acp with an additional two - carbon unit .
the second step is the reduction of -ketoacyl - acp to -hydroxyacyl - acp catalysed by the -ketoacyl - acp reductase ( fabg ) enzyme .
water is then removed by -hydroxydecanoyl - acp dehydratase / isomerase ( faba ) and/or -hydroxyacyl - acp dehydratase ( fabz ) to produce 2-trans - enoyl - acp .
the final step in the elongation process is the nadh - dependent reduction of the double bond to produce fatty acyl acp , which is catalysed by fabi , the only enoyl - acp reductase ( enr ) enzyme in plasmodium ( figure 1 ) .
successive elongation cycles typically lead to the formation of decanoic ( c-10 ) , lauric ( c-12 ) , and myristic ( c-14 ) acids as the major fatty acyl acps in plasmodium , whereas palmitic ( c-16 ) acid is the most predominant fatty acid synthesized in humans .
enr from mycobacterium tuberculosis ( mtinha ) was first identified as being the primary target for isoniazid ( inh ; figure 2(a ) ) , the most prescribed drug to treat active tuberculosis .
the enzyme is an nadh - dependent enoyl - acp reductase specific for long - chain enoyl thioester substrates .
mtinha is a member of the mycobacterial fas ii , which elongates acyl fatty acid precursors of mycolic acids .
inh is a prodrug that is activated by the de mycobacterial katg - encoded catalase - peroxidase enzyme in the presence of manganese ions , nad ( h ) and oxygen [ 19 , 20 ] .
approximately 50% of m. tuberculosis inh - resistant clinical isolates harbor deletions of , or missense mutations in , the katg gene [ 21 , 22 ] .
the katg - produced acytylpyridine fragment of inh is covalently attached to the c4 position of nadh and forms a binary complex with the wild - type mtinha .
this isonicotinoyl - nad adduct has been characterized spectroscopically and shown to bind to the enzyme with a dissociation constant value lower than 0.4 nm .
the isonicotinoyl - nad adduct has been shown to be a slow , tight - binding competitive inhibitor of wild - type mtinha .
the initial rapidly reversible weak binding ( ki = 16 nm ) is followed by a slow isomerization , leading to a tighter enzyme - inhibitor complex with an overall dissociation constant ( ki * ) value of 0.75 nm .
the mechanism of action of inh requires its conversion , by the mycobacterial katg , into a number of electrophilic intermediates .
the formation of the adduct has been proposed to be formed through the addition of either an isonicotinic acyl anion to nad or an isonicotinic acyl radical to an nad free radical .
rapid freeze - quench electron paramagnetic resonance spectroscopy experiments have shown that hydrogen atom abstractions may be initiated by a katg tyrosil radical .
furthermore , it has been shown that the yield of isonicotinoyl - nad adduct is about the same after oxidation of inh by katg or mn .
accordingly , oxidation by mn - pyrophosphate has been proposed as an alternative method for nonenzymatic inh activation for simple chemical synthesis of various inh derivatives that mimic the isonicotinoyl - nad adduct .
based on the activation mechanism proposed for inh , via an electron transfer reaction , an alternative self - activation inner - sphere electron transfer reaction route was proposed for designing new drugs for the treatment of inh - resistant and inh - sensitive tuberculosis .
these drugs would be activated by electron transfer reactions before interacting with their cellular targets , and a redox reversible metal complex coordinated to the prodrug was employed as a prototype .
accordingly , we have recently demonstrated that pentacyano(isoniazid)ferrateii or [ fe(cn)5(inh ) ] ( figure 2(b ) ) inhibits the activity of both wild - type and i21v mutant ( inh - resistant ) mtinha enzymes .
the in vitro kinetics of inactivation indicates that this process requires no activation by katg , no need for the presence of nadh , and is also effective against inh - resistant inha mutants .
an mic value of 0.2 g ml for this inorganic complex was determined by the radiometric bactec afb system for the m. tuberculosis h37rv strain , and toxicity assays in hl60 leukemia and mcs-7 breast cancer cells yielded an ic50 value greater than 25 g ml , thereby indicating a good selectivity index ( si = ic50/mic > 125 ; the tuberculosis antimicrobial acquisition and coordinating facility of usa suggests that in order to a compound move forward through screening programs , si should be larger than 10 ) .
more recently , we have shown that the [ fe(cn)5(inh ) ] complex is a slow - onset inhibitor of mtinha enzyme activity , with a true overall dissociation constant value of 70 nm . in this mechanism of action an initial enzyme - inhibitor complex is rapidly formed , which then undergoes a slow isomerization reaction of an enzyme - inhibitor binary complex , where the inhibitor is more tightly bound to enzymes .
the weakness in the use of classical enzyme inhibitors as drugs for clinical conditions is that inhibition results in the upstream accumulation of the substrate for the enzyme , which may overcome the inhibition .
by contrast , the build - up of substrate can not have any effect on the isomerization of the enzyme - inhibitor complex typical of the slow - onset mechanism and hence reversal of the inhibition .
the structural similarity between mtinha and pfenr stimulated us to test the [ fe(cn)5(inh ) ] against the malarial enzyme .
in addition , we propose an enzyme mechanism for pfenr based on steady - state kinetics , pre - steady - state kinetics , and fluorescence spectroscopy data .
crotonoyl - coa , nadh , and butyryl - coa were from sigma ( steinheim , germany ) .
, new jersey , usa . the [ fe(cn)5(inh ) ] compound was a kind gift of dr .
sousa from universidade federal do cear , departamento de qumica orgnica e inorgnica , fortaleza , ce , brazil .
the steady - state activity assays were carried out in a uv-2550 uv - visible spectrophotometer , and fluorescence binding experiments were carried out in an rf-5301pc spectrophotometer , both from shimadzu ( kyoto , japan ) .
all enzyme activity assays were performed under initial rate conditions at 25c in 100 mm sodium phosphate buffer ph 7.5 in 500 l total reaction volumes , and each individual initial rate datum was the average of duplicate or triplicate measurements .
crotonoyl - coa , nadh , butyryl - coa , [ fe(cn)5(inh ) ] , and the enzyme were dissolved in 100 mm sodium phosphate buffer ph 7.5 .
the reactions were monitored by the decrease in absorbance at 340 nm due to the conversion of nadh ( 340 = 6220 m cm ) to nad . in order to determine the true steady - state kinetic parameters and initial velocity patterns ,
pfenr activity was measured in the presence of varying concentrations of crotonoyl - coa ( 10240 m ) and several fixed - varied nadh concentrations ( 20200 m ) , and reactions were initiated by the addition of 0.12 m of pfenr into the reaction mixture .
fluorescence titration was performed to assess the dissociation constant at equilibrium for binary complexes formed between pfenr and either substrate(s ) or product(s ) at 25c .
crotonoyl - coa , nadh , butyryl - coa , and pfenr were dissolved in 100 mm sodium phosphate buffer ph 7.5 .
fluorescence titration with crotonoyl - coa was carried out by making microliter additions of 0.5 mm and 5 mm crotonoyl - coa stock solutions ( 0.50425.97 m crotonoyl - coa final concentrations ) to 1 ml of 3 m pfenr , keeping the dilution to a maximum of 10.1% .
measurements of changes in intrinsic protein fluorescence of pfenr upon crotonoyl - coa binding employed excitation wavelength at 305 nm , and the emission wavelengths ranged from 325 to 370 nm ( maximum pfenr em = 348 nm ) ; the slits for excitation and emission wavelengths were , respectively , 15 and 5 nm .
fluorescence titration with nadh was carried out by making microliter additions of 0.5 mm , 1 mm , and 5 mm nadh stock solutions ( 0.50119.21 m nadh final concentrations ) to 1 ml of 3 m pfenr , keeping the dilution to a maximum of 4.1%
. measurements of changes in nadh fluorescence upon binding to pfenr employed excitation wavelength at 360 nm , and the emission wavelengths ranged from 360 to 560 nm ( maximum em = 460 nm ) ; the slits for excitation and emission wavelengths were , respectively , 1.5 and 5 nm .
fluorescence titration with butyryl - coa was carried out by making microliter additions of 0.5 mm , 2.5 mm , 5 mm , and 10 mm butyryl - coa stock solutions ( 0.50550.85 m butyryl - coa final concentrations ) to 1 ml of 3 m pfenr , keeping the dilution to a maximum of 8.4% .
measurements of changes in intrinsic protein fluorescence upon butyryl - coa binding to pfenr employed excitation wavelength at 300 nm , and the emission wavelengths ranged from 320 to 400 nm ( maximum pfenr em = 337 nm ) ; the slits for excitation and emission wavelengths were , respectively , 5 and 10 nm . control experiments were employed to both determine the maximum ligand concentrations to be used with no inner filter effect and determine dilution effects on protein fluorescence .
no fluorescence titration with nad could be performed due to a large inner filter on protein fluorescence .
pre - steady - state kinetic measurements of nadhnad conversion by pfenr were carried out to confirm the proposed kinetic mechanism and assess whether the apparent rate constant for this conversion contributes to the rate - limiting step . the decrease in absorbance upon nadh conversion to nad
was monitored at 340 nm ( 1 mm slit width = 4.65 nm spectral band ) , at 25c , using a split time base ( 0.22 s ; 200 data points for each time base ) .
the experimental conditions were 3.14 m pfenr and 200 m nadh in 100 mm sodium phosphate buffer ph 7.5 ( mixing chamber concentrations ) .
fe(cn)5(inh ) ] time - dependent inhibition of pfenr was performed since it had been observed in mtinha .
the enzyme was preincubated with the inhibitor for 15 min ( 2.77 : 2455 m enzyme : inhibitor solution ) , and the activity was monitored by adding 0.12 : 54 m enzyme : inhibitor mixture aliquots , with different times of exposure , to the reaction mixtures which contained crotonoyl - coa and nadh concentrations close to their km values .
typical slow - binding experiments were carried out in the presence of various [ fe(cn)5(inh ) ] concentrations as an attempt to elucidate the kinetic mechanism of the time - dependent inhibition observed for pfenr . in the case of slow - binding inhibitors ,
the binding , which is comprised by the establishment of the equilibrium between enzyme , inhibitor , and enzyme - inhibitor complexes , occurs slowly in a time scale of seconds to minutes .
these experiments were conducted under conditions in which [ fe(cn)5(inh ) ] concentrations were significantly larger than enzyme concentration , and measurements were made within a time range over which there was no significant substrate depletion .
the assay mixtures contained 80 m crotonoyl - coa , 120 m nadh , 2% ( vol / vol ) glycerol , 49.7 g ml bsa , and 035 m [ fe(cn)5(inh ) ] .
the reactions were initiated by adding enzyme ( 0.69 nm ) to the assay mixture and were monitored by following the decrease in nadh absorbance at 340 nm for 21600 s ( 6 hours ) .
enzyme activity was stabilized by the addition of glycerol and bsa to the reaction mixture as described for mtinha [ 25 , 31 ] . the two - step mechanism observed for mtinha inhibition by [ fe(cn)5(inh ) ] was investigated for pfenr .
the latter was preincubated in the presence of various concentrations of [ fe(cn)5(inh ) ] , and the decrease of pfenr activity was assessed by withdrawing aliquots from the preincubation mixtures at appropriate intervals , followed by rapid measurement of initial velocity .
reactions of time - dependent inactivation of pfenr were carried out in mixtures containing 12 m pfenr and various concentrations of inhibitor .
control experiments were performed with no inhibitor . at various times , the residual enzyme activity was determined by diluting an aliquot ( 100-fold ) of the reaction mixture into the assay solution , which contained 40 m crotonoyl - coa and 50 m nadh . the kinetic parameter values and their respective standard errors were obtained by fitting the data to the appropriate equations by using the nonlinear regression function of sigmaplot 2004 ( spss , inc . ) .
apparently parallel family of lines of initial velocity data were tentatively fitted to ( 1 ) , which describes a double displacement mechanism . for ( 1 )
, v is the measured reaction velocity , v is the maximal velocity , a and b are the concentrations of the substrates ( nadh and crotonoyl - coa ) , and ka and kb are their respective michaelis constants :
( 1)v = vabkab+kba+ab .
data from equilibrium fluorescence spectroscopy were fitted either to a hyperbolic function ( ( 2 ) ; ) or to the hill equation ( ( 3 ) ; ) . for ( 2 ) and ( 3 )
, f is the observed fluorescence signal , fmax is the maximal fluorescence , f / fmax ratio represents the degree of saturation , a is the concentration of the ligand ( either nadh or butyryl - coa ) , n represents the total number of binding sites , kd is the overall dissociation constant , and k represents a mean dissociation constant for enzyme : ligand binary complex formation , which is comprised of interaction factors and the intrinsic dissociation constant [ 36 , 37 ] :
( 2)ffmax = akd+a ,
( 3)ffmax = ank+an .
the equilibrium fluorescence data representing the fractional saturation ( 0.100.90 ) for the homotetrameric form of pfenr were fitted to ( 4 ) ( the hill plot ) , in which y is the fraction of substrate binding sites occupied by the substrate and h is the hill coefficient :
( 4)log ( y1y)=hlog alog k.
the pre - steady - state time course of nadhnad conversion was fitted to ( 5 ) for a single exponential decay , in which a is the absorbance at time t , a0 is the absorbance at time zero , and k is the apparent first - order rate constant for product formation : apparent pseudofirst - order rate constants were calculated by fitting the progress curves for time - dependent inactivation of pfenr to integrated equation ( 5 ) for slow - binding inhibition [ 25 , 32 , 39 ]
. for ( 6 ) , at and a0 are the 340-nm absorbances at time t and time 0 , v0 , vs , and kobs represent , respectively , the initial velocity , the final steady - state velocity , and the apparent first - order rate constant for the establishment of the equilibrium between ei and ei * complexes ( the observed rate constant for formation of ei * or the apparent isomerization rate constant ) :
( 6)at = vst+(v0vs)(1ekobst)kobs .
an estimate of the apparent dissociation constant ( ki ) was obtained by fitting the variation of initial rate ( v0 ) with inhibitor concentration ( i ) to ( 7 ) for competitive inhibition , in which v0max is the initial velocity in the absence of inhibitor :
( 7)v0=v0max 1+[i]/kiapp
.
a true value for the apparent dissociation constant ki for the initial enzyme - inhibitor binary complex ( ei ) can be calculated by ( 8) , in which knadh and kcrotonoyl - coa are the values for the michaelis - menten constants and [ nadh ] and [ crotonoyl - coa ] are the concentration values used in the slow - onset inhibition assays :
( 8)ki = kiapp1+[nadh]/knadh+[crotonoyl - coa]/kcrotonoyl - coa .
the data from two - step mechanism studies of pfenr inhibition by [ fe(cn)5(inh ) ] were fitted to ( 9 ) , in which ki is the dissociation constant for the initial enzyme - inhibitor binary complex ( ei ) , k5 is the forward isomerization rate constant for conversion of ei to ei * , k6 is the reverse isomerization rate constant ( conversion of ei * to ei ) , i is the concentration of inhibitor ( [ fe(cn)5(inh ) ] ) , and kinact is the apparent first - order constant for enzyme inhibition :
( 9)kinact = k6+k5iki+i .
initial velocity patterns were determined using either crotonoyl - coa or nadh as the variable substrate to distinguish between a sequential and a double displacement ( ping - pong ) mechanism , providing information regarding the order of substrate binding .
double - reciprocal plot analysis revealed a parallel line pattern for both substrates ( figure 3 ) , which is consistent with a double displacement mechanism . in this mechanism ,
a catalytic event can occur when only one of the two substrates is bound to the enzyme .
a catalytic event occurs after the binding of substrate a to the enzyme ( e ) , but prior to the binding of substrate b. after the first catalytic event , a portion of substrate a remains in the active site of the enzyme , whereas the other portion denoted as product p dissociates .
the first catalytic event produces a different stable enzyme form ( f ) to which substrate b can bind and proceed with catalysis to generate product q and regenerate free enzyme ( e ) .
the data were tentatively fitted to ( 1 ) , yielding the following values for the true steady - state kinetic constants : vmax = 5.8 ( 0.3 ) u mg , kcat = 6.2 ( 0.4 ) s , knadh = 85 ( 9 ) m , kcrotonoyl - coa = 63 ( 6 ) m , kcat / knadh = 7.3 ( 0.9 ) 10 m s , and kcat / kcrotonoyl - coa = 9.8 ( 1.1 ) 10 m s. there are , however , some conditions where lines that seem parallel really are not . for an ordered bi bi system in which the dissociation constant for substrate a ( kia ) is very much smaller than the michaelis - menten constant for a ( ka or kma ) , the family of reciprocal plots intersect far to the left of the 1/v axis and far below the 1/[a ] axis .
in addition , seemingly parallel initial velocity patterns can also occur in rapid equilibrium random systems where the binding of one substrate strongly inhibits the binding of the other .
equilibrium binary complex formation experiments were employed to confirm , or refute , the proposed enzyme mechanism .
there was an enhancement in nucleotide fluorescence upon nadh binding to pfenr , whereas butyryl - coa binding to pfenr resulted in intrinsic protein fluorescence quench .
titration of pfenr with nadh was hyperbolic ( figure 4(a ) ) , and the data were fitted to ( 3 ) yielding a value of 398 ( 53 ) m for the overall dissociation constant ( kd ) of nadh .
enzyme titration with butyryl - coa was sigmoidal ( figure 4(b ) ) , and the data were best fitted to ( 4 ) yielding a value for k for butyryl - coa of 1151 ( 638 ) m .
the value for k represents a mean dissociation constant for pfenr : butyryl - coa binary complex formation , which is comprised of interaction factors and the intrinsic dissociation constant [ 36 , 37 ] .
a hill plot of the data ( figure 4(c ) ) fitted to ( 4 ) yielded a value larger than one for h ( 1.6 0.1 ) .
the latter indicates moderate positive homotropic cooperativity on butyryl - coa binding to tetrameric pfenr , which is further supported by the upward - curved double - reciprocal plot ( figure 4(c)inset ) .
no intrinsic protein fluorescence change upon addition of crotonoyl - coa to pfenr could be observed , suggesting that this compound can not bind to free pfenr enzyme .
protein fluorescence titration with nad could not be performed due to large inner filter effect .
steady - state kinetics data suggested that pfenr follows a double displacement ( ping - pong ) mechanism .
fluorescence spectroscopy data showed that nadh binds to free enzyme whereas it appears that crotonoyl - coa can not .
although butyryl - coa binding to free enzyme resulted in protein fluorescence quench , it is not certain whether crotonoyl - coa can not bind to free enzyme or its binding results in no change in protein fluorescence .
hence , the ordered bi bi and rapid equilibrium random mechanisms could not be ruled out . to try to address this uncertainty ,
pre - steady - state kinetics measurements were carried out to show whether or not there is hydride transfer in the absence of crotonoyl - coa as expected for the double displacement mechanism with nadh binding first . moreover , determination of a value for the apparent first - order rate constant for the course of nadhnad conversion could also indicate whether or not this step is rate limiting .
it should be pointed out that nadh concentration was in excess as compared to pfenr concentration , so that any burst in product formation could be reliably assessed [ 41 , 42 ] .
fitting the pre - steady - state trace showing a single exponential decay upon nadhnad conversion ( figure 5 ) to ( 5 ) yielded a value of 1100 ( 29 ) s for the apparent first - order rate constant for product formation ( 1 cm pathlength ) .
the nadhnad conversion step has thus no contribution to rate - limiting step(s ) as its value is much larger than the catalytic rate constant value .
there was an apparent accumulation of observable product ( nad ) on the enzyme active sites when high concentration of pfenr ( 3.14 m ) was mixed with nadh ( 200 m ) .
taken together , steady - state kinetics , pre - steady - state kinetics , and equilibrium binding data are consistent with a double displacement mechanism for pfenr ( figure 6 ) . in this mechanism ,
a catalytic event occurs after the binding of nadh to free enzyme ( e ) , generating and releasing nad into solution prior to the binding of crotonoyl - coa to a modified form of the enzyme ( f ) , followed by formation of butyryl - coa and regeneration of free enzyme ( e ) ( figure 6 ) .
the enzyme mechanism has been shown to be ordered sequential with enoyl - acp substrate binding first for staphylococcus aureus enoyl reductase and ordered sequential with nadh binding first for fabv - encoded enoyl reductase from burkholderia mallei . although uncommon for reductases / dehydrogenases , double displacement mechanisms have been described for enterobacter cloacae nitroreductase , salmonella typhimurium histidinol dehydrogenase , bovine liver mitochondrial dihydroorotate dehydrogenase , and mammalian pyruvate dehydrogenase complex .
of course , no mechanism is ever proved ; at best one can say that given the current data the likely mechanism for pfenr is double displacement .
further efforts are needed to provide support for either the double displacement mechanism or any other that may give a better description of the experimental results .
based on substrate binding to immobilized pfenr by surface plasmon resonance ( spr ) results , it has been proposed that pfenr enzyme mechanism is random order of substrate addition .
although there have been reports on determination of apparent steady - state kinetics parameters for pfenr [ 38 , 50 , 51 ] , to the best of our knowledge , no data have been presented for determination of true steady - state kinetic parameters on which to base a proposal for pfenr enzyme mechanism in solution . to determine the true steady - state kinetic parameters and initial velocity patterns for distinguishing mechanisms
, enzyme activity measurements have to be carried out in the presence of varying concentrations of one substrate and several fixed - varied concentrations of the other substrate .
interestingly , the kd value of 398 m for nadh is approximately 8-fold larger than a previously reported value ( 51.6 m ) by spr .
these results are somewhat puzzling as equilibrium constants determined by spr usually match well the values obtained by solution techniques ( fluorescence spectroscopy , isothermal titration calorimetry ) .
in addition , a kd value of 62.5 m for crotonoyl - coa has been determined by spr measurements , whereas no change in intrinsic pfenr protein fluorescence could be detected upon addition of crotonoyl - coa to free enzyme . incidentally ,
the true steady - state value for kcat ( 6.2 s ) reported here is approximately 4-fold larger as compared to an apparent value ( 1.62 s ) described elsewhere .
we have previously shown that [ fe(cn)5(inh ) ] compound ( figure 2(b ) ) is a slow - onset inhibitor of wild - type and drug - resistant mtinhas [ 30 , 31 , 52 ] .
we then anticipated that [ fe(cn)5(inh ) ] may also be an effective inhibitor of pfenr enzyme activity .
prior to embarking on a full analysis of the mode of inhibition of pfenr activity by [ fe(cn)5(inh ) ] compound , the enzyme was preincubated with inhibitor , and aliquots were withdrawn at different times to measure the remaining residual activity .
pfenr inhibition by [ fe(cn)5(inh ) ] was time dependent ( data not shown ) .
thereby , pfenr slow - binding experiments were carried out in the presence of various concentrations of [ fe(cn)5(inh ) ] to try to elucidate the mechanism of the observed time - dependent enzyme inhibition .
the progress curves for enzyme activity indicate that [ fe(cn)5(inh ) ] is a slow - onset inhibitor of pfenr ( figure 7 ) .
three basic slow - onset inhibition mechanisms have been described by morrison and walsh ( figure 8 ; ) . in mechanism
a ( figure 8) , it is assumed that the time - dependent enzyme inactivation observed is resultant of a slow bimolecular enzyme - inhibitor interaction , because the inhibitor concentration is low and/or due to barriers that the inhibitor encounters in its binding at the enzyme 's active site . in mechanism b ( figure 8) , it is assumed that there is an initial rapid enzyme - inhibitor interaction to form ei , which then undergoes a slow isomerization ( conformational change ) to ei * . for inhibitors that conform to this mechanism ,
the overall dissociation constant ( ki * ) must be lower than the dissociation constant for initially formed complex ei ( ki ) , a condition that can be satisfied only when k6
< k5 ( ki * = kik6/(k5 + k6 ) and when k5 and k6 are slower than all other steps . according to mechanism b , in the presence of a fixed concentration of substrate ( a ) , the value of kobs increases hyperbolically as a function of inhibitor concentration with limiting values of k6 and k5 + k6 at , respectively , zero and infinite concentrations of inhibitor .
as described in section 2 , kobs in ( 6 ) represents the apparent first - order rate constant for the establishment of the equilibrium between ei and ei * complexes ( the observed rate constant for formation of ei * or the apparent isomerization rate constant ) .
a third mechanism ( mechanism c ) has also been described to account for slow - binding inhibition of enzyme - catalysed reactions ( figure 8) .
this mechanism involves a slow initial isomerization of two forms of free enzyme in solution ( e and e * ) , followed by a rapid binding of inhibitor to form e*i complex .
the kf and kr stand , respectively , for the forward and reverse rate constants for the conversion of free enzyme ( e ) to a form of free enzyme ( e * ) to which the inhibitor binds .
however , fitting the kobs values as a function of [ fe(cn)5(inh ) ] inhibitor concentration yielded a plot that did not allow to draw solid conclusions on the mechanism . on the other hand , fitting the initial rate values ( v0 ) as a function of [ fe(cn)5(inh ) ] inhibitor concentration to ( 7 ) yielded a value of 40 ( 11 ) m for ki .
a true value of 11 ( 3 ) m for the apparent dissociation constant ki for the initial enzyme - inhibitor binary complex ( ei ) could thus be calculated by employing ( 8) .
the two - step mechanism of pfenr inhibition by [ fe(cn)5(inh ) ] was assessed by an experiment in which the enzyme was preincubated in the presence of various concentrations of inhibitor and the decrease of pfenr activity was assessed by withdrawing aliquots of enzyme from preincubation mixtures at appropriate intervals , followed by rapid measurement of initial velocity .
these results were analyzed in terms of a two - step inhibition mechanism in which the initial rapid binding of the inhibitor to enzyme is followed by a second slow step that results in the final enzyme - inhibitor complex . since the value of ki is much larger than ki * , the inhibitor concentration in the assay solution is lower than the ki value ( [ i ] < ki ) , and there is a large excess of the competitive substrates nadh and crotonoyl - coa over [ fe(cn)5(inh ) ] , the steady - state concentration of ei in the assay solution is kinetically insignificant .
the apparent first - order rate constants for pfenr inactivation ( kinact ) could be calculated from a plot of residual enzyme activity against time for each inhibitor concentration . a plot of kinact values versus inhibitor concentration shows that the kinact values increase hyperbolically as a function of inhibitor concentration ( figure 9 ) , which is consistent with mechanism b ( figure 8) .
these data were thus fitted to ( 9 ) , which describes the slow - binding inhibition for mechanism b , yielding values of 58 ( 12 ) m for ki , 53 ( 2 ) s for k5 , and 4 ( 2 ) s for k6 .
a value of 4 m can thus be calculated for the overall inhibition constant ( ki * = kik6/(k5 + k6 ) .
it should be pointed out that an independent estimate for the ki * value could be obtained by plotting the final steady - state velocity ( vs ) as a function of [ fe(cn)5(inh ) ] inhibitor concentration and fitting the data to ( 7 ) for competitive inhibition .
however , the data were not consistent as the experimental data appear to depart somewhat from the values predicted by fitting the data to ( 6 ) . the two - step mechanism followed by [ fe(cn)5(inh ) ]
the enzyme isomerization could involve , for instance , a protein conformational change or alteration of water structure at the active site .
for the inhibitor , the change could be hydration / dehydration of a carbonyl , change in ionization state , or a conformational change .
the weakness in the use of classical enzyme inhibitors as drugs for clinical conditions is that inhibition results in the upstream accumulation of the substrate for the enzyme , which may overcome the inhibition .
by contrast , the build - up of substrate can not have any effect on the isomerization of enzyme - inhibitor complex typical of the slow - onset mechanism and hence reversal of the inhibition .
moreover , the low half - time value for the limiting step for inhibitor dissociation from the binary complex is a desirable feature since it may be expected to enhance inhibitor 's effectiveness .
it has recently been proposed by scientists at glaxosmithkline ( usa ) that measurement of the dissociative half - life is a crucial metric of compound optimization and could be a key indicator of in vivo duration of efficacy and target selectivity .
it has recently been shown that [ fe(cn)5(inh ) ] compound has a promising profile in toxicological assays and that it is amenable to large - scale production .
these features indicate that [ fe(cn)5(inh ) ] compound is a viable candidate for further studies ( including in vivo efficacy ) and that it may represent a prototype for the development of chemotherapeutic agents to treat malaria . | malaria continues to be a major cause of children 's morbidity and mortality worldwide , causing nearly one million deaths annually .
the human malaria parasite , plasmodium falciparum , synthesizes fatty acids employing the type ii fatty acid biosynthesis system ( fas ii ) , unlike humans that rely on the type i ( fas i ) pathway .
the fas ii system elongates acyl fatty acid precursors of the cell membrane in plasmodium .
enoyl reductase ( enr ) enzyme is a member of the fas ii system .
here we present steady - state kinetics , pre - steady - state kinetics , and equilibrium fluorescence spectroscopy data that allowed proposal of p. falciparum enr ( pfenr ) enzyme mechanism .
moreover , building on previous results , the present study also evaluates the pfenr inhibition by the pentacyano(isoniazid)ferrateii compound .
this inorganic complex represents a new class of lead compounds for the development of antimalarial agents focused on the inhibition of pfenr . |
hepatitis b virus ( hbv ) and hepatitis c virus ( hcv ) infections cause morbidity and mortality in haemodialysis patients . prolonged vascular exposure and multiple blood transfusions
contaminated devices , equipments , and supplies , environmental surfaces , and attending personnel may also play a crucial role in the nosocomial transmission of these infections .
infections with hepatitis viruses in haemodialysis patients are further promoted by the significant immune status dysfunction developing due to irreversible renal compromise [ 13 ] .
furthermore , hepatitis viral infections in haemodialysis patients cause liver disease in renal failure patients undergoing replacement therapy .
they also pose a significant problem in the management of these cases as patients with renal failure can not clear the viruses effectively .
patients with coinfections with these viruses develop severe clinical presentations and resistance to interferon treatment .
there are very limited data available on the occurrence of such infections in haemodialysis patients from this part of the country .
the present study aimed to investigate the occurrence of hbv and hcv infections in haemodialysis patients and the risk factors associated with such infections .
this study was conducted as a retrospective case - control study involving the haemodialysis patients at a dialysis centre of a tertiary care hospital .
all the patients who underwent haemodialysis from january 2004 to june 2012 were included in the study .
patients receiving haemodialysis were considered as a case for the study if their serum tested positive for either hbv or hcv .
in contrast , the patients receiving haemodialysis were considered as a control if their serum tested negative for all the three viruses . for every case ,
patients ' medical records were reviewed to obtain details like age , gender , clinical diagnosis , and duration and frequency of dialysis , history of blood transfusions , and past surgeries and these details were recorded in a preformed questionnaire for all cases and controls .
further , the results of serological tests ( hbsag , anti - hcv antibodies ) , renal function tests ( serum urea and creatinine ) , and liver function tests were recorded for both groups .
duration of dialysis , frequency of dialysis , and the results of biochemical parameters were compared for infected cases and non - infected controls and were analyzed statistically using nonparametric tests and chi - square tests .
the present retrospective study was conducted for a period of 102 months , that is , from january 2004 to june 2012 .
a total of 180 patients were diagnosed to have chronic kidney disease and were on maintenance haemodialysis during the study period .
also , our dialysis unit caters to approximately 15 in - patients per month referred from other departments or hospitals for haemodialysis .
therefore , the total number of patients who received haemodialysis at our centre during the study period was 1710 . among these patients , 45/1710 cases ( 2.63% )
were found to be infected with either hbv or hcv and were included as cases for our study .
out of 45 cases studied , 26/1710 ( 1.52% ) tested positive for hbsag and 19/1710 ( 1.11% ) tested positive for anti - hcv antibodies .
out of 26 cases infected with hbv , 22 ( 84.6% ) were males and 4 ( 15.4% ) were females . among hcv positive cases , 18 ( 94.7% ) were males and 1 ( 5.2% ) was female . among the cases ,
dual infection with hbv and hcv was seen in two patients , 1 male and 1 female .
hbv infection was seen most commonly in the age group of 5060 yrs , whereas hcv was commonest in the age group of 3040 years .
mean duration of dialysis for infected cases was 4.8 years while that of the non - infected controls was 3.18 years .
there was a statistically significant ( p = 0.008 ) difference between the cases and controls with respect to duration of dialysis ( mann - whitney u test since mean was less than 2 standard deviations , non - parametric tests were used ) .
when comparing frequency of dialysis between cases and controls , as it was a categorical variable , chi - square test was applied and there was no statistically significant ( p value = 0.228 ) correlation of the frequency of dialysis between cases and controls . with respect to liver function tests , a significant elevation of ast ( aspartate aminotransferase ) and alt ( alanine transaminase ) values among infected cases ( p value = 0.001 ) was observed in this study .
most of the patients ( 80% ) on haemodialysis in our study were anaemic with haemoglobin concentration < 10 g% as shown in figure 1 .
the comparison of haemoglobin values among cases and controls is depicted in figure 2 . with respect to renal function tests in cases and controls ,
mean value of blood urea for cases was 99.85 and for controls was 103.1 , and mean value of creatinine was 22.3 for cases and 8.65 for controls , respectively .
no statistically significant difference was observed between both groups with respect to serum levels of urea ( p value = 0.6 ) , creatinine ( p value = 0.228 ) , and haemoglobin ( p value = 0.6 ) .
risk factors for acquiring hbv and hcv infections were studied for both groups . among the infected cases ,
multiple blood transfusions were seen only in 3 cases ( 2 positive for hbsag and 1 for hcv ) . among the three , one case with positive anti - hcv underwent multiple blood transfusions and in the remaining two cases ,
the history of undergoing surgeries was obtained in nine cases . among the 9 cases ,
hbv infection was seen in 4 , hcv in 4 , and dual infection was seen in one case .
twelve cases were diabetic , with hbv infection in 6 , hcv in 5 cases , and dual infection in 1 case ( figure 3 ) . with respect to similar risk factors in the control group , 14 control patients had diabetes and 4 of them had history of undergoing surgery in the past
. only 1 control patient had history of undergoing blood transfusion ( figure 3 ) .
patients diagnosed with chronic renal failure ( crf ) on maintenance haemodialysis pose a higher risk for acquiring hbv or hcv infections due to frequent use of blood and blood products and multiple invasive procedures performed in these patients .
the literature review points to the fact that viral hepatitis is a serious threat for haemodialysis patients as 1.9% of all deaths among this population were related to the consequence of viral hepatitis .
the results from our study demonstrate that the occurrence of hbv and hcv infections in haemodialysis patients is 1.52% and 1.11% , respectively , which is lower than the rates reported from different studies all over the world and india [ 58 ] .
an indian study has reported the occurrence of hbv in haemodialysis to vary from 3.4% to 42% , much higher than that seen in our study .
the lower rates of infection in our study might be due to decreased transfusion requirements owing to the availability of erythropoietin and better screening of blood and blood products for blood - borne infections .
introduction of vaccination for hbv , isolation of hepatitis b virus ( hbv ) positive patients , and regular surveillance for hbv infection at our centre could also have contributed for the lesser rates of infection .
occurrence of hcv infection was much less than hbv in our study which was in accordance with a study conducted in spain which reported lesser prevalence rates for hcv infection in haemodialysis ( hd ) patients .
another study showed a significant decline of hepatitis c infection among end - stage renal disease patients in central brazil , highlighting the importance of public health strategies such as screening for anti - hcv in blood banks and infection control measures for control and prevention of hepatitis c in the haemodialysis environment .
since both of these viruses share a common mode of transmission , we looked for the occurrence of coinfections among the cases studied . among the cases , dual infection with hbv and hcv was seen in two patients , 1 male and 1 female ( 2/45 = 4.4% ) .
study from the same centre on the occurrence of coinfections in the general population reported lower rates ( 1.68% ) of dual infections .
studies from other centres in india have reported a varying prevalence of coinfections with hbv and hiv ( 940% ) and for hcv and hiv ( 28% ) .
the higher occurrence in our study may be due to the study population being restricted to haemodialysis patients and also due to the lesser sample size in the current study .
another factor contributing to the higher rates is the enhanced risk of coinfections among chronic renal failure patients on haemodialysis due to multiple transfusions and invasive procedure performed in these patients .
however , dual infection with hbv and hcv in our study which was 4.4% , similar to the study conducted in hyderabad .
the present study highlights the duration of dialysis as an important risk factor for infection among haemodialysis patients .
this observation was in agreement with previous reports in palestine , moldavia , and other studies from different regions of the world [ 1517 ] .
duration of dialysis is an important risk factor for acquiring infections as it is related to nosocomial transmission and dissemination of the infections in the dialysis units . on comparison of ast and alt values among cases and controls ,
this can be explained on the basis of destruction of hepatocytes caused by the immune reaction of body responding to the hepatitis viruses leading to excessive release of the aminotransferases .
these findings were in accordance with the studies conducted in karachi and jenin district and gaza strip in palestine [ 1 , 18 ] .
in our study , when compared with the control group , 67% of the patients on haemodialysis with hbv / hcv were found to have higher hemoglobin levels .
this interesting finding was supported by few studies and was attributed to the increase in rbc counts after infection with hepatitis viruses and also to increased erythropoietin production after hepatic stimulation by chronic infection with hepatitis virus .
however , the exact cause for the same is not yet established [ 1922 ] .
a study from our centre brings to light that hbv and hcv infections , though less common , continue to remain as an important causes of infection in haemodialysis patients .
the risk of exposure increases with the number of dialysis sessions and is maximum in patients on maintenance haemodialysis .
interventions to reduce the occurrence of these infections are of utmost need to reduce the risk of long - term complications . | hepatitis b ( hbv ) and hepatitis c ( hcv ) viruses are the most important causes of chronic liver disease in patients with end stage renal disease on hemodialysis .
the prevalence of hepatitis infection among hemodialysis patients is high and varies between countries and between dialysis units within a single country .
this case - control study was undertaken to estimate the occurrence of hbv and hcv infections in patients undergoing hemodialysis in our tertiary care center .
all patients receving hemodialysis at our centre with hcv or hbv infection were included in the study .
the total number of patients admitted for hemodialysis during the study period was 1710 . among these , 26 patients were positive for hbv , 19 were positive for hcv , and 2 were positive for both hcv and hbv .
mean age of the infected cases in our study was 48.63 years .
mean duration of dialysis for infected cases was 4.8 years while that of the noninfected controls was 3.18 years .
the mean dialysis interval was twice a week .
interventions to reduce the occurrence of these infections are of utmost need to reduce the risk of long - term complications among hemodialysis patients . |
these are worsened by curettage and so a high index of suspicion is necessary for diagnosis .
only two cases of hysteroscopic findings in arteriovenous malformations have been reported in literature so far .
we present a case of acquired uterine arteriovenous malformation , which showed a characteristic pulsatile vascular mass on hysteroscopy .
a 36-year - old lady , p2l2 ( para 2 , living 2 ) underwent medical termination of pregnancy one month ago outside for failure of contraception .
she was admitted at our hospital with heavy bleeding . on admission , she was pale , pulse was 110/min , b.p .
was 100/70 mm hg and vaginal examination revealed a bulky soft uterus with open os and clots in the vagina .
a transvaginal ultrasound showed parenchymal inhomogeneity in the uterus in the anterior wall with several interspersed sonolucent spaces of varying size [ figure 1 ] .
her cbc showed a hb of 7 gm% , serum beta hcg was 50 iu / ml . a provisional diagnosis of uterine arteriovenous malformation or organized retained products of conception was made and hysteroscopy proceed was planned .
transvaginal ultrasound showing parenchymal inhomogeneity in the uterus in the anterior wall with several interspersed sonolucent spaces of varying size transvaginal ultrasound color doppler showing increased vascularity hysteroscopy revealed a clot in the uterine cavity .
there was a pulsatile bluish vascular mass in the fundus extending to anterior wall of uterus .
mr angiography confirmed an arteriovenous malformation at the fundus and anterior wall of uterus being fed by mainly by left uterine artery [ figure 4 ] .
patient was taken for percutaneous arterial embolization and bilateral uterine artery embolization was done with polyvinyl alcohol particles .
patient had cessation of bleeding after the embolization and she resumed menstrual cycle 5 weeks after the procedure with scanty menstrual flow .
transvaginal scan revealed reduced vascularity with embolic particles seen in uterine myometrium [ figure 5 ] .
hysteroscopy showing bluish vascular mass in anterior and right lateral wall of uterus mr angiography showing arteriovenous malformation at the fundus and anterior wall of uterus being fed by mainly by left uterine artery transvaginal scan revealed reduced vascularity with embolic particles seen in uterine myometrium six months later , patient continues to have cyclic menstrual bleeding with moderate flow .
acquired arteriovenous malformations are known to occur after uterine curettage , medical abortion , gestational trophoblastic disease , cesarean section and endometrial carcinoma .
they usually present with torrential bleeding requiring blood transfusion and this bleeding is worsened by uterine curettage .
the differential diagnosis is retained products of conception , polyps , adenomyosis , endometrial carcinoma and gestational trophoblastic disease .
abnormal uterine bleeding is an important cause of morbidity in the midlife and uterine arteriovenous malformations are a rare albeit important cause of such bleeding .
transvaginal sonography with color doppler may point to the diagnosis but the condition may be confused with retained products of conception . in our case ,
the serum beta hcg was 50 iu / ml , which was suggestive of retained products of conception or placental polyp .
hysteroscopic picture of arteriovenous malformation has been described in literature as a pulsatile vascular mass in the uterine cavity . in this case
there was a clot in the uterine cavity and typical vascular mass , which showed pulsations on reducing the intrauterine pressure .
recognition of this rare hysteroscopic picture is important , as arteriovenous malformations are being increasingly diagnosed and treated .
hysteroscopy if often the investigation of choice for abnormal uterine bleeding and arteriovenous malformations are a rare but potentially treatable cause of abnormal uterine bleeding .
transcatheter embolization of feeding vessels is an established treatment of arteriovenous malformations other being hysterectomy .
there are reports of failed angiographic embolization in which definitive treatment i.e. , hysterectomy was offered .
, have also been described . acquired arteriovenous malformations arise mostly after pregnancy or pregnancy related complications and they may coexist with conditions like retained placental tissue , retained products of conception and gestational trophoblastic disease . while the treatment of retained products of conception is curettage , in arteriovenous malformations it can lead to torrential hemorrhage .
hysteroscopy has an important potential role in the diagnosis of arteriovenous malformation in selected patients .
recognition of typical hysteroscopic picture can aid in diagnosis and the coexisting confounding problems like polyps , retained products of conception can also be tackled at the same sitting . however ,
sometimes even putting in a small hysteroscope can cause torrential hemorrhage in cases with a_v malformations and this should be kept in mind while tacking a suspected case .
transcatheter embolization of feeding vessels is a minimally invasive treatment for such lesions and affords an opportunity to manage these cases and salvage the uterus as well . | arteriovenous malformations are a rare but important cause of abnormal uterine bleeding in the midlife . acquired uterine
arteriovenous malformations are being increasingly diagnosed by transvaginal ultrasound , color doppler and magnetic resonance angiography .
we present a case where the suspected diagnosis was placental polyp or retained products of conception and hysteroscopy showed typical findings of uterine arteriovenous malformation , which was later , confirmed by magnetic resonance angiography .
the patient underwent arterial embolization and recovered satisfactorily with resolution of hemorrhage and resumption of normal menstrual cycles .
abnormal bleeding due to arteriovenous malformations is worsened by curettage and hence its recognition is important in the management of abnormal uterine bleeding in the midlife . |
stomach cancer is the fifth most common cancer in the world and approximately one million new cases were reported in 2012 .
the current global trend of stomach cancer incidence has shown a substantial decline since 1975 , when stomach cancer was the world s most common cancer .
although stomach cancer incidence rates have declined in east asian countries following the global trend , stomach cancer is still the second most common cancer in east asia , accounting for approximately 50% of all cases of stomach cancer worldwide . among the east asian countries , the republic of korea ( hereafter korea ) has the highest incidence of stomach cancer in the world , and the incidence of stomach cancer in china accounts for more than 40% of all new cases .
likewise , high stomach cancer incidence remains a major public health problem in japan despite efforts to create early detection screening programs .
in contrast , the us has one of the lowest incidence rates of stomach cancer ; stomach cancer is not one of the top 15 most common cancers .
based on recent reports , the stomach cancer incidence rate in korea was 11.7 times higher than that in the us .
the us is a heterogeneous country with many immigrants , and approximately 14.7 million americans are asian american , representing 4.8% of the total us population in 2010 .
the state of california has the largest asian population of any of the us states .
many asian americans live in los angeles , california . in particular , chinese americans comprise most of the asian population in california ( 4.2% of the total population ) followed by filipino , korean , and japanese people . owing to this racially diverse population
, the state of california makes special efforts to collect data on the cancer incidence rates of each specific ethnic group .
since stomach cancer incidence in the us is significantly lower than that in asia , comparing the cancer incidence rates of asian americans living in los angeles with those among native asians in their own respective countries might provide an important clue into the etiology of the disease .
the asian countries with a high - risk of stomach cancer , specifically korea , japan , china , and the philippines , were selected due to their geographical proximity and cultural similarities .
several studies have reported risk factors of stomach cancer , and some of the established risk factors include helicobacter pylori infection , smoking , and diet . among these three risk factors ,
diet habits change most dramatically among those who move from an asian country to a western country , a phenomenon referred to as
numerous studies have indicated the negative impacts of dietary acculturation such as obesity and diabetes . however , in the present study , we hypothesized that diet acculturation among asian americans might have a positive impact on stomach cancer incidence and that those immigrants may benefit from assimilating westernized foods into their diet , which might then contain fewer traditional asian foods that tend to be salted and/or pickled .
thus , the objective of the present study was to compare the incidences of stomach cancer among asian immigrants living in los angeles , california with those among native asians by sex and age group .
a comparative study evaluating countries with high and low incidences is necessary because it could provide valuable insight into the etiology and possible preventive methods that should be promoted to reduce stomach cancer incidence rates and ease the related public health burden .
cancer incidence in five continents ( ci5 ) is an international cancer incidence report generated by the international agency for research on cancer and the international association of cancer registries .
the international classification of disease ( icd , 10th revision ) code for stomach cancer , c16 , was used .
the ci5plus database provides race - specific , age - specific , sex - specific , and cancer site - specific estimates for 118 selected populations .
the data source for asian ethnic groups in the us was only available from the state of california for the city of los angeles .
the composition of asian populations in los angeles included 4.2% chinese , 3.3% filipino , 2.2% korean , and 1.0% of japanese people .
in addition , the rate per 100,000 individuals was used to compare the annual incidence rates of stomach cancer by 5-year interval age groups starting from 10 years of age .
the stomach cancer incidence rates for native asian countries were also collected from the ci5 dataset to allow for reliable data comparisons .
since the ci5 did not have data on entire countries , the data available from specific regions of each country were used to compare native asian groups .
the well - known cities of each country were selected : osaka for japan , shanghai for china , and manila for the philippines .
however , data from statistics korea were used for the data analysis on native koreans because no such data were available in the ci5 .
since the data from korea were standardized based on the entire korean population , the direct standardization method was used to convert these data into a segi world population dataset for consistency .
for males , the overall age - standardized cancer incidence rate for native koreans ( average 65.4 per 100,000 ) was the highest among all of the races ; however , this rate was the lowest among filipino americans ( average 5.9 per 100,000 ) ( figure 1 ) [ 11 , 12 ] . among the native groups , the incidence demonstrated a decreasing trend in japanese , chinese , filipino , and non - hispanic white males , whereas the incidence was constant in korean males . for all us immigrant ethnic populations , the overall stomach cancer incidence rates were lower than those among the native groups
the incidence rate of korean american males was one - third less than the incidence rates of native korean males .
the incidence rate of japanese american males and filipino american males was two - thirds less than the incidence rates of native japanese males and native filipinos males .
the incidence rate of chinese american males was three - quarters less than the incidence rates of native chinese males . among asian male immigrants ,
the incidence rates for korean americans , chinese americans , and filipino americans remained constant , but the incidence rate of japanese americans tended to decrease over time . among all females ,
the overall age - standardized cancer incidence rate among native koreans ( average 24.7 per 100,000 ) was the highest , but filipino americans had the lowest incidence rate ( average 2.7 per 100,000 ) . among the native groups , the incidence rates of japanese , chinese , filipino , and non - hispanicwhite females tended to decrease over time , yet the incidence rate among korean females remained constant .
for all ethnicities , the overall stomach cancer incidence rates of female immigrants were lower than those among the female native groups .
the incidence rate of korean american females was two - thirds less than the incidence rates of native korean females .
the incidence rate of japanese american and chinese american females was one - half lower than the incidence rates of native japanese and native chinese females .
the incidence rate of filipino american females was one - third less than the incidence rates of native filipino females . among the asian female immigrant groups , the incidence rate among korean american , japanese american , and filipino american females remained constant , while the incidence rate among chinese american females decreased ( figure 2 ) . between males and females
the incidence rates of males were significantly higher than those for females across all groups .
the highest disparity was found between the incidence rates of native korean males and females ; native korean males had a 2.6 times higher incidence than their female counterparts did .
for both males and females in all age groups , the incidence of stomach cancer increased with age . among all native koreans ,
native japanese , native chinese , korean americans , and japanese americans , the incidence rates of stomach cancer increased steeply with age . among the different racial groups in los angeles ,
the incidence of stomach cancer among korean americans demonstrated the steepest increase , which is expected based on the rate of stomach cancer among native koreans .
from age 70 and older , the incidence tended to decrease for all groups , except for all age groups in the native japanese population where the incidence rates continued to increase ( figures 3 and 4 ) .
for males , the overall age - standardized cancer incidence rate for native koreans ( average 65.4 per 100,000 ) was the highest among all of the races ; however , this rate was the lowest among filipino americans ( average 5.9 per 100,000 ) ( figure 1 ) [ 11 , 12 ] . among the native groups , the incidence demonstrated a decreasing trend in japanese , chinese , filipino , and non - hispanic white males , whereas the incidence was constant in korean males . for all us immigrant ethnic populations , the overall stomach cancer incidence rates were lower than those among the native groups
the incidence rate of korean american males was one - third less than the incidence rates of native korean males .
the incidence rate of japanese american males and filipino american males was two - thirds less than the incidence rates of native japanese males and native filipinos males .
the incidence rate of chinese american males was three - quarters less than the incidence rates of native chinese males . among asian male immigrants ,
the incidence rates for korean americans , chinese americans , and filipino americans remained constant , but the incidence rate of japanese americans tended to decrease over time . among all females ,
the overall age - standardized cancer incidence rate among native koreans ( average 24.7 per 100,000 ) was the highest , but filipino americans had the lowest incidence rate ( average 2.7 per 100,000 ) . among the native groups , the incidence rates of japanese , chinese , filipino , and non - hispanicwhite females tended to decrease over time , yet the incidence rate among korean females remained constant .
for all ethnicities , the overall stomach cancer incidence rates of female immigrants were lower than those among the female native groups .
the incidence rate of korean american females was two - thirds less than the incidence rates of native korean females .
the incidence rate of japanese american and chinese american females was one - half lower than the incidence rates of native japanese and native chinese females .
the incidence rate of filipino american females was one - third less than the incidence rates of native filipino females . among the asian female immigrant groups , the incidence rate among korean american , japanese american , and filipino american females remained constant , while the incidence rate among chinese american females decreased ( figure 2 ) . between males and females
the incidence rates of males were significantly higher than those for females across all groups .
the highest disparity was found between the incidence rates of native korean males and females ; native korean males had a 2.6 times higher incidence than their female counterparts did .
for both males and females in all age groups , the incidence of stomach cancer increased with age . among all native koreans ,
native japanese , native chinese , korean americans , and japanese americans , the incidence rates of stomach cancer increased steeply with age . among the different racial groups in los angeles ,
the incidence of stomach cancer among korean americans demonstrated the steepest increase , which is expected based on the rate of stomach cancer among native koreans .
from age 70 and older , the incidence tended to decrease for all groups , except for all age groups in the native japanese population where the incidence rates continued to increase ( figures 3 and 4 ) .
a steady decline in stomach cancer incidence has been observed worldwide over the last several decades even in historically high - risk countries such as japan and china . in the us ,
likewise , the same decreasing trends were observed in the present study except for native koreans .
the incidence tended to be lower among asian populations in the us where the overall incidence rate of stomach cancer is also low .
these data suggest that environmental factors play a role in the development of stomach cancer .
the factors that are known to have accounted for this decline include antibiotics against h. pylori , early detection programs , improved medical treatments , and changes in dietary habits by consuming more fresh vegetables and fruits rather than the traditional pickled and salty foods .
nevertheless , no change in the incidence of stomach cancer in korea was found ; therefore , further study is needed .
in addition , the incidence rates among males were significantly higher than those among females for all groups
. unfortunately , a detailed analysis and explanation of sex - related differences are not available in the literature ; instead , only vague theories that assume behavioral differences especially among males such as tobacco smoking have been speculated to explain the etiology behind this unchanging , high incidence of stomach cancer .
one interesting study conducted by researchers at the massachusetts institute of technology reported that estrogen might protect females from developing stomach cancer .
furthermore , the results of the present study indicate that the incidence of stomach cancer tends to increase with age , but peak at age 70 .
one study found that the incidence tends to rise progressively with age , and the highest incidence occurs between 50 and 70 years .
however , in the present study , this trend decreased for most groups aged 80 and older , which might have resulted from a selective survival bias and the low reliability of the incidence data on older adults .
the overall stomach cancer incidence rate for native koreans was the highest among all groups for both males and females , whereas both non - hispanic whites and filipino americans showed the lowest rate .
interestingly , the pattern of incidence rates within asian american ethnic groups resembled the pattern of incidence rates in their native countries .
several studies have demonstrated that cancer incidence rates among immigrants follow a similar direction to those in their respective countries of origin .
one study that examined the incidence of stomach cancer among asian american immigrants from 1973 to 1986 reported that the incidence among chinese immigrants was similar to that among whites .
another study investigated the effect of diet and place of birth on stomach cancer incidence and demonstrated that second generation hawaiian japanese people had lower stomach cancer incidence than first generation hawaiian japanese people did .
in general , smoking , dietary patterns , and having a h. pylori infection have been considered major risk factors for stomach cancer .
the differences in the prevalence of smoking seem to reflect the geographical variations between the incidence rates of stomach cancer in the us and asia . the prevalence of smoking is lower in the us than that in asian countries is ; the overall smoking prevalence in the us was 17.8% , while that in china , the philippines , korea , and japan was 28.1 , 28 , 25.8 , and 19.7% , respectively [ 19 - 23 ] .
this trend in smoking is also reflected in the incidence rate of stomach cancer , as the us had the lowest stomach cancer incidence among the five countries in the present study .
therefore , the variability in tobacco use between the residents of these countries might explain the association between smoking prevalence and stomach cancer incidence .
furthermore , the prevalence of smoking might explain the large difference between the stomach cancer incidences of males and females .
the prevalence of smoking in the us was 20.5% for males and 15.3% for females .
however , the difference between the prevalence of smoking among males and females was higher in asian countries compared to the prevalence in the us .
for example , the prevalence of smoking was estimated to be 43.7% for males and 7.9% for females in korea . therefore , smoking is related to stomach cancer incidence and varies by country and gender .
regarding dietary patterns , traditional asian meals tend to have a high salt content and contain preserved foods including soups ; therefore , asians tend to consume a high - salt diet .
in addition , the geographical variations of stomach cancer incidence might also reflect differences in the prevalence of h. pylori infections , which are considered to cause more than 50% of all gastric cancer cases worldwide .
the prevalence of h. pylori infection exceeds 50% among the general population in korea , japan , and china .
interestingly , the incidence of stomach cancer in the philippines was the lowest among all of the asian countries , despite their tendency to consume a high - salt diet and the high prevalence of h. pylori infections in the philippines .
first , the incidence of stomach cancer calculated for korean , japanese , chinese , and filipino americans was possibly less accurate than that calculated for the native populations because some data were missing over several study years in the american dataset .
this might have been because a limited number of asian americans reside in los angeles and because these ethnic groups were surveyed within a specific geographic area .
in addition , different immigration patterns over the data collection period might have resulted in missing data .
second , the high incidence observed in korea and japan may reflect good practice provided by screening programs and a high level of accessibility to relevant early detection services . these factors could have led to the underestimation of the actual incidence rates in china , the philippines , and the us . finally , the data obtained from the ci5 might have misclassified some individuals based on their race .
for example , vietnamese people could have classified themselves as chinese , and persons of mixed race were likely asked to choose only one racial background in the original survey .
third , the length of residence in california could have distorted the results of this study .
fourth , these findings might not be generalizable to other populations since we only analyzed data on asian americans living in los angeles .
last , the data on the incidence of stomach cancer in native koreans were only available from 1999 . in summary , this study compared the incidence rates of stomach cancer between asian immigrants and native asians .
the migratory effect on stomach cancer incidence suggests that lifestyle plays an important role in the development of stomach cancer .
it is expected that increased overall socioeconomic status as well as improved hygienic conditions could have accelerated the decreasing trend we observed in the stomach cancer incidence among immigrants to the us . moreover
, an increased availability of fresh vegetables and fruits could have modified these immigrants dietary habits .
it is important to note that korea has had the highest incidence of stomach cancer since 1999 and continues to today , while the incidence in neighboring asian countries has demonstrated a decreasing trend .
this implies that efforts should be made to reduce the incidence of stomach cancer in korea by looking to other countries that have lowered their national incidence of stomach cancer . | stomach cancer is the second most common cancer in eastern asia , accounting for approximately 50% of all new cases of stomach cancer worldwide .
our objective was to compare the stomach cancer incidence rates of asian americans in los angeles with those of native asians to assess the etiology of stomach cancer from 1988 to 2011 . to examine these differences , asian americans ( korean
, japanese , chinese , and filipino americans living in los angeles , california , usa ) and native asians ( from korea , japan , china , and the philippines ) were selected for this study . using the cancer incidence in five continents database ,
stomach cancer incidence rates were examined .
data from the national cancer registry of korea were used for native koreans . between native countries , the incidence rates in japan , china , the philippines , and
the us declined over time , but the incidence in korea has remained constant .
the incidences among asian immigrants were lower than those among native asians .
the incidence rates of males were approximately 2 times higher than those among females in asian countries were .
the effect of immigration on stomach cancer incidence suggests that lifestyle factors are a significant determinant of stomach cancer risk .
however , the incidence in korea remains the highest of these countries |
rauwolfia serpentina ( family : apocynaceae ) is a medicinally important herb commonly known as indian snake root .
it has been used for centuries in ayurvedic medicines because of their valuable therapeutic actions especially in the treatment of hypertension and psychotic disorders like schizophrenia , anxiety , insomnia , insanity , and so forth [ 1 , 2 ] .
rauwolfia serpentina has been also used for the treatment of skin cancers , burns , eczema , and snake bite [ 3 , 4 ] .
rauvolfia verticillata is another traditional chinese medicinal plant that also belongs to the same apocynaceae family and also was been used for hypertension and cardiac disorders .
both rauwolfia serpentine and rauvolfia verticillata contains pharmacologically active indolic alkaloids , namely , reserpine , rescinnamine , and yohimbine ( their chemical structures are given in figure 1 ) [ 5 , 6 ] .
various analytical methods have been reported for the determination of reserpine , rescinnamine , and yohimbine in their plant extracts , pharmaceutical preparation , and biological fluids .
two methods based on high - performance liquid chromatography ( hplc ) have been reported for the analysis of rauwolfia serpentine being one using absorption photometric detection on a photodiode array detector ( to determine reserpine ) and the other using florescence detection ( to determine both reserpine and rescinnamine ) .
besides , two hplc methods with uv absorption photometric detection were also reported for the determination of yohimbine in plant bark and in commercial preparation or together with reserpine in a rauvolfia verticillata preparation . hptlc method for yohimbine and phosphorimetric method for rescinnamine and reserpine were also available in literature for determination in pharmaceutical preparations . for the determination of reserpine in biological fluids
the first one employed hplc method to analyze in horse plasma whereas two other employed tandem mass spectroscopy detection to analyze in equine and mice blood plasma .
one lcms method is also reported for the pharmacokinetic study of yohimbine in horse plasma . in order to better use the therapeutic relevant herbs ,
no analytical method has been reported in the literature for the simultaneous determination of reserpine , rescinnamine , and yohimbine in biological fluids .
therefore , a method with minimal sample treatment procedure is required for the simultaneous determination of these alkaloids in plasma . among the currently available bioanalytical techniques ,
ultraperformance liquid chromatography ( uplc ) has gained a considerable attention in recent years and has been emerged as the preeminent analytical tool for pharmaceutical and biomedical analysis .
the van deemter equation indicates that , as the particle size decreases to less than 2.5 m , there is a significant improvement in efficiency that will not degrade with the increasing of mobile phase flow rates .
therefore , uplc by utilizing 1.7 m particle produces chromatographic peaks narrower than the ones obtained by conventional hplc . by using multiple reaction monitoring ( mrm ) as the ms detection , uplc - ms / ms method can offer a more sensitive and selective detection , which is suitable for the pharmacokinetic study at the therapeutic dose .
the present study describes a sensitive uplc - ms / ms method using one step sample preparation process for the simultaneous determination of reserpine , rescinnamine , and yohimbine in human plasma which could be applied for the pharmacokinetic study .
reserpine ( 99% purity ) and yohimbine ( 98% purity ) were obtained from fluka and merck ( germany ) , respectively .
rescinnamine ( 98% ) and papaverine ( is , 99% purity , structure in figure 1 ) were obtained from acros organics .
all aqueous solutions were prepared using water from a milli - qr gradient a10 water ultrapurification unit ( millipore , france ) .
milford , ma , usa ) . the uplc system , consisted by a quaternary solvent manager , binary pump , degasser , an autosampler , and a column oven .
the separation was performed on acquity uplc beh c18 column ( 50 2.1 mm , i.d .
the mobile phase consisting of a mixture of acetonitrile and water acidified with formic acid ( 60/40/0.1 , v / v / v ) pumped at a flow rate of 0.2 ml / min .
the yohimbine and is were eluted at 0.64 min , whereas reserpine and rescinnamine were eluted at 0.74 min with a total run time of 2 min .
the injection volume was 5 l in partial loop mode and the temperature of the autosampler was kept at 15c . a triple - quadrupole tandem mass spectrometer ( micromass quattro micro waters corp . ,
milford , ma , usa ) equipped with electrospray ionization ( esi ) interface was used for analytical detection .
the transitions of m / z 609.32 > 195.01 , m / z 635.34 > 221.03 , m / z 355.19 > 144 , and m / z 340.15 > 202.02 were selected for the quantification of reserpine , rescinnamine , yohimbine , and is , respectively .
the optimized ionization conditions were as follows : capillary voltage 3.3 kv , cone voltage 64 v , source temperature 150c , and desolvation temperature 350c .
nitrogen was used as desolvation and cone gas with the flow rate at 650 and 50 l / hr , respectively .
argon was used as the collision gas at a flow rate of 0.10 ml / min .
the collision energies for reserpine , rescinnamine , yohimbine , and is were 48 ev , 34 ev , 30 ev , and 28 ev , respectively , with the dwell time of 0.077 s. the mass lynx software ( version 4.1 , scn 714 ) was used to control the uplc - ms / ms system and data was collected and processed using targetlynx program .
a standard stock solution of reserpine , rescinnamine , yohimbine , and is was prepared by dissolving in methanol to give a final concentration of 500 g / ml . the solutions were kept in the refrigerator and could be used for 15 days from the date of preparation .
stock solution of reserpine , rescinnamine , and yohimbine was used for calibration standards and quality control ( qc ) samples , respectively .
the standard stock solutions of reserpine , rescinnamine , and yohimbine was then serially diluted in methanol : water ( 50 : 50% , v / v ) to provide standard working solution in the following concentration range : 3.6 to 4000 ng / ml ( reserpine ) , 2.7 to 3000 ng / ml ( rescinnamine ) , and 2.3 to 2500 ng / ml ( yohimbine ) . a 20 l aliquot of each working solutions was added to blank human plasma to yield effective calibration standards ranging from 0.36 to 400 ng / ml ( reserpine ) , 0.27 to 300 ng / ml ( rescinnamine ) , and 0.23 to 250 ng / ml ( yohimbine ) .
the effective plasma concentrations of low ( lqc ) , medium ( mqc ) , and high ( hqc ) qc samples were prepared in a similar way for reserpine ( 0.96 , 24 , and 300 ng / ml ) , rescinnamine ( 0.80 , 20 , and 250 ng / ml ) , and yohimbine ( 0.64 , 16 , and 200 ng / ml ) , respectively . analytes spiked plasma used for calibration standards and quality control samples were kept at 80c until assayed or used for validating the assay procedures . the is working solution
( 1 g / ml ) for routine use was prepared by diluting the papaverine stock solution in methanol : water ( 50 : 50% , v / v ) .
plasma samples stored at around 80c were thawed , left for 1 h , and vortex for 30 s at room - temperature before extraction to ensure homogeneity . to 200 l of blank plasma sample 20 l of working standard solution and 10 l ( 1 g / ml ) of is ( except blank sample ) were added .
the samples were vortex - mixed for about 30 s and 770 l of acetonitrile was added .
the samples were again vortex mixed gently for 1.5 min and then cold - centrifuged for 12 min at 10000 rpm .
after centrifugation , 600 l of supernatant was transferred into hplc vial , and 5 l volumes ( in partial loop with needle over fill mode ) of the sample were subjected to the analysis by uplc - ms / ms .
a full method validation was performed according to guidelines set by the united states food and drug administration ( us - fda ) and european medicines agency ( emea ) guidelines [ 19 , 20 ] .
the validation of this procedure was performed in human plasma in order to evaluate the method in terms of selectivity , linearity of response , accuracy , precision , recovery , and stability of analytes during both short - term sample processing and long - term storage .
the selectivity of the method towards endogenous plasma matrix components , metabolites , and component medications were assessed in human plasma . among the analyzed batch of samples , human plasma samples showing not significant interference at the retention time of analytes ( lloq level ) and
is ( 50 ng / ml ) were selected as calibration matrix and qc matrix .
these samples were analyzed and no relevant interferences were found at the retention time of the analytes and papaverine .
the linear response of the method was determined by the evaluation of eight points analytical curve : 0.36 to 400 ng / ml ( reserpine ) 0.27 to 300 ng / ml ( rescinnamine ) , and 0.23 to 250 ng / ml ( yohimbine )
. analytical curves from accepted three precision and accuracy validation method were used to establish linearity .
analytical curves were plotted using a least square linear regression model y = mx + b , weighted by 1/x , in which y is the peak area ratio ( analyte / is ) , m is slope of the analytical curve , b is the y - axis intercept of the analytical curve , and x is the analyte ( reserpine , rescinnamine , and yohimbine ) concentration .
the concentrations of reserpine , rescinnamine , and yohimbine were calculated from each analytical curve and the resulting calculated parameters were used to determine concentrations of these analytes in quality control samples .
the lowest concentration on the analytical curve was to be accepted as the lower limit of quantification ( lloq ) , if the analyte response was at least five times more than that of drug free ( blank ) extracted plasma and highest concentration was to be accepted as upper limit of quantification ( uloq ) .
in addition , the analyte peak of lloq sample should be identifiable , discrete , and reproducible with accuracy within 20% and a precision 20%
. the deviation of standards other than lloq from the nominal concentration should not be more than 15.0% .
precision of an analytical method describes the closeness of two or more measurements to each other whereas the accuracy of an analytical method describes the closeness of mean test results obtained by the method to the true value ( concentration ) of the analyte .
intra- and interday accuracies are expressed as a percentage of deviation from the respective nominal value .
the precision of the assay was measured by the percent coefficient of variation ( % cv ) at three different concentrations in human plasma .
intraday precision and accuracy were assessed by analyzing six replicates of the quality control samples at three different levels ( reserpine ( 0.96 , 24 , and 300 ng / ml ) , rescinnamine ( 0.80 , 20 , and 250 ng / ml ) , and yohimbine ( 0.64 , 16 , and 200 ng / ml ) ) during a single analytical run . similarly the interday precision and accuracy were assessed by analyzing 18 replicates of the quality control samples at different concentration levels through three precision and accuracy batches runs on 3 consecutive validation days . the deviation at each concentration level from the nominal concentration was expected to be 15.0% for precision whereas the mean accuracy should not deviate by 15.0% .
the recoveries of reserpine , rescinnamine , and yohimbine at three qc levels were determined by comparing peak area ratios of the indolic alkaloid in analyte spiked plasma samples before and after proceeding the extraction of the added analytes .
calculation of the matrix effects was conducted by comparing the ratio of the indolic alkaloid signals obtained from analyte spiked plasma with the one obtained from aqueous solution with the same concentration of the indolic alkaloids .
deviation in analyte concentration of a maximum of 15% was considered acceptable and is in line with current emea guideline .
the extraction recovery and matrix effects of is were also simultaneously evaluated using the same method .
stability of reserpine , rescinnamine , and yohimbine in plasma was assessed by analyzing six replicates of qc samples at low and high concentrations under a variety of storage and processing conditions .
bench - top stability was assessed after exposure of the plasma samples to room temperature for 6 h , which exceeds the residence time of the sample processing procedures .
the freeze - thaw stability was evaluated after undergoing three freeze ( at around 80c)-thaw ( room temperature ) cycles .
the processed sample stability was determined by storing the reconstituted qc samples for 48 h under autosampler condition ( maintained at 15c ) before being analyzed .
long - term stability was assessed after storage of the test samples at around 80c for 30 days .
the samples were considered stable in plasma at each concentration if the deviation from the mean calculated concentration of stability quality control samples was within 15% .
initial feasibility experiments of various mixtures of organic solvents such as acetonitrile and methanol along with pure water , both having 0.1% formic acid along with altered flow - rates ( in the range of 0.200.4 ml / min ) , were performed to optimize an effective chromatographic resolution of reserpine , rescinnamine , yohimbine , and is . mobile phase containing methanol and water with 0.1% formic acid did not produce satisfactory separation results
. then acetonitrile with water having 0.1% formic acid was tried starting from 50 : 50 ratio with altered flow rate in range from 0.2 to 0.4 ml / min .
results was found to be satisfactory and the best resolution of peaks was achieved with an isocratic elution by a mobile phase comprising acetonitrile : water : formic acid ( 60 : 40 : 0.1,v / v / v ) at a flow rate of 0.2 ml / min , on a c18 column ( 50 2.1 mm , i.d . 1.7
the selected conditions were found to be suitable for the adequate analytes response using electrospray .
uplc - ms / ms operation parameters were carefully optimized for the determination of indole alkaloids and is . a standard solution ( 200 ng / ml ) of reserpine , rescinnamine , yohimbine , and is was directly infused along with the mobile phase into the mass spectrometer with esi as the ionization source .
the mass spectrometer was tuned initially in both positive and negative ionization modes for the indole alkaloids and papaverine .
it was observed that the signal intensities of positive ions were much more intense than those of negative ions .
parameters , such as capillary and cone voltage , desolvation temperature , esi source temperature , and flow rate of desolvation gas and cone gas , were optimized to obtain the optimum intensity of deprotonated molecules of indole alkaloids and papaverine for quantification . among the parameters , capillary voltage and especially the cone voltage
lastly , analytes produced the strongest ion signals when cone voltage was set up at 64 v. while cone voltage exceeded 64 v , the ion signals decreased rapidly .
the collision energy was investigated from 10 to 50 ev to optimize the response of product ion , and the best values were selected for each product ions .
the ms spectra ( parent ion ) and ms / ms spectra ( daughter ion ) for reserpine , rescinnamine , yohimbine , and papaverine are shown in figures 2 and 3 , respectively .
protein precipitation can be helpful in producing a clean sample and avoiding endogenous substances in plasma with the analytes and is onto the column and ms system .
clean samples are essential for minimizing ion suppression and matrix effect in uplc - ms / ms analysis .
two organic solvents , methanol and acetonitrile , in presence or absence of formic acid were evaluated .
finally methanol was found to be optimal , which can produce a clean chromatogram for a blank plasma sample and yield the highest recovery for the analytes from the human plasma .
selectivity of the method was assessed by comparing thechromatograms of human blank plasma with the corresponding spiked lloq sample .
only those lots which are under the acceptance criteria ( < 20% in comparison to the spiked lloq and < 5% in comparison to is area ) were selected .
this infers that there were no potential significant endogenous substances in plasma that interfered with the peaks of analyte and is .
thus the method seems to be selective enough for determination of reserpine , rescinnamine , yohimbine , and is in plasma .
representative mrm chromatograms obtained from blank plasma showing no interference at the retention time of analytes and is are shown in figure 4 .
representative chromatograms at lloq and uloq level are shown in figures 5 and 6 , respectively .
the linearity of the method was determined by a weighted least square regression analysis of standard plot associated with an eight - point standard curve .
the analytical curves were generated by plotting area ratio ( analytes / is ) as a function of analyte concentration .
analytical curve was found to be linear in range of 0.36400 , 0.27300 , and 0.23250 ng / ml for reserpine , rescinnamine , and yohimbine , respectively , in human plasma .
the determination coefficients ( r ) were consistently greater than 0.995 during the course of validation .
the lloqs value for this assay was 0.36 , 0.27 , and 0.23 ng / ml for reserpine , rescinnamine , and yohimbine , respectively , in plasma .
representative lloq is sensitive enough to investigate the pharmacokinetic behavior of these analytes in plasma sample .
table 1 summarizes the inter- and intraday precision and accuracy values for qc samples in human plasma .
for reserpine , the intra- and interday precisions were 8.3 , whereas the intra- and interday accuracies were in the range of 89.8111.1% .
for rescinnamine , the intra- and interday precisions were 9.6 , whereas the intra- and interday accuracies were in the range of 91.2108.1% .
similarly , for yohimbine , the intra- and interday precisions were 9.8 , whereas the intra- and interday accuracies were in the range of 91.1110.4% .
these results indicate that the method has good precision and accuracy was also adequate for the level of analyte concentration in the samples and are within the acceptance limit of 15% and <15% for precision and accuracy , respectively [ 19 , 20 ] . the percentage recoveries ( mean sd ) of reserpine , rescinnamine , and yohimbine obtained from plasma at three different concentration levels ( reserpine ( 0.96 , 24 , and 300 ng / ml ) , rescinnamine ( 0.80 , 20 , and 250 ng / ml ) , and yohimbine ( 0.64 , 16 , and 200 ng / ml ) ) were 69.3 to 72.9% , 64.9 to 81.7% , and 69.8 to 75.8% , respectively . the mean recovery for the papaverine ( is ) at the concentration employed was 76.9% .
this result indicates that the extraction efficiency for reserpine , rescinnamine , and yohimbine using protein precipitation method was satisfactory , consistent , and concentration independent .
the ionization suppression or enhancement effects were between 15% , indicating no relative matrix effects .
the stabilities of reserpine , rescinnamine , and yohimbine were investigated at two concentrations of qc samples ( low and high concentrations ) to cover expected conditions during analysis , storage , and processing of all samples , which include the stability data from various stability exercises like bench - top , freeze / thaw , processed sample , and long - term stability tests .
the stability results summarized in table 2 indicate that reserpine , rescinnamine , and yohimbine spiked into human plasma were stable for at least 6.0 h at room temperature , for at least 48 h in final extract at 15c under autosampler storage condition and during three freeze - thaw cycles when stored at around 80c and thawed to room temperature .
an efficient , reliable , and novel uplc - ms / ms method was successfully developed for the simultaneous determination of indolic alkaloids ( reserpine , rescinnamine , and yohimbine ) in human plasma sample . the developed method is validated according to official guideline .
proposed method is advantageous in term of sensitivity , one step sample preparation , and short runtime ( 2.0 min ) and hence suitable for high - throughput analysis .
the analytical method presented here will be useful for the quantitative investigation of these indolic alkaloids in pharmacokinetic and toxicological analysis . | a sensitive and selective uplc - ms / ms method was developed and validated for the determination of three indolic alkaloids ( reserpine , rescinnamine , and yohimbine ) in human plasma using papaverine as internal standard ( is ) .
after a one step protein precipitation with acetonitrile , separation was carried out using c18 column ( 50 2.1 mm , i.d .
1.7 m ) and mobile phase consisting of acetonitrile : water : formic acid ( 60 : 40 : 0.1% , v
/ v / v ) pumped at a flow rate of 0.2 ml / min .
the mass spectrometric determination was carried out using an electrospray interface operated in the positive mode with multiple reaction monitoring ( mrm ) mode .
the precursor to product ion transitions of m / z 609.32 > 195.01 , m / z 635.34 > 221.03 , m / z 355.19 > 144 , and m / z 340.15 > 202.02 were selected for the quantification of reserpine , rescinnamine , yohimbine , and is , respectively .
the analytical response was found to be linear in the range of 0.36400 , 0.27300 , and 0.23250 ng / ml with lower limit of quantification of 0.36 , 0.27 , and 0.23 ng / ml for reserpine , rescinnamine , and yohimbine , respectively .
validation was made following official guidelines .
the proposed method enabled reproducible results and hence could be reliable for pharmacokinetic and toxicological analysis . |
we enrolled 54 patients ( 25 men and 29 women ; mean age : 29.3 years ; age range : 13 - 80 years ) who underwent a cr , followed by a ssdr of the lower extremities at our hospital between january 2003 and march 2007 .
the crs were performed between january 2003 and june 2006 , whereas the ssdrs were performed between february 2007 and march 2007 ( we started the ssdr method at our hospital in july 2006 ) .
the mean time interval between performing the cr and ssdr for each patient was 20 months ( range : 2 - 75 months ) .
the underlying diseases of the patients included previous trauma such as traffic accidents and fall injuries ( n = 15 ) , osteoarthritis of the knee joints ( n = 11 ) , previous infectious arthritis ( n = 5 ) , osteochondromatosis ( n = 4 ) , congenital vascular malformation ( n = 3 ) , legg - calv - perthes disease ( n = 2 ) , a tumor ( n = 2 ) , fragile x syndrome ( n = 1 ) , and unknown causes ( n = 11 ) .
the crs were performed using the dhf-158h2 ( hitachi , tokyo , japan ) and cxdi-40 g ( canon digital radiography systems , tokyo , japan ) units ( table 1 ) .
the cr method yielded two image sets ( standing anteroposterior radiography of the lower extremities [ sapl ] and scanogram of the lower extremities [ sl ] ) of the lower extremities .
the sapl was performed to evaluate the alignment of the knee joints and to visualize the detailed anatomy of the bone and soft tissue .
the sapl was performed with the patients facing the radiographic tube and both patellae pointing forward .
the minimum distance between the patient and the tube was 200 cm , and an image was obtained using 80 kvp , 200 mas and 0.25 sec .
the images were recorded on a cxdi-40 g , which had a amorphous silicone flat sensor , using a vertical cassette holder with three individual 36 43 cm standard cassettes .
the three images were then stitched together to form a composite image that was distributed to a picture achieving and communication systems ( pacs ) workstation ( 9 ) .
the sl was performed in the supine position and its purpose was to measure the length of both legs and to compare the differences in the length .
the lower extremities were positioned with both patellae pointing toward the ceiling and a radiopaque ruler taped on the table between both legs .
three separate anteroposterior images were obtained with the center over the hip , knee , and ankle joints using 3 separate standard - sized ( 36 43 cm ) cassettes at 80 kvp and 50 mas for the hips , 66 kvp and 16 mas for the knees , as well as 60 kvp and 10 mas for the ankles .
the images of lower extremities performed on sapl of cr are more magnified than a radiopaque ruler , since the distance between the ruler and cassette is shorter than the distance between the patient and the cassette ( 10 ) .
the ssdr method was performed to evaluate the alignment of the knee joints as well as measure the lengths of lower extremities . to obtain an ssdr image
, we used a scan - dx ( advanced digital technology , seoul , korea ) ( table 1 ) .
the position of the patient for ssdr was the same as for the sapl of the cr method .
the ssdr was obtained with a slot - scan in such a way that the x - rays were collimated into a narrow , horizontally oriented , and fan - shaped beam that closely matched the rectangular detector ( 2 ) .
the ssdr was performed using 110 kvp and 200 mas at all levels , scanning from the anterior superior iliac spine to the foot , to cover the entirety of the lower extremity .
usually , 8 to 12 seconds were required to scan the lower extremities , including the pelvis ( average scanning time per line of scan : 0.005 sec ) .
we did not obtain a separate scanogram using the ssdr method because we could analyze the deformities of the lower extremities as well as simultaneously measure the exact lengths of lower extremities using the ssdr method exclusively without the average magnification of images ( 11 ) . in this study ,
the digital radiography system was applied with a multi - channel ionization chamber ( mic ) detector , which provided high quality imagery and very short image processing time .
this was attributed to direct conversion , low radiation scatter , and high fill factor .
two musculoskeletal radiologists evaluated all of the lower extremities images by consensus , using a pacs ( centricity 2.0 , ge healthcare , milwaukee , wi ) , which displayed all the image data on two monitors ( two monitors , 1,536 2,048 image matrices , 8-bit viewable gray scale , 60-foot - lambert luminescence ) . to avoid any recall bias ,
the ssdr and sapl were evaluated in a different order and at two - week intervals . before we evaluated each image in detail , we optimized the window width and level of each image on the pacs monitor to allow for optimal image evaluation .
we evaluated the visibility of four features , including the outer margin of the cortex of the bone , the inner margin of the cortex , the trabeculae of the medullary bone ( visibility of an individual trabeculae ) , and the intermuscular fat plane . in turn , this evaluation was performed at six different levels of the lower extremity region , including the pelvis , hip , femur , knee , tibia and ankle ( 4 ) .
a visibility grading scale was used for assessing the image quality of each feature ( 0 : inadequately visualized , 1 : sufficiently visualized , 2 : well visualized ) .
the visibility score was calculated as the sum of the grades for each of the four features .
we compared the mean grading scores for the ssdr and cr methods at each level and for each feature .
finally , we compared the mean of the overall scores for each method to determine whether one of the methods was a significantly superior diagnostic tool for evaluation of the lower extremities .
we measured the entrance skin dose using an x - ray anatomical phantom ( victoreen , moedling , austria ) .
the phantom was composed of three sections , including a hip ( model 76 - 642 ) , knee ( model 76 - 675 ) , and ankle ( model 76 - 659 ) .
the unit consisted of radiopaque isocyanate rubber that had approximately the same absorption and secondary radiation - emitting characteristics as living tissue .
similarly , the unit was homogeneous and cast shadows similar to the shadows cast by tissue .
no spongy portions were present in the phantom and bone marrow was simulated with tissue - equivalent material , thereby allowing for a critical detailed study of bone structure and sharpness comparison using x - rays .
we evaluated the radiation exposure of the cr and ssdr methods at the hip , knee , and ankle on two occasions .
in addition , we measured each entrance skin dose for sapl and sl using the cr method , and then summated the doses of both sapl and sl at each site .
last , we measured the entrance skin dose for the ssdr method at the same sites .
the equipment used for dose measurement was a model solidose 400 dosimeter ( rti electronics , mlndal , sweden ) .
we placed the dosimeter on a cassette for the cr method or a detector for the ssdr method , respectively . before obtaining the images ,
the image quality score was analyzed using the paired t - test ( spss , version 14 ; spss , chicago , il ) .
we compared the mean scores of the features and overall scores for the six levels and for the two methods .
a p - value of less than 0.05 indicated a statistically significant difference . the measured entrance skin dose between for each method was not statistically analyzed .
we enrolled 54 patients ( 25 men and 29 women ; mean age : 29.3 years ; age range : 13 - 80 years ) who underwent a cr , followed by a ssdr of the lower extremities at our hospital between january 2003 and march 2007 .
the crs were performed between january 2003 and june 2006 , whereas the ssdrs were performed between february 2007 and march 2007 ( we started the ssdr method at our hospital in july 2006 ) .
the mean time interval between performing the cr and ssdr for each patient was 20 months ( range : 2 - 75 months ) .
the underlying diseases of the patients included previous trauma such as traffic accidents and fall injuries ( n = 15 ) , osteoarthritis of the knee joints ( n = 11 ) , previous infectious arthritis ( n = 5 ) , osteochondromatosis ( n = 4 ) , congenital vascular malformation ( n = 3 ) , legg - calv - perthes disease ( n = 2 ) , a tumor ( n = 2 ) , fragile x syndrome ( n = 1 ) , and unknown causes ( n = 11 ) .
the crs were performed using the dhf-158h2 ( hitachi , tokyo , japan ) and cxdi-40 g ( canon digital radiography systems , tokyo , japan ) units ( table 1 ) .
the cr method yielded two image sets ( standing anteroposterior radiography of the lower extremities [ sapl ] and scanogram of the lower extremities [ sl ] ) of the lower extremities .
the sapl was performed to evaluate the alignment of the knee joints and to visualize the detailed anatomy of the bone and soft tissue .
the sapl was performed with the patients facing the radiographic tube and both patellae pointing forward .
the minimum distance between the patient and the tube was 200 cm , and an image was obtained using 80 kvp , 200 mas and 0.25 sec .
the images were recorded on a cxdi-40 g , which had a amorphous silicone flat sensor , using a vertical cassette holder with three individual 36 43 cm standard cassettes .
the three images were then stitched together to form a composite image that was distributed to a picture achieving and communication systems ( pacs ) workstation ( 9 ) .
the sl was performed in the supine position and its purpose was to measure the length of both legs and to compare the differences in the length .
the lower extremities were positioned with both patellae pointing toward the ceiling and a radiopaque ruler taped on the table between both legs .
three separate anteroposterior images were obtained with the center over the hip , knee , and ankle joints using 3 separate standard - sized ( 36 43 cm ) cassettes at 80 kvp and 50 mas for the hips , 66 kvp and 16 mas for the knees , as well as 60 kvp and 10 mas for the ankles .
the images of lower extremities performed on sapl of cr are more magnified than a radiopaque ruler , since the distance between the ruler and cassette is shorter than the distance between the patient and the cassette ( 10 ) .
the ssdr method was performed to evaluate the alignment of the knee joints as well as measure the lengths of lower extremities . to obtain an ssdr image
, we used a scan - dx ( advanced digital technology , seoul , korea ) ( table 1 ) .
the position of the patient for ssdr was the same as for the sapl of the cr method .
the ssdr was obtained with a slot - scan in such a way that the x - rays were collimated into a narrow , horizontally oriented , and fan - shaped beam that closely matched the rectangular detector ( 2 ) .
the ssdr was performed using 110 kvp and 200 mas at all levels , scanning from the anterior superior iliac spine to the foot , to cover the entirety of the lower extremity .
usually , 8 to 12 seconds were required to scan the lower extremities , including the pelvis ( average scanning time per line of scan : 0.005 sec ) .
we did not obtain a separate scanogram using the ssdr method because we could analyze the deformities of the lower extremities as well as simultaneously measure the exact lengths of lower extremities using the ssdr method exclusively without the average magnification of images ( 11 ) . in this study ,
the digital radiography system was applied with a multi - channel ionization chamber ( mic ) detector , which provided high quality imagery and very short image processing time .
this was attributed to direct conversion , low radiation scatter , and high fill factor .
two musculoskeletal radiologists evaluated all of the lower extremities images by consensus , using a pacs ( centricity 2.0 , ge healthcare , milwaukee , wi ) , which displayed all the image data on two monitors ( two monitors , 1,536 2,048 image matrices , 8-bit viewable gray scale , 60-foot - lambert luminescence ) . to avoid any recall bias ,
the ssdr and sapl were evaluated in a different order and at two - week intervals . before we evaluated each image in detail , we optimized the window width and level of each image on the pacs monitor to allow for optimal image evaluation .
we evaluated the visibility of four features , including the outer margin of the cortex of the bone , the inner margin of the cortex , the trabeculae of the medullary bone ( visibility of an individual trabeculae ) , and the intermuscular fat plane . in turn , this evaluation was performed at six different levels of the lower extremity region , including the pelvis , hip , femur , knee , tibia and ankle ( 4 ) .
a visibility grading scale was used for assessing the image quality of each feature ( 0 : inadequately visualized , 1 : sufficiently visualized , 2 : well visualized ) .
the visibility score was calculated as the sum of the grades for each of the four features .
we compared the mean grading scores for the ssdr and cr methods at each level and for each feature .
finally , we compared the mean of the overall scores for each method to determine whether one of the methods was a significantly superior diagnostic tool for evaluation of the lower extremities .
we measured the entrance skin dose using an x - ray anatomical phantom ( victoreen , moedling , austria ) .
the phantom was composed of three sections , including a hip ( model 76 - 642 ) , knee ( model 76 - 675 ) , and ankle ( model 76 - 659 ) .
the unit consisted of radiopaque isocyanate rubber that had approximately the same absorption and secondary radiation - emitting characteristics as living tissue .
similarly , the unit was homogeneous and cast shadows similar to the shadows cast by tissue .
no spongy portions were present in the phantom and bone marrow was simulated with tissue - equivalent material , thereby allowing for a critical detailed study of bone structure and sharpness comparison using x - rays .
we evaluated the radiation exposure of the cr and ssdr methods at the hip , knee , and ankle on two occasions .
in addition , we measured each entrance skin dose for sapl and sl using the cr method , and then summated the doses of both sapl and sl at each site .
last , we measured the entrance skin dose for the ssdr method at the same sites .
the equipment used for dose measurement was a model solidose 400 dosimeter ( rti electronics , mlndal , sweden ) .
we placed the dosimeter on a cassette for the cr method or a detector for the ssdr method , respectively . before obtaining the images ,
the image quality score was analyzed using the paired t - test ( spss , version 14 ; spss , chicago , il ) .
we compared the mean scores of the features and overall scores for the six levels and for the two methods .
the ssdr method demonstrated a better image resolution and contrast when compared against the sapl of the cr method ( fig .
a comparison of the image quality using the two methods was shown in table 2 .
the overall scores for the ssdr method , for all levels , were significantly higher than those obtained by the cr method ( p < 0.05 ) .
however , the mean scores of outer margin of the cortex at the levels of the pelvis , femur , knee and tibia , as well as those of inner margin of cortex at hip , tibia and ankle , were not significantly different between the two methods .
the entrance skin dose of the ssdr method was less than half of the entrance dose of the sapl for the cr method at the level of the hip and knee , but was similar at the level of the ankle ( table 3 ) .
similarly , it is also lower ( much lower in some cases ) for the comparison of the entrance skin dose for ssdr method compared to the sl and the sapl and sl combined of the cr method ( table 3 ) .
several studies pertaining to the advantages of the ssdr method with respect to other digital techniques have already been reported for chest imaging and mammography exist in the literature ( 1 , 2 , 12 , 13 ) . the ssdr method was determined to be advantageous over the full - field digital radiography based on significant scatter reduction , high detective quantum efficiency , and improved image quality ( 1 ) .
further , the ssdr method has allowed for dose - efficient scatter rejection and the ability to use small detectors to produce a large - area image during a mammography ( 14 ) . as a result
, some studies have suggested that the ssdr method could be an alternative to other digital techniques ( 2 ) .
some studies have also cited the advantages of using a mic detector ( 15 , 16 ) .
since the mic detector is a direct conversion type , it enables a high counting rate , high spatial resolution , low sensitivity to gas impurity , and low cost ( 16 ) . in our study , we applied both a slot - scan type and mic detector to obtain a digital radiography of better image quality with lower radiation dose of the lower extremities , compared to the cr method . in terms of evaluating each level ,
most levels showed differences in image quality when comparing the cr and ssdr methods ; however the images at the level of the tibia showed less of a difference in comparison to the other levels ( table 2 ) .
reasons for this include a relatively shallower thickness of the lower extremities below the knees , which resulted in a higher spatial resolution below the knees , in comparison to the pelvis and thigh for the cr method ( 1 ) .
the image quality at the level of the pelvis and hip were relatively worse than at the other levels due to the presence of thick soft tissue and multiple pelvic organs ( 17 ) .
the mean scores of the outer margin of the cortex at the levels of the pelvis , femur , knee , and tibia were not significantly different between the sapl of the cr method and the ssdr method .
the outer margin of the cortex has a higher contrast due to the surrounding soft tissue , which is relatively radiolucent as compared with the medullary bone .
therefore , despite its lower contrast resolution , the cr method imagery for the outer margin of the cortex is as good as the ssdr method .
the scanning time required to obtain the ssdr of the lower extremities was about 10 seconds . as a result , the ssdr method had a high probability of producing a motion artifact .
however , a previous study demonstrated that motion in a brief period degraded only a small segment of slot scanner image , while it affects the entire portion of cr image ( 18 ) .
continuous motion throughout the image acquisition period caused an artifact that was less apparent with the ssdr method than with the cr method .
actually , in our cases , none of the ssdr images had any significant motion artifacts leading to a difficulty in reaching the correct diagnosis .
the lower extremities are not as affected by spontaneous body movement , which usually occur as a result of respiration , heartbeat , and bowel movement . in this study
, we measured the entrance skin dose instead of the effective radiation dose to evaluate radiation exposure .
generally , the effective radiation dose is used because it reflects the absorption of radiation ( 6 ) . to obtain the effective radiation dose , a thermo - luminescent dosimeter - loaded phantom and commercially available software for calculation are required .
however , we did not have access to such equipment ; especially for the lower extremities . therefore , we had no choice but to measure the entrance skin dose as an alternative method for measuring radiation exposure .
the entrance skin dose for the ssdr method was much lower than that of the cr method ( table 3 ) .
the largest difference in entrance skin dose was recorded at the level of the pelvis , which contained multiple pelvic organs ( urinary bladder , bowel , and large arteries as well as the uterus in females ) , large bones ( pelvic bones , sacrum and femoral heads ) and a fair amount of soft tissue ( subcutaneous and intrapelvic fat , muscles ) . as the body mass of a patient increases , the scatter fraction also increases ( 1 ) .
therefore the entrance skin dose for both sapl and the sl of the cr method was much larger than the entrance skin dose of the ssdr method .
when the sl was performed , the patient was exposed to x - rays on three occasions .
one of the three x - ray exposures was intended to obtain an image at the targeted level , whereas the other two x - ray exposures were caused unnecessarily by a scatter fraction to the other two levels . despite the conclusive results , the study had some inherent limitations ; first
, the images obtained by the cr and ssdr methods could not be obtained at same time .
the mean time interval between first performing the cr , followed by the ssdr was 20 months with a maximum interval time of 75 months .
second , we measured the entrance skin dose to evaluate radiation exposure instead of the effective radiation dose , because we did not have access to a commercially available phantom for the lower extremity region . in conclusion , the data suggest that the ssdr method can provide a better image quality than the cr method in the lower extremity region , as evidenced by a more detailed anatomy in the medullary bone and soft tissue of the lower extremity region . in addition ,
the ssdr method , for the most part , exposes the patient to a lower entrance skin dose , especially for imaging at the level of the hip and knee . | objectiveto compare the slot - scan digital radiography ( ssdr ) of the lower extremity region and the computed radiography ( cr ) method with respect to the image quality and radiation exposure.materials and methodswe enrolled 54 patients who underwent both the ssdr and cr of the lower extremities .
the study evaluated and statistically compared the image quality of four features ( outer cortex , inner cortex , trabeculae and intermuscular fat ) at six different levels ( pelvis , hip , femur , knee , tibia and ankle ) between each method .
the image quality was evaluated using a visibility scale , and the entrance skin dose was measured using a dosimeter at three different levels of a phantom ( hip , knee , and ankle).resultsthe mean image visibility scale values for the ssdr method were significantly higher than for the cr method .
the entrance skin dose for the ssdr method was 278 gy at each level , compared to the entrance skin doses of the cr method , which were 3,410 gy for the hip , 1,152 gy for the knee , and 580 gy for the ankle.conclusionboth the image quality and patient entrance skin dose data suggest that the ssdr method is superior to the cr method for the lower extremity musculoskeletal examination . |
the human oral cavity , much like the rest of the human body , is home to its own microbiota consisting of over 750 species of commensal bacteria , viruses , and fungi , including mutans streptococci ( mainly streptococcus mutans and streptococcus sobrinus ) , which are associated with caries initiation and progression [ marsh , 2003 ; avila et al . ,
the oral microbiota is a dynamic entity and is greatly impacted by oral hygiene practices and diet [ bullon et al .
for example , mutans streptococci upregulate virulence genes when a high level of carbohydrate , especially sucrose , becomes available in the environment .
major virulence genes of streptococcus mutans include gtfb , gtfc , and gtfd , which encode glucosyltransferases ( gtfs ) that bind sucrose as a substrate and form soluble and insoluble glucans [ yamashita et al . , 1993 ; vacca - smith et al . ,
current methods of combating caries - associated bacteria are mostly broad - spectrum antimicrobials [ allaker and douglas , 2009 ] .
it acts by reducing viability of all bacteria in the oral cavity , resulting in a decreased incidence of caries .
antibiotics that alter oral microbiota can put selective pressure on microorganisms to become resistant to drugs , kill symbiotic bacteria , and allow proliferation of other disease - causing pathogens [ banas et al .
, 2007 ; cegelski et al . , 2008 ] . over the past few years
, great advances have been made in identifying specific virulence properties unique to s. mutans .
it is known that it produces insoluble glucans using gtfs and is both aciduric and acidogenic due to f - atpase proton - translocating pumps [ gibbons and qureshi , 1980 ; marsh , 2005 ] .
targeting these unique virulence factors of s. mutans without threatening its existence , or the existence of other species in the oral cavity , is a more attractive approach compared to bactericidal methods because it inhibits pathogenesis and formation of biofilms without stimulating evolution of resistant bacteria [ cegelski et al . ,
there has been a rising interest in naturally derived biologically active compounds that may have potential therapeutic uses in medicine and dentistry [ groppo et al . , 2008 ; newman , 2008 ] .
plants have been used in folk medicine for thousands of years , and even with the advent of modern medicine , products derived from medicinal plants have been the basis for development of many new lead chemicals for pharmaceuticals [ cowan , 1999 ; newman , 2008 ; palombo , 2009 ] .
many currently used antibiotics were discovered by screening natural products and compound libraries against whole organisms , which identified bacteriostatic and/or bacteriocidal properties .
dental medicine has become especially amenable to plant - derived products , driven by evidence that shows populations that regularly incorporate foods or beverages containing certain phytochemicals into their diet have better oral health [ palombo , 2009 ] .
for example , japanese tea drinkers have been known to have decreased incidence of caries compared to other populations [ hamilton - miller , 2001 ; taylor et al . , 2005 ] .
italian researchers examined the oral health of groups of people with specific , long - standing drinking habits ( regular consumption of either coffee , barley coffee , tea , or red wine ) .
they found that those with the drinking habits had significantly better plaque scores and had lower bacterial counts of mutans streptococci and lactobacilli compared to those who did not have drinking habits , implying that these plant - based beverages must have some active compound(s ) that maintain oral health with regular consumption [ signoretto et al .
some major classes of phytochemicals that are known to have biological activity are simple phenols and phenolic acids , quinones , flavonoids ( including flavonols and flavones ) , and terpenoids [ cowan , 1999 ] .
all of the aforementioned categories , with the exception of terpenoids , can be classified under the broader class of
polyphenols. polyphenols are one of the most common , widespread groups of substances produced in plants and it has long been known that they have a broad range of health - promoting activities , including antimicrobial activity [ tapiero et al . ,
various in vivoand in vitro studies have shown that polyphenols are able to selectively interfere with specific virulence traits of s. mutans , preventing adherence to tooth surfaces and inhibiting gtf enzymes , without having a marked effect on bacterial viability [ koo et al .
since diet is a key factor that defines oral health , much research has focused on widely consumed food products .
tea ( camellia sinensis ) and american cranberry fruit ( vaccinium macrocarpon ) have received particular attention because of their unique polyphenol compositions and their prevalence in the human diet [ hamilton - miller , 2001 ; taylor et al . ,
an abundance of studies have reported bacteriostatic or bactericidal activities for compounds derived from hundreds of higher plants , but this review will mainly focus on studies conducted using cranberry fruit and the various forms of tea due to ( i ) their high rate of consumption , ( ii ) relative lack of toxicity , and ( iii ) tendency to be bacteriostatic rather than bactericidal , and discuss their applicability in the context of clinical oral health and caries management . table 1 summarizes the biological activities of polyphenol compounds isolated from natural sources discussed in this review .
a polyphenol , in its simplest definition , is any substance that contains at least one aromatic ring with one or more hydroxyl groups ( other substituents can be present ) .
polyphenols are sorted into approximately 15 major classes [ harborne , 1964 ] , and of those classes , the flavonoids family contains the largest variety of compounds that show pharmacological activities [ cushnie and lamb , 2005 ; tavares et al . , 2010 ;
the basic structure of a flavonoid consists of two aromatic rings linked by three carbons , forming a heterocyclic ring ( fig .
. exact classification of flavonoid compounds varies depending on the literature ; according to bennick , there are two major subclasses within the flavonoid family : 3-desoxyflavonoids , also referred to as flavones due to the presence of a ketone group at the c4 carbon ( fig .
1e ) , which resemble flavonols in structure , but do not have the ketone group at the c4 carbon .
variations in position , number , and type of substituents give rise to a great diversity of flavonoids .
flavan-3-ol units ( such as catechin ) can polymerize to form proanthocyanidins ( sometimes referred to as condensed tannins ) of varying molecular weights .
it is important to note here that proanthocyanidins and polymers of flavan-3-ols are sometimes referred to as flavanols , not to be confused with flavonols .
proanthocyanidins can exist as oligomers ( containing up to 6 catechin units ) , which are soluble , or polymers ( more than 6 catechin units ) , which are insoluble .
plants contain varying degrees and ratios of low - molecular - weight polyphenol monomers , dimers , and trimers to high - molecular - weight polymers .
molecular weight and/or solubility of these polyphenols , in addition to number and location of substituents , seem to be correlated with biological activity [ hemingway et al . , 1992 ; haslam and cai , 1994 ; gross et al . , 1999 ] .
through in vitro testing , many biological and pharmacological activities have been demonstrated for this family of compounds , ranging from direct bacteriocidal action to selective inhibition of specific enzymes .
it is generally believed that one of the major modes of action of polyphenols is the ability to complex with other molecules [ haslam and cai , 1994 ] .
tea is one of the most widely consumed beverages in asian countries and it was recently reported that tea is also the major source of flavan-3-ols and flavonols in the us diet [ song and chun , 2008 ] .
tea is known to be a rich source of polyphenols , but polyphenol type and levels vary depending on how the tea has been processed [ astill et al . ,
there are various ways a tea can be processed ( green , black , oolong ) , but all tea comes from the plant camellia sinensis .
green tea is unfermented and contains mostly monomeric polyphenols such as epicatechin , epicatechin gallate , and epigallocatechin ( fig .
1f ) ; black tea has been fermented and many of the simple catechins have been oxidized to larger molecules of higher molecular weight . oolong tea is semifermented and contains a mixture of monomeric and oligomeric polyphenols [ yam et al . ,
hamilton - miller claims that tea , in any of its varieties , does not contain
tannins , or what has been defined earlier in this paper as high - molecular - weight polymeric polyphenols . however , certain studies ( discussed below ) claim that oolong tea does contain unique , unspecified polymeric polyphenols not present in green tea , and that these polyphenols show significant inhibitory activity . a possible explanation for this inconsistency may be due to the fact that authors referring to the polymeric polyphenols in oolong tea technically meant oligomeric polyphenols ( the technical difference being that oligomers are smaller and soluble and polymers are larger and insoluble ) .
tannin must be established . further complicating the matter is the existence of evidence suggesting that certain polyphenolic components of tea , particularly monomeric catechins , may directly be bactericidal .
sunphenon , a crude green tea extract , was able to cause a significant inhibition in adherence of s. mutans to saliva - coated hydroxyapatite , as well as to inhibit gtf activity in vitro .
the same study showed that when rats were fed a diet enriched with sunphenon ( in both food and water ) , caries formation was greatly inhibited .
otake et al . attributed the results of the study to sunphenon 's ability to prevent adherence and inhibit gtf enzyme , but also mentioned that sunphenon ( or any of its constituents ) may be bactericidal .
the authors did not offer possible mechanisms as to how it is bactericidal nor did they corroborate such claims with bacterial viability assays .
preliminary chemical analyses of the polyphenolic substances in oolong tea extract showed that oolong tea extract contained significant quantities of unknown polyphenols that are not detected in green tea .
a fraction of oolong tea extract , designated otf10 , did not in fact contain any low - molecular - weight compounds and showed the strongest inhibition of s. sobrinus gtf activity .
the authors did not identify the exact structure of the polymeric polyphenols in oolong tea but suggested that polymerization of monomeric catechins during the fermentation process may be critical to the inhibitory effects on gtf .
ooshima et al . examined the effects of a monomeric - catechin - free fraction of oolong tea ( referred to as ot-6 ) on bacterial growth as well as the effects in vivo .
ot-6 did not affect growth of s. mutans or s. sobrinus ; however it did significantly inhibit insoluble glucan synthesis by gtfs .
when rats infected with s. mutans were given food and water treated with ot-6 , plaque index and caries score were significantly reduced [ ooshima et al .
matsumoto et al . attempted to elucidate exactly how polymeric polyphenols found in oolong tea may exert inhibitory activities on bacterial gtfs by cloning functional domains of a recombinant gtf enzyme the n - terminal catalytic domain and the c - terminal glucan - binding domain .
matsumoto et al . found that the polymeric polyphenol fraction specific for oolong tea ( devoid of low - molecular - weight catechins ) hindered glucan synthesis by noncompetitively inhibiting the glucan - binding domain region of the enzyme , not the n - terminal catalytic domain .
sasaki et al . also studied the effects of oolong tea , but looked at the fractions of oolong tea that did not contain the high - molecular - weight compounds .
in other words , the fraction that was studied included entirely monomeric compounds ( gcg , egcg , cg ) .
this monomer - rich fraction , referred to as ot-2 , exhibited significant antibacterial activity against mutans streptococci ; however , when ot-2 was pretreated with bovine serum albumin , all bactericidal activity was abolished [ sasaki et al . ,
the authors postulated that the monomeric polyphenols have a high tendency to complex with proteins , and such reactions neutralize any bactericidal potential .
in the human oral cavity , the monomeric polyphenols affinity for proline - rich salivary proteins may diminish their bactericidal potential . as mentioned earlier , oolong tea contains a mixture of both monomeric and oligo-/polymeric polyphenols so it is possible that these exert distinctive cariostatic effects . however , there are still unanswered questions regarding the activities and structure - function relationships of tea polyphenols
both generated strong evidence that polymeric polyphenols inhibit gtf activity , but they did not provide any insight into whether they may or may not have any bactericidal potential by conducting bacterial viability assays .
also did not address the possibility of protein interaction of the polymeric polyphenol - rich fraction of oolong tea .
the fact that ot-6 exhibited inhibitory effects against gtf in vivo [ ooshima et al . ,
1993 ] suggests that polymeric polyphenol activity may be preserved even in the presence of protein - rich saliva , but more evidence is necessary to corroborate this .
furthermore , monomeric catechins such as ec , egc , and egcg were shown to inhibit gtf in some studies [ otake et al . ,
it is worth exploring whether or not they share similar modes of action , as well as to what extent monomeric catechins affect cell viability .
teas offer a great variety of bioactive compounds but characterization of specific compounds and structure - activity relationships remain elusive .
green teas , with their high monomeric , potentially bactericidal polyphenol content , may hold less use than oolong teas , which contain known polymeric enzyme inhibitors .
more information is needed on how monomeric catechins of tea will interact with salivary proteins .
few studies have been conducted on black teas , mainly due to the inability of chromatographic methods to characterize its chemical components .
cranberry fruit ( vaccinium macrocarpon ) has well - known antiadhesive properties and holds great potential as an antiadhesion agent against cariogenic mutans streptococci .
the therapeutic potential of cranberry was first appreciated with escherichia coli , when it was observed that cranberry juice prevented urinary tract infections by inhibiting of adherence of bacteria [ avorn et al . ,
1994 ] ; it was also found to be capable of inhibiting the adherence of helicobacter pylori to host gastric cells [ burger et al . , 2000 ] .
many fruits that are a part of the human diet contain polyphenols , but cranberry is the only fruit that has consistently and effectively been shown to inhibit biofilm formation [ johnson - white et al . ,
johnson - white et al . attempted to simulate the antiadhesive properties of cranberry juice and could not find such effects in apple , orange , or grape juices .
yamanaka et al . treated s. mutans and s. sobrinus cells with cranberry juice and found that it reduced cell surface hydrophobicity of the cells , interfering with adhesion and the initial stages of biofilm formation .
the authors hypothesized that cranberry juice components may interact with hydrophobic proteins on the surface of the bacterial cell . since hydrophobicity is an important factor in the initial attachment of the bacteria to a surface , reducing hydrophobicity decreases likelihood of adhesion .
steinberg et al . used a high - molecular - weight , nondialyzable constituent ( ndm ) of cranberry juice to study its effects on s. mutans gtf activity and found that ndm significantly reduced the activities of gtf as well as fructosyltransferase , both in solution and immobilized on saliva - coated hydroxyapatite .
interestingly , the antiadhesion effect of ndm was less pronounced , though still present , when sucrose was not present in the reaction mixture . the precise structure of any active components within the ndm could not be resolved by chromatographic methods due to the high molecular weight [ steinberg et al . ,
in addition to cell surface hydrophobicity and ability to synthesize glucans using gtfs , mutans streptococci are also acidogenic and aciduric .
high metabolic activity creates an acidic environment , but the bacterium is able to maintain an alkaline intracellular ph by removing protons through the use of an f - atpase proton pump .
conducted a study comparing the efficacies of the three main polyphenolic components of cranberry : flavonols ( such as quercetin , kaempferol , and myricetin ) , anthocyanins ( flavan compounds containing a glycoside moiety ) , and proanthocyanidins ( flavan-3-ol oligomers )
. results showed that , out of all the compounds tested , proanthocyanidins most efficiently inhibited bacterial gtfs [ duarte et al . ,
more importantly , flavonols and proanthocyanidins were shown to interfere with the bacteria 's f - atpase activity , preventing ph drop and subsequent acidification of extracellular environment .
flavonols such a quercetin are known noncompetitive inhibitors of f - atpases , but this was the first study that demonstrated similar capabilities in proanthocyanidins .
it is unknown whether or not proanthocyanidins share the same inhibitory mechanism as non - proanthocyanidin flavonols .
ultimately , cranberry polyphenols were able to suppress multiple virulence traits without affecting bacterial viability .
flavonols and their glycosides such as myricetin and myricetin 3-rhamnoside exhibited significant but moderate effects .
monomer flavan-3-ols and phenolic acids such as epicatechin , caffeic acid , and chlorogenic acid showed negligible inhibitory activity ; however , procyanidin a2 , a dimer of epicatechin , did show inhibitory activity against gtfs [ gregoire et al .
the epicatechin units of the dimer are linked by an a - type interflavan linkage which is believed to make the molecule rigid .
2 ) , which may possibly explain the lack of inhibitory activity of similar polyphenols in non - cranberry fruits such as apple , which contains procyanidin b2 lacking the a - type interflavan linkage [ johnson - white et al . , 2006 ; gregoire et al . , 2007 ] .
the authors also noted that the degree of polymerization may be a critical factor that determines biological activity .
none of the polyphenols tested had bactericidal effects on s. mutans , which is noteworthy since low - molecular - weight components in tea were reported to have an effect on bacterial viability [ otake et al . , 1991 ] . in the aforementioned studies , cranberry flavonols demonstrated moderate inhibitory activity but proanthocyanidins seemed to demonstrate the most significant inhibitory effects against bacterial gtf and f - atpase .
koo et al . exclusively examined proanthocyanidins of various degrees of polymerization and their effects of gtf activity and ph drop .
all other proanthocyanidins were able to inhibit gtf ; proanthocyanidins that had a degree of polymerization between 4 and 12 were the most potent inhibitors [ koo et al . ,
proanthocyanidin oligomers that had degrees of polymerization greater than 3 only had modest inhibitory effects of acid production ; however , the proanthocyanidin dimer procyanidin a2 was more inhibitory [ duarte et al . , 2006 ; gregoire et al . , 2007 ] .
it is important to note that none of the proanthocyanidins tested showed bactericidal activity [ duarte et al . , 2006 ; gregoire et al . , 2007 ] . in animal studies , proanthocyanidins was able to significantly reduce the incidence of smooth surface caries only and fluoride ( or fluoride in conjunction with proanthocyanidin ) was more effective than the treatment with proanthocyanidins alone [ koo et al . , 2010 ] .
it defined a precise range of degree of polymerization for which these compounds would have activity , and it also demonstrated that degree of polymerization is an important factor in determining what aspect of virulence would be targeted ( gtf or f - atpase ) .
in other words , it is the combination of polyphenols of varying sizes , and not any single compound , that is able to exert multitarget - inhibitory activities .
2010 ] proposed that proanthocyanidins with degrees of polymerization between 4 and 12 were optimal for interaction and inhibition of gtf ; any greater polymerization actually diminished inhibitory activity . as with tea polyphenols , there are still questions that need to be addressed .
what still remains unclear is the exact mechanism in which proanthocyanidins disrupt acid production and acid tolerance in s. mutans , a major virulence trait .
low - molecular - weight polyphenols ( such as flavonols and procyanidin a2 ) are thought to partially inhibit the f - atpase membrane - associated enzyme .
the role of oligomeric proanthocyanidins in ph drop is more elusive , and the degree of polymerization does not seem to be related to any effects in glycolytic ph drop .
nevertheless , it is evident that proanthocyanidin oligomers directly interact with bacterial membrane and though precise mechanisms are unknown , they can cause membrane disturbances which affect the glycolytic pathway .
proanthocyanidins and other compounds derived from cranberries , an already popular and sustainable fruit , have promising cariostatic properties and may potentially be developed as a therapeutic supplement to fluoride , the only proven anticaries agent currently in use .
traditional medicinal plants help researchers narrow their focus , so as to avoid a haphazard , random search for useful compounds [ kingston and newman , 2005 ; newman , 2008 ] . in that sense ,
cranberry and tea polyphenols are the most sensible sources for polyphenols in that they are already widely consumed as part of the human diet .
such factors explain why much of the research conducted is disproportionately devoted to these two plants . nevertheless , there are some natural bioactive compounds that are not derived from common foods per se , but have demonstrated remarkable potential to inhibit virulence traits of cariogenic bacteria .
one unusual source for such compounds is propolis , produced by the apis mellifera species of bees .
propolis is a resinous substance consisting of various plant products mixed with beeswax [ ghisalberti , 1979 ] .
flavonoids ( particularly aglycones ) have been detected in propolis , but non - polyphenol compounds , such as terpenoids , have also demonstrated biological activity .
preliminary studies found that propolis was a potent inhibitor of plaque formation and caries induction in rats [ koo et al . , 1999 ] , possibly due to a complex mixture of bioactive compounds .
selected and tested 30 distinct compounds occurring in propolis on the activity of gtf enzymes of s. mutans and streptococcus sanguinis .
flavones and flavonols were potent inhibitors of gtfs ; of these , a compound called apigenin ( 4,5,7-trihydroxyflavone ) was the most effective inhibitor of gtfs ( fig .
some compounds exhibited bactericidal activity ; of these , tt - farnesol ( a sesquiterpene ) was the most effective antibacterial agent .
when apigenin and tt - farnesol were isolated and tested on s. mutans biofilms in vitro either separately or in combination [ koo et al . , 2003 ] , all experimental groups showed some reduction in plaque accumulation , but only the combination of test agents resulted in a significant effect .
further investigation showed that tt - farnesol has bactericidal activity in planktonic cells of mutans streptococci , but has limited bactericidal effects on biofilms at highest therapeutic doses tested ; tt - farnesol seemed to also disrupt glucan synthesis and acid product , but in a mechanism different from its chemically distinct partner apigenin .
it seems most like that tt - farnesol affects the membrane integrity of bacteria by changing the permeability or fluidity of the cell membrane [ koo et al . ,
koo et al . tested the possible supplementary effects that apigenin and/or tt - farnesol might have on fluoride 's anticaries abilities in vitro as well as in vivo . according to results , a combination of all three agents ( apigenin , tt - farnesol , and fluoride ) was most effective in the reduction of insoluble glucans .
novel , multifaceted combination therapies represent a more efficient method of fighting caries - causing plaque compared to what is widely used in practice now ( use of only one agent , fluoride ) [ koo , 2008 ] .
the critical factor in discovering novel therapeutic compounds is that they should not have significant effects on cell viability . because apigenin and tt - farnesol are both inhibitory without threatening cell viability , they are potential therapeutic compounds that can be evaluated for human safety and clinical efficacy .
the two main polyphenol sources discussed , camellia sinensis and vaccinium macrocarpon , both contain high levels of bioactive compounds ; however , studies conducted on cranberry polyphenols have been more conclusive .
identification of specific structure - activity relationships , such as the unique a - type interflavan linkage in some cranberry polyphenols , has not been achieved for tea .
studies involving tea polyphenols are also limited in that there is little information regarding the extent to which the low - molecular - weight polyphenols are bactericidal .
low - molecular - weight components of cranberry , such as catechin ( which is also found in green and oolong teas ) , did not seem to affect bacterial viability , as was the case in studies using tea catechins .
this could be due to confounding factors such as experimental design , or there could be some inherent , uncharacterized difference between the two sources that affects biological activity .
for example , size ( molecular weight and/or degree of polymerization ) seems to be a significant determinant of biological activity .
data for both tea and cranberry extracts indicate that inhibitory activity increases with increase in molecular weight and degree of polymerization .
studies conducted with apigenin and tt - farnesol derived from propolis support the idea that it is not one specific compound , but more likely a combination of compounds , that will have optimal , multilevel inhibitory activity against s. mutans .
finally , even if some phytochemicals , such as tea monomeric catechins and tt - farnesol , do possess some degree of bactericidal activity , if there is a possible dose that maximizes inhibitory efficiency of such compounds without significantly affecting bacterial cell viability , they should not be ruled out .
traditional antibacterial approaches are falling out of favor due to increased incidence of resistant organisms and virulence - targeted therapies are gaining increased interest . since a great deal
is already known about the critical virulence traits of s. mutans , researchers can focus on how to manage caries by finding ways to target virulence enzymes such as gtfs and f - atpases .
natural products discovered from medicinal plants have provided numerous clinically used medicines and there is an abundance of evidence that naturally occurring compounds , especially from foods that we already consume , may have therapeutic potential in managing caries .
once a potential plant - based therapy has been proposed , certain factors need to be taken into consideration , such as cost , sustainability of the source , effectiveness in comparison to ( as well as in conjunction with ) fluoride , and possible toxicity to humans .
there should also be rigorous testing to ensure that the compound or derivative does not have significant bactericidal effects .
a major goal of studying plant - derived polyphenols is possibility for chemical synthesis of an analog(s ) ; but even if this turns out to be unfeasible , we know from studies in human populations that simple incorporation of these foods is correlated with better oral health and based on that knowledge , certain dietary habits can be advocated .
| there are over 750 species of bacteria that inhabit the human oral cavity , but only a small fraction of those are attributed to causing plaque - related diseases such as caries .
streptococcus mutans is accepted as the main cariogenic agent and there is substantial knowledge regarding the specific virulence factors that render the organism a pathogen .
there has been rising interest in alternative , target - specific treatment options as opposed to nonspecific mechanical plaque removal or application of broad - spectrum antibacterials that are currently in use .
the impact of diet on oral health is undeniable , and this is directly observable in populations that consume high quantities of polyphenol - rich foods or beverages .
such populations have low caries incidence and better overall oral health .
camellia sinensis , the plant from which various forms of tea are derived , and vaccinium macrocarpon ( american cranberry fruit ) have received notable attention both for their prevalence in the human diet as well as for their unique composition of polyphenols .
the biologically active constituents of these plants have demonstrated potent enzyme - inhibitory properties without being bactericidal , a key quality that is important in developing therapies that will not cause microorganisms to develop resistance .
the aim of this review is to consider studies that have investigated the feasibility of tea , cranberry , and other select plant derivatives as a potential basis for alternative therapeutic agents against streptococcus mutans and to evaluate their current and future clinical relevance . |
as science and technology advance providing health , medical , and social benefits to much of society the ethical considerations related to how society uses these advances become apparent . for example , progress in genetic sequencing technologies has led to improved diagnosis , and in some cases treatment , of certain medical conditions .
however , these advances also have raised ethical questions , including when to report increased genetic risk of conditions with no treatments or late onset , and how to protect individuals privacy in large - scale genetic data - sharing environments , among others ( 14 ) .
important societal questions such as these need to be considered and addressed by the public and informed by the scientists most familiar with the technology .
recently , personalized genetic testing ( pgt ) has been used by some educational institutions as a pedagogical tool for teaching human genetics . citing the importance of integrating science and ethics
, we argue that pgt can be an effective educational tool for incorporating ethics into the biology classroom , and provide suggestions for instructors using this approach .
there is consensus among science educators that it is necessary to educate students about the ethical and societal consequences of science to produce well - informed citizens and future scientists ( 4 ) .
current students will come of age in the genomic era , and their decisions will likely affect how personal genetic information is viewed and used in society ; it is important to ensure they understand the scientific concepts and the ethical , legal , and societal issues related to genetic information , creating a significant educational need for both the scientific and ethical aspects of genetics ( 10 ) .
this need can be addressed in part by integrating ethics into undergraduate and graduate science courses directly , for example , by incorporating discussion of relevant ethical considerations into biology and genetics courses .
the presidential commission for the study of bioethical issues asserts that bioethics is fully consonant with a liberal arts and sciences education , and emphasizes the need to develop and evaluate models and curricula that integrate bioethics and science education at all levels of education ( 13 , 15 ) . just as it takes students years to learn the language and concepts of science , students require adequate time to learn the vocabulary and foundational principles of bioethics and develop ethical reasoning skills ( 12 ) . to this end
, ethics can be integrated throughout the science curriculum , exposing students to relevant bioethical issues each semester through case studies , discussions , or assignments that supplement course materials , and giving students the opportunity to build their ethical fluency ( 27 ) . integrating ethics into existing undergraduate curricula does not sacrifice scientific content , but rather enriches it .
advantages to incorporating bioethics into science classes include the potential to spark student interest in both science and ethics , and the opportunity to teach students how to make direct connections between what is being learned in the classroom with the bioethical challenges that are being presented in the public press and in professional journals
this enhanced analysis prepares future scientists and policymakers to deal with ethical questions , increases awareness among future health professionals about the importance of ethical judgments , and enhances the scientific and ethical literacy of college - educated citizenry ( 27 ) .
many have argued that educators have a responsibility to expose students to diverse perspectives on course content , and that consideration of relevant ethical and societal issues broadens students views and allows them to think about the implications of the science they study ( 4 , 7 , 21 , 23 ) .
it is important that students considering careers in science and health care are able to apply basic ethical principles to address and contribute to the resolution of current and future bioethical concerns ( 12 ) . while early integration of ethics and science in education provides exposure to the principles that inform ethical reasoning which helps students build skills to address ethical issues in future professional life
one survey of core requirements for undergraduate biology programs at 104 colleges and universities found that none included a bioethics requirement ( 27 ) .
another survey of introductory genetics instructors found that only 13% of undergraduate institutions in the united states require ethics for biology majors , and only a third of those require an ethics of science course ( 4 ) .
in the same survey , although 99% of instructors indicated a belief that students should be exposed to ethical issues , and 55% felt this should take place within the science classroom , most instructors reported spending less than 5% of class time on ethical issues ( 4 ) .
instructors can integrate ethics into regular coursework and minimize time lost to scientific content by selecting topics closely aligned with the syllabus ( 7 ) .
one strategy is to incorporate ethical concepts into discussion of real - world applications of the science being taught , which helps contextualize ethical concepts and guidelines , since their interpretation relies on situational details ( 18 ) .
an area in which this integration method can be applied is genetics : as the scientific field evolves , education addressing relevant ethical and societal issues is needed to complement the growth of genetics education ( 8) .
scholars suggest that considering relevant ethical issues engages and motivates students to learn more about genetics ( 10 ) .
for example , an instructor might teach a module simulating genetic testing , including consent forms and relevant regulations about informed consent to prompt consideration of ethical issues ( 7 ) .
a simulation module helps students to consider case studies that raise the ambiguities faced by genetic counselors and physicians
some instructors and educational institutions have begun conducting genetic testing with consenting students to generate real - world experiences using technology relevant to course content .
facilitating pgt , whether conducted in an academic or commercial laboratory , can increase student interest in course content , but educators have a responsibility to teach students about the many facets of genetic testing , including the interpretation , limitations , and potential impact of genetic information ( 21 , 23 ) .
although this obligation has been articulated , more evidence and guidance are needed for how instructors using pgt in their teaching can integrate ethics into their classes .
decreased cost and increased accessibility has enabled a growing number of colleges and universities in the united states to offer pgt in courses at the undergraduate , graduate , or professional school levels , including colleges at pennsylvania state university , university of california , berkeley , and university of rhode island ; schools of medicine at harvard university , mount sinai , stanford university , and university of pennsylvania ; schools of pharmacy at ohio state university and temple university ; and the school of nursing at duke university ( 5 , 6 , 7 , 11 , 25 ) .
there is ongoing discussion about the ethical implications of pursuing this pedagogical strategy ( 5 , 19 , 21 , 23 , 26 ) .
many have raised issues of student vulnerability and coercion and concerns as to how to ensure fully informed consent in the classroom pgt process ( 5 , 19 , 21 , 23 ) .
learning about disease risk might cause psychological harm , and require schools to provide genetic counseling services . maintaining privacy and confidentiality
is important as students access personal genetic information and engage in open classroom discussions ( 19 , 21 , 23 , 26 ) .
some also have discussed issues of justice and fairness , including the cost and accessibility of pgt for students ( 19 , 21 , 23 ) .
the literature on pgt as a pedagogical tool focuses on undergraduate and higher levels of education rather than the high school level
. the strategy might be less appropriate in high school classes due to the ethical implications of pgt and additional challenges inherent to obtaining parental permission and child assent to participate .
one way schools have offered students access to their own genetic information is through the use of direct - to - consumer ( dtc ) genetic testing services .
food and drug administration action might curtail dtc genetic testing as it relates to health information , genetic testing delivered through medical professionals will continue and grow ( 28 ) , offering new avenues of incorporating pgt into the classroom and raising additional issues .
for example , if future pgt requires a physician order , must students visit their own doctors ? will there be a separate referring physician affiliated with the class ? and what level of involvement should clinicians have in the course ?
instructors of courses incorporating pgt suggest that students who undergo self - testing might benefit more from lessons and gain a better understanding of the science ( 5 , 22 ) .
by having their own dna analyzed , students gain a personal stake that might encourage them to engage more deeply with the material ( 5 , 25 ) .
several studies examining the use of pgt in classrooms support these claims . in one survey , students indicated they were more likely to enroll in a genetics course if it offered pgt ( 60.2% ) , and that personalized course material is more interesting ( 94.6% ) and easier to learn ( 87.3% ) ; professors agreed that adding pgt increases student interest , participation , and learning ( 6 ) . another study surveying pgt s impact on student learning found that the method enhanced both self - reported and assessed knowledge of genomics ( 20 ) .
interviews conducted with students who chose to undergo pgt indicated that it could improve students self - reported motivation and engagement with course content ( 25 ) .
these findings are important in light of calls to enhance genetic literacy among students and young adults ( 8 , 10 )
. however , pgt also might be an innovative and useful tool to integrate ethical discussion and learning , in response to parallel calls to educate students on the ethical and societal aspects of genetics ( 7 , 8 , 10 , 19 , 23 ) .
done well , pgt in the classroom might increase knowledge about viewing genetic information critically , help students make better use of such technologies in the future , train them to recognize the limits and benefits of genetic testing , and engender consideration of the broader ethical implications of genetic testing ( 5 , 6 ) .
educating students about ethical and societal issues surrounding genetics should be an essential part of pgt in the classroom , given the personal nature of these exercises ( 23 ) .
for instructors considering pgt in their classes , previous courses provide informative lessons and helpful examples of ways to integrate ethics .
the following suggestions for integrating ethics into pgt courses can be applied broadly at the undergraduate , graduate , and professional school levels .
the incorporation of pgt into classrooms deserves thoughtful discussion within the academic community ( 26 ) .
a multidisciplinary approach to course planning , perhaps undertaken by a high - level committee or task force , can anticipate and address the ethical issues incumbent in offering pgt in a classroom setting ( 25 , 26 ) .
a diverse group including scientists , clinicians , genetic counselors , psychologists , bioethicists , legal counsel , and students should be consulted ( 1 , 2 , 5 , 22 ) .
such a committee could evaluate explicitly the ethical issues raised by pgt in the educational setting and propose safeguards .
in addition , the committee could offer advice on specific approaches for integrating ethics into the course .
for example , when tufts university school of medicine conducted this type of deliberative process , the multidisciplinary faculty group recommended a smaller pilot course that would meet regularly to discuss topics including ethical issues and scientific limitations of pgt , early engagement with the institutional review board , and curriculum committees to explore ways to enrich educational content with a discussion of benefits and harms ( 26 ) .
similarly , the icahn school of medicine at mount sinai assembled a multidisciplinary group of experts in genetic counseling , medical genetics , health psychology , and bioinformatics to develop a genomics course in which students , residents , and fellows had the option to access and interpret their whole genome sequences ( 22 ) .
stanford school of medicine also convened a diverse task force comprising faculty in genetics , genetic counseling , law , ethics , education , and clinical departments to evaluate its course before implementation , considering ethical issues and crafting safeguards which led to its approval ; the course gave students the option to have their genomes sequenced by dtc genetic testing companies ( 3 , 21 , 25 ) .
include diverse faculty and pedagogical resources in the course curriculum . including faculty in law , bioethics , genetic counseling , and other fields
can be an enriching educational experience , and is a practical way to integrate ethics into the biology classroom ( 12 ) .
parts of the stanford university course were taught by a clinical geneticist , a genetic counselor , and a bioethicist and lawyer ( 20 ) .
this approach also can help address science instructors concerns about lack of preparation for teaching ethics .
for example , students in the stanford university course screened in the family , a documentary about a woman s struggle to cope with her brca test results ( 21 ) .
in addition , the presidential commission for the study of bioethical issues has developed publicly accessible pedagogical materials to facilitate ethics integration , including modules on informed consent and a guide to incidental findings for consumers of dtc genetic testing services , which might help instructors guide discussion on consent in genetic testing ( 16 ) .
structured , faculty - led discussion is important to facilitate a basic understanding and subsequent application of ethical principles ; students should be encouraged to participate actively in classroom dialogue ( 12 ) .
the first lecture of the icahn school of medicine course focused on ethical and societal issues in genetic testing , and another lecture covered accuracy of results and informed consent in genetics ( 22 ) .
the stanford university course included two weeks of instruction and discussion about the risks , benefits , uses , and limitations of pgt to provide students the necessary background to make an informed decision ( 20 , 21 ) .
topics for discussion include those identified by the stanford university task force : confidentiality , conflicts of interest , coercion , informational risks , informed decision - making , financial accessibility , genetic counseling needs , and the experimental nature of the course offering ( 25 ) . at stanford university
, the debates that started in the high - level review percolated into the classroom , as faculty presented arguments for and against pgt to students ( 5 ) ; such deliberation and debate could very well be the best way to thoroughly explore the many ethical issues raised by [ pgt ] and to engage students in thinking about this controversial and cutting edge application of genomics ( 5 ) .
courses also could address foundational ethical theories and the regulatory landscape of genetic testing ( 7 , 24 ) .
these topics are more extensive than what is typically covered by research or professional ethics courses .
for example , a pennsylvania state university course gave undergraduates the opportunity to undergo genetic sequencing using a commercial dtc testing service and addressed research ethics as a lecture topic , but the course syllabus does not mention ethical or societal issues of genetic testing ( 17 ) .
similarly , temple university school of pharmacy offered a laboratory course allowing students to extract their own dna and perform genotyping analysis ; it taught students the ethical aspects of collecting and handling genetic information , but not the broader ethical considerations of pgt ( 9 ) .
it is important to introduce ethical and societal considerations and scientific limitations before students decide whether to get tested , as studies have indicated that students informed decision making can be influenced and enhanced by these discussions ( 1 , 2 , 20 , 22 ) .
however , it also is useful to integrate broader discussions of ethical and societal issues throughout the course before , during , and after students undergo pgt .
sustained engagement is important , as ethical issues arise at each step of the pgt process .
instructors should consider formalizing the consideration of ethical and societal issues as a course objective , and include ways to evaluate learning in student assessments .
one way to measure awareness of and engagement with bioethical issues is through a writing assignment , in which students can reflect upon relevant issues and how consideration of these issues influenced their decision of whether to undergo testing ( 19 , 23 , 24 ) .
many have called for additional research on pgt as an educational tool to confirm its effectiveness and better understand student perspectives ( 6 , 20 , 21 ) . however , these calls have not emphasized evaluating the pedagogical usefulness of pgt for teaching ethics .
the few existing studies focus primarily on whether pgt enhances scientific understanding or whether students made informed decisions regarding self - testing , and research is limited to the medical or graduate school contexts .
for example , while post - course surveys at temple university indicated increased student comprehension of the relevance of genetic analysis ( 9 ) , and student questionnaires at the icahn school of medicine indicated that providing introductory education about genetic testing might facilitate informed decision making ( 22 ) , neither assessment addressed whether students gained a deeper understanding of ethical and societal issues .
a stanford university study offers one example of post - course evaluation which includes ethical issues in open response questions ( 25 ) .
interviews with students who chose to undergo pgt indicate that using personal genetic information motivated learning ; the course provided them with insight into what future patients might experience ; they thought more deeply about the risks and benefits of testing ; and they benefited from reflecting on ethical and societal issues presented in class ( 25 ) .
students valued discussion of ethical issues because it allowed them to consider dimensions to their decision that they had not identified prior to the course ( 25 ) .
these responses suggest that using pgt in an educational setting can help students gain a greater understanding of broader issues related to genetic testing ( 25 ) .
combined with another study reporting improved grasp of biological concepts by students who elected to undergo pgt ( 20 ) , these data point to the usefulness of pgt for teaching both science and ethics .
investigations into the utility of pgt as a method for integrating ethics are necessary to ensure that all educational goals for students including an increased understanding of ethical considerations are well served ( 6 , 12 ) .
robust evaluation of the pedagogical impact of pgt as a tool for teaching ethics in various educational settings is needed to identify best practices and set standards for future courses .
scientific and technological advances such as those made in genetics give rise to important and complex ethical issues .
it is critical to educate students to become scientifically and ethically literate researchers , health professionals , and citizens who can engage effectively in discussions and decision making about science in personal , professional , and societal contexts .
science educators should work to ensure that students learn about the many aspects of genetic testing , including its benefits , risks , limitations , and relevant ethical and societal issues .
although further research on its pedagogical effectiveness is needed , the use of pgt in the classroom is an opportunity to incorporate ethics in an innovative , participatory , and engaging format . done well | personalized genetic testing ( pgt ) has been used by some educational institutions as a pedagogical tool for teaching human genetics .
while work has been done that examines the potential for pgt to improve students interest and understanding of the science involved in genetic testing , there has been less dialogue about how this method might be useful for integrating ethical and societal issues surrounding genetic testing into classroom discussions . citing the importance of integrating ethics into the biology classroom
, we argue that pgt can be an effective educational tool for integrating ethics and science education , and discuss relevant ethical considerations for instructors using this approach . |
the california women 's health survey is an ongoing , monthly telephone survey that collects information on health - related behavior and attitudes from randomly selected adult women ( 8,9 ) .
trained staff interviewed participants in 2000 ( n = 4,012 ) and 2003 ( n = 4,004 ) in english ( n = 7,058 ) or spanish ( n = 958 ) .
participants reported race , ethnicity , country of birth , year of entrance into the united states , and current age , county of residence , number and age(s ) of children , educational attainment , household income , and access to healthcare coverage .
we defined the key misconception as an affirmative answer to the following dichotomized question : " antibiotics , such as penicillin , doxycycline , or amoxicillin , are used to treat a variety of medical conditions ; do you believe that a cold or flu should usually be treated with antibiotics ? "
we calculated the crude proportion of women who held this misconception by year with weighted proportions and mantel - haenszel chi - square statistics that adjusted each sample to the age and race distribution of the 1990 california population ( 8,9 ) .
we examined the relationship between the misconception and the characteristics listed in table 1 by year of study by using prevalence proportions and unconditional logistic regression . to produce comparable results , multivariate models for 2000 and 2003 included all variables that demonstrated a significant association with the misconception in either year in univariate analyses .
we developed a third multivariate model that combined data from both years to assess the independent association between the misconception and year of study .
while we used logistic regression to assess for confounding , we present unadjusted prevalence ratios and calculate relative prevalence reductions to measure changes in the misconception over time .
we did so because the unadjusted and adjusted results by logistic regression were equivalent ( which suggests an absence of confounding ) and because the odds ratio did not reasonably approximate the prevalence ratio for women without a high school education ( 10 ) .
we considered p values 0.01 to be significant , but we present 95% confidence intervals ( cis ) to facilitate comparing our results to those from other studies .
we restricted all models to participants who gave usable responses to all variables ( n = 7,430 ) and assessed models for potential 2-way interactions , goodness of fit , and potential colinearity . * or , odds ratio ; ci , confidence interval ; ged , general equivalency diploma .
percentages for race / ethnicity and age are weighted to the 1990 california population ; all other percentages are unweighted .
central = fresno , kern , kings , madera , merced , monterey , san joaquin , san luis obispo , santa barbara , santa cruz , stanislaus , and tulare counties ; southern = imperial , los angeles , orange , riverside , san bernardino , san diego , and ventura counties ; northern = all remaining counties . in california , 21.0% of
women surveyed in 2003 believed a cold or flu should usually be treated with an antibacterial drug .
table 1 lists factors independently associated with this key misconception among california women . in both 2000 and 2003 , the unadjusted prevalence of the misconception was greatest among women who were hispanic , 1834 years of age , had an annual household income $14,999 , had < 12 years of education , resided in central or southern california , had lived in the united states for 5 years , had children < 6 years of age in the household , or had no access to a healthcare plan . we observed a 26.3% decline in the prevalence of the misconception in all women surveyed in 2003 compared with those surveyed in 2000 ( 21.0% vs. 28.5% , p<0.001 ) .
first , we detected a strong statistical interaction between education and year ( p = 0.007 ) ; women with a college diploma were 47.0% less likely and women without a high school diploma were 17.0% less likely to report the misconception in 2003 than those surveyed in 2000 ( table 2 ) .
women with a high school diploma were as likely to report the misconception in 2000 and 2003 .
second , although not significant , after adjusting for other associated factors , asian women were 55.7% less likely to report the misconception in 2003 compared with those surveyed in 2000 .
other women were , on average , 24.0% less likely to report the misconception in 2003 than in 2000 . among women
55 years of age , african american women were as likely to report the misconception in 2003 as 2000 , and their adjusted odds ratio for the misconception increased from 3.1 ( 95% ci 1.705.67 ) in 2000 to 5.3 ( 95% ci 2.6710.52 ) in 2003 ( detailed data on race stratified by age and year not shown ) . *
pr , unadjusted prevalence ratio ; ci , confidence interval , ged , general equivalency diploma . number respondents with misconception divided by total number respondents .
our survey results show a 26.3% decline from 2000 to 2003 in the misconception among california women that a cold or flu should usually be treated with an antibacterial drug .
this finding suggests that general educational campaigns before and during this period may have contributed to moderate reductions in the prevalence of misconceptions about antibacterial drug use .
although this study was not designed to evaluate specific interventions , our study period and population - based sample can provide an ecologic assessment of changing beliefs .
the decline in the misconception was greatest among women who were non - hispanic white or asian , had at least some college education , or had higher household incomes . among levels of education ,
this finding may reflect a true unexplained finding , an artifact of the sample ( selection or information bias ) , or chance .
the inverse relationship between the misconception and the presence of children < 6 years of age in the household in 2000 may indicate success in initial educational campaigns focused on pediatricians and mothers with young children ; that this relationship becomes less significant in 2003 may indicate that other women are " catching up . "
our findings , drawn from a large , ethnically diverse , population - based sample , show that 21.0% of california women in 2003 still held the misconception , particularly women who were younger , black , or hispanic ; had lower educational attainment or household income ; resided in central and southern california ; or had no healthcare coverage .
although not restricted to women , other studies identified similar demographic and socioeconomic risk groups ( 11,12 ) .
social services intended to reach persons with these characteristics may be important venues for education on appropriate antibacterial drug use .
cultural and social perspectives of disease , including differences in antibacterial drug regulations that affect the availability and cost of drugs in immigrants ' countries of birth , can play an important role in misconceptions about antibacterial drugs ( 13,14 ) .
we documented racial and ethnic variation in california women with the misconception , despite adjusting for selected socioeconomic factors ; the remaining variation likely reflects unmeasured socioeconomic or cultural factors .
we note , in particular , the persistent prevalence of the misconception in african american women and the decreased prevalence of it in asian women .
while this study could not evaluate specific interventions , some california aware activities were focused on asian physicians during 2003 .
other research has documented racial and ethnic variation in parental expectations of antibacterial drugs ( 15 ) .
new intervention studies and campaigns aimed at educating and influencing women in specific minority , education , and income subgroups about appropriate antibacterial drug use are needed .
first , although women may be more likely to be the health decisionmakers for young children and for the household , we do not know if our results can be generalized to men .
second , we did not have sufficient sample size to explore specific subgroups within the asian race category .
third , we only included women in households with telephones who spoke either spanish or english and chose to participate .
fourth , we were only able to interview women in spanish or english , which may limit comparability between immigrant hispanic and asian women .
in conclusion , our surveys document a decline in a key misconception in california women about appropriate antibacterial drug use and support continuation of educational campaigns to address this public health problem .
further refining and focusing of these educational efforts are needed to reach minority women and women with lower educational attainment and lower household incomes . | to our knowledge , previous population - based surveys have not assessed misconceptions about antibacterial drug use over time .
we documented a 26.3% decline in a key misconception in california women in 2003 compared to 2000 ; declines varied significantly by education level . educational campaigns specifically designed to influence important subpopulations are needed . |
tuberculosis ( tb ) remains the most widespread and deadly infectious disease worldwide , and it has been recently declared as a great emergency by the world health organization ( who ) . unfortunately , 8.99.9 million new cases of tb were reported in 2008 , while 1.11.7 million human immunodeficiency virus ( hiv)-negative patients died from this disease .
the factors which are mainly responsible for this evolving incidence are : ( i ) the migration of individuals from regions with high tb incidence to those with low incidence , ( ii ) the increasing number of hiv - positive individuals , and ( iii ) the emergence of multidrug - resistant tb due to unwise use of antibiotics . in a total population of approximately
11 million people , the incidence of tb in greece is estimated at 18/100,000 persons and the mortality rate at 2 /100,000 annually [ 24 ] .
the spread of mycobacterium tuberculosis in the majority of humans is inhibited after the development of an effective immune response .
interestingly , only a minority of these patients have an identifiable risk factor , such as hiv infection , advanced age , alcohol abuse , chronic corticosteroid therapy , diabetes and poor nutrition / socio - economic status . in the remaining patients ,
studies in twins strongly suggest that host genetic factors play a major role in the susceptibility to development of tb .
intense efforts are therefore being made to discover any possible genetic factors that may have a role in the host defense system and explain the increased susceptibility to overt m. tuberculosis infection . in 1981 a gene was found in mice that played an essential role in the determination of resistance to mycobacteria and other intracellular pathogens .
the human homologue of the nramp1 gene , designated nramp1 , now known as slc11a1 , is located on the long arm of chromosome 2 ( 2q35 ) .
nramp1 protein seems to play an important role in regulation of cytoplasmic cation levels , especially iron .
iron is an essential mycobacterial nutrient , and has a major role in generating reactive oxygen and nitrogen intermediates in macrophages ; hence , it affects the intracellular growth of mycobacteria .
although it was believed that the gene is expressed solely in reticuloendothelial cells , it was recently shown to be expressed in cd11c dendritic cells , and exerts its pleiotropic effects on cytokine transcription , major histocompatibility complex ( mhc ) class ii molecule expression , and presentation of protein antigens to t lymphocytes .
the possible association of nramp1 polymorphisms with susceptibility to development of tb has been studied in different countries .
a case - control study , conducted in west africans , showed a positive association of the gene with pulmonary tb .
a similar study , conducted in koreans , detected a significant association for the 3utr polymorphism , but not for the int4 locus . on the other hand ,
studies conducted in denmark , mexico , morocco , japan , brazil and china suggested that the gene is not associated with an increased risk for developing tb [ 1517 ] . in a study conducted in the chinese population ,
nramp1 allelic polymorphisms at the int4 and d543n loci were each significantly associated with severe forms of pulmonary tb .
the aim of the present study was to explore the association between nramp1 polymorphisms and the susceptibility to develop active pulmonary tb after a latent m. tuberculosis infection , in the greek population .
the inclusion criteria for the patients group were : ( i ) greek origin ; ( ii ) all patients should be genetically unrelated ; and ( iii ) diagnosis of pulmonary tb based on suggestive radiological findings , a microscopic examination of sputum smears from an experienced microscopist , and documented in all patients with the presence of positive sputum cultures for m. tuberculosis infection .
the exclusion criteria for the patients group were : ( i ) presence of extrapulmonary tb ; ( ii ) patients known to have an hiv infection were not recruited ( all the other patients who agreed to participate were screened for hiv antibodies and hiv - positive patients were not enrolled in the study ) ; and ( iii ) presence of any immunodeficiency syndrome or systemic disease .
a 5 ml blood sample was drawn from 142 greek patients with pulmonary tb who were hospitalized in the sotiria general hospital of thoracic diseases from january 2004 until december 2008 .
the ethics committee of sotiria general hospital , athens , greece , accepted the study protocol .
currently , in the athens area of greece , sotiria general hospital of thoracic diseases is the health institution where the majority of tb cases are diagnosed and monitored .
the sample size was considered to be adequate for the study , taking into consideration the size of the greek population and the incidence of tb in greece . moreover , during the study period , cases of pulmonary tb were diagnosed , but were excluded from the study because most of these tb cases were diagnosed in immigrants of foreign origin and due to the patient s co - morbidities .
the inclusion criteria for the control group were : ( i ) ethnically matched volunteers who visited sotiria hospital for a routine check - up ; and ( ii ) the presence of a latent m. tuberculosis infection .
latent m. tuberculosis infection was defined as a positive tuberculin skin and at least 2 negative sputum cultures , combined with the absence of suggestive radiological findings for pulmonary tb .
, healthy subjects who visited sotiria hospital for a routine check - up were enrolled .
a 5 ml blood sample was drawn from every healthy volunteer , and was handled identically to the patients group . in all controls
specifically , 0.1 ml of purified protein derivative ( ppd ) , which contained 5 tuberculin units , was injected intradermally in the volar surface of the forearm .
the transverse width of induration in millimetres ( mm ) at the skin test site was measured after 4872 hours .
a positive tuberculin skin test was defined as skin transverse width 12 mm . in all of the controls enrolled , a chest - x - ray ( cxr ) and sputum cultures for m. tuberculosis
one hundred forty - four ethnically matched volunteers with latent m. tuberculosis infection were included .
the exclusion criteria for the control group were : ( i ) hiv - positive subjects ; ( ii ) history of prior tb ; ( iii ) evidence of current or prior tb in the cxr ; and ( iv ) presence of any immunodeficiency syndrome or systemic disease .
the kit used for the extraction was purelink genomic dna kit catalog no.s k1820 - 01 , k1820 - 02 , k1821 - 04 , from invitrogen .
the nramp1 polymorphisms typed were : ( i ) a single - nucleotide change in intron 4 ( int4 ) ( 469 + 14g / c ) ; ( ii ) d543n , a non - conservative single - base substitution at codon 543 , which changes aspartic acid to asparagine ; and ( iii ) 3utr , a tgtg deletion in the 3untralsated region ( 1729 + 55del4 ) .
the pcr primers for the int4 polymorphism were 5ctctggctgaaggctctcc3 and 5tgtgctatcagttgagcctc3. the primers for d543n and 3utr were 5gcatctccccaattcatggt3 and 5aactgtcccactctatcctg3 , respectively .
real - time pcr studies were performed in a lightcycler 480 instrument , roche . for each of the 3 polymorphisms analyzed ,
a lightsnip kit was designed from the constructor company , tib - molbiol , germany .
the pcr reaction mixture contained 14.410.4 l pcr grade h20 , 1.0 l reagent mix 2.0 l lightcycler faststart dna master hyprobe 1.6 l mgcl2 and 1.05.0 l dna template .
initially , variants at 3 polymorphisms ( 3 utr , int4 , d543n ) in the nramp1 gene were compared between tuberculosis cases and controls with fisher s exact test . to assess the differences in demographic characteristics , we used chi - square ( for categorical data ) and t test ( for continuous data ) .
deviations from hardy - weinberg equilibrium ( hwe ) were assessed using the chi - square test .
subsequently , the 3 nramp1 polymorphisms were each independently associated with tuberculosis through logistic regression analysis after adjustment for age and sex .
the sas statistical package ( version 9.1 , sas institute inc , cary , nc ) was used to analyze the data .
the inclusion criteria for the patients group were : ( i ) greek origin ; ( ii ) all patients should be genetically unrelated ; and ( iii ) diagnosis of pulmonary tb based on suggestive radiological findings , a microscopic examination of sputum smears from an experienced microscopist , and documented in all patients with the presence of positive sputum cultures for m. tuberculosis infection .
the exclusion criteria for the patients group were : ( i ) presence of extrapulmonary tb ; ( ii ) patients known to have an hiv infection were not recruited ( all the other patients who agreed to participate were screened for hiv antibodies and hiv - positive patients were not enrolled in the study ) ; and ( iii ) presence of any immunodeficiency syndrome or systemic disease .
a 5 ml blood sample was drawn from 142 greek patients with pulmonary tb who were hospitalized in the sotiria general hospital of thoracic diseases from january 2004 until december 2008 .
the ethics committee of sotiria general hospital , athens , greece , accepted the study protocol .
currently , in the athens area of greece , sotiria general hospital of thoracic diseases is the health institution where the majority of tb cases are diagnosed and monitored .
the sample size was considered to be adequate for the study , taking into consideration the size of the greek population and the incidence of tb in greece . moreover , during the study period , cases of pulmonary tb were diagnosed , but were excluded from the study because most of these tb cases were diagnosed in immigrants of foreign origin and due to the patient s co - morbidities .
the inclusion criteria for the control group were : ( i ) ethnically matched volunteers who visited sotiria hospital for a routine check - up ; and ( ii ) the presence of a latent m. tuberculosis infection .
latent m. tuberculosis infection was defined as a positive tuberculin skin and at least 2 negative sputum cultures , combined with the absence of suggestive radiological findings for pulmonary tb .
, healthy subjects who visited sotiria hospital for a routine check - up were enrolled .
a 5 ml blood sample was drawn from every healthy volunteer , and was handled identically to the patients group . in all controls
specifically , 0.1 ml of purified protein derivative ( ppd ) , which contained 5 tuberculin units , was injected intradermally in the volar surface of the forearm .
the transverse width of induration in millimetres ( mm ) at the skin test site was measured after 4872 hours .
a positive tuberculin skin test was defined as skin transverse width 12 mm . in all of the controls enrolled , a chest - x - ray ( cxr ) and sputum cultures for m. tuberculosis
one hundred forty - four ethnically matched volunteers with latent m. tuberculosis infection were included .
the inclusion criteria for the patients group were : ( i ) greek origin ; ( ii ) all patients should be genetically unrelated ; and ( iii ) diagnosis of pulmonary tb based on suggestive radiological findings , a microscopic examination of sputum smears from an experienced microscopist , and documented in all patients with the presence of positive sputum cultures for m. tuberculosis infection .
the exclusion criteria for the patients group were : ( i ) presence of extrapulmonary tb ; ( ii ) patients known to have an hiv infection were not recruited ( all the other patients who agreed to participate were screened for hiv antibodies and hiv - positive patients were not enrolled in the study ) ; and ( iii ) presence of any immunodeficiency syndrome or systemic disease .
a 5 ml blood sample was drawn from 142 greek patients with pulmonary tb who were hospitalized in the sotiria general hospital of thoracic diseases from january 2004 until december 2008 .
the ethics committee of sotiria general hospital , athens , greece , accepted the study protocol .
currently , in the athens area of greece , sotiria general hospital of thoracic diseases is the health institution where the majority of tb cases are diagnosed and monitored .
the sample size was considered to be adequate for the study , taking into consideration the size of the greek population and the incidence of tb in greece . moreover , during the study period , cases of pulmonary tb were diagnosed , but were excluded from the study because most of these tb cases were diagnosed in immigrants of foreign origin and due to the patient s co - morbidities .
the inclusion criteria for the patients group were : ( i ) greek origin ; ( ii ) all patients should be genetically unrelated ; and ( iii ) diagnosis of pulmonary tb based on suggestive radiological findings , a microscopic examination of sputum smears from an experienced microscopist , and documented in all patients with the presence of positive sputum cultures for m. tuberculosis infection .
the exclusion criteria for the patients group were : ( i ) presence of extrapulmonary tb ; ( ii ) patients known to have an hiv infection were not recruited ( all the other patients who agreed to participate were screened for hiv antibodies and hiv - positive patients were not enrolled in the study ) ; and ( iii ) presence of any immunodeficiency syndrome or systemic disease .
a 5 ml blood sample was drawn from 142 greek patients with pulmonary tb who were hospitalized in the sotiria general hospital of thoracic diseases from january 2004 until december 2008 .
the ethics committee of sotiria general hospital , athens , greece , accepted the study protocol .
currently , in the athens area of greece , sotiria general hospital of thoracic diseases is the health institution where the majority of tb cases are diagnosed and monitored .
the sample size was considered to be adequate for the study , taking into consideration the size of the greek population and the incidence of tb in greece . moreover , during the study period , cases of pulmonary tb were diagnosed , but were excluded from the study because most of these tb cases were diagnosed in immigrants of foreign origin and due to the patient s co - morbidities .
the inclusion criteria for the control group were : ( i ) ethnically matched volunteers who visited sotiria hospital for a routine check - up ; and ( ii ) the presence of a latent m. tuberculosis infection .
latent m. tuberculosis infection was defined as a positive tuberculin skin and at least 2 negative sputum cultures , combined with the absence of suggestive radiological findings for pulmonary tb .
, healthy subjects who visited sotiria hospital for a routine check - up were enrolled .
a 5 ml blood sample was drawn from every healthy volunteer , and was handled identically to the patients group . in all controls
specifically , 0.1 ml of purified protein derivative ( ppd ) , which contained 5 tuberculin units , was injected intradermally in the volar surface of the forearm .
the transverse width of induration in millimetres ( mm ) at the skin test site was measured after 4872 hours .
a positive tuberculin skin test was defined as skin transverse width 12 mm . in all of the controls enrolled , a chest - x - ray ( cxr ) and sputum cultures for m. tuberculosis
one hundred forty - four ethnically matched volunteers with latent m. tuberculosis infection were included .
the inclusion criteria for the control group were : ( i ) ethnically matched volunteers who visited sotiria hospital for a routine check - up ; and ( ii ) the presence of a latent m. tuberculosis infection .
latent m. tuberculosis infection was defined as a positive tuberculin skin and at least 2 negative sputum cultures , combined with the absence of suggestive radiological findings for pulmonary tb .
, healthy subjects who visited sotiria hospital for a routine check - up were enrolled .
a 5 ml blood sample was drawn from every healthy volunteer , and was handled identically to the patients group . in all controls
specifically , 0.1 ml of purified protein derivative ( ppd ) , which contained 5 tuberculin units , was injected intradermally in the volar surface of the forearm .
the transverse width of induration in millimetres ( mm ) at the skin test site was measured after 4872 hours .
a positive tuberculin skin test was defined as skin transverse width 12 mm . in all of the controls enrolled , a chest - x - ray ( cxr ) and sputum cultures for m. tuberculosis
one hundred forty - four ethnically matched volunteers with latent m. tuberculosis infection were included .
the exclusion criteria for the control group were : ( i ) hiv - positive subjects ; ( ii ) history of prior tb ; ( iii ) evidence of current or prior tb in the cxr ; and ( iv ) presence of any immunodeficiency syndrome or systemic disease .
dna samples were extracted from human whole blood collected into tubes containing edta . the kit used for the extraction was purelink genomic dna kit catalog no.s k1820 - 01 , k1820 - 02 , k1821 - 04 , from invitrogen .
the nramp1 polymorphisms typed were : ( i ) a single - nucleotide change in intron 4 ( int4 ) ( 469 + 14g / c ) ; ( ii ) d543n , a non - conservative single - base substitution at codon 543 , which changes aspartic acid to asparagine ; and ( iii ) 3utr , a tgtg deletion in the 3untralsated region ( 1729 + 55del4 ) .
the pcr primers for the int4 polymorphism were 5ctctggctgaaggctctcc3 and 5tgtgctatcagttgagcctc3. the primers for d543n and 3utr were 5gcatctccccaattcatggt3 and 5aactgtcccactctatcctg3 , respectively .
real - time pcr studies were performed in a lightcycler 480 instrument , roche . for each of the 3 polymorphisms analyzed ,
a lightsnip kit was designed from the constructor company , tib - molbiol , germany .
the pcr reaction mixture contained 14.410.4 l pcr grade h20 , 1.0 l reagent mix 2.0 l lightcycler faststart dna master hyprobe 1.6 l mgcl2 and 1.05.0 l dna template .
initially , variants at 3 polymorphisms ( 3 utr , int4 , d543n ) in the nramp1 gene were compared between tuberculosis cases and controls with fisher s exact test . to assess the differences in demographic characteristics , we used chi - square ( for categorical data ) and t test ( for continuous data ) .
deviations from hardy - weinberg equilibrium ( hwe ) were assessed using the chi - square test .
subsequently , the 3 nramp1 polymorphisms were each independently associated with tuberculosis through logistic regression analysis after adjustment for age and sex .
the sas statistical package ( version 9.1 , sas institute inc , cary , nc ) was used to analyze the data .
all patients who were enrolled had a culture - positive pulmonary tb , combined with suggestive radiological findings .
positive acid - fast bacilli ( afb ) sputum smear for m. tuberculosis was observed in 108 ( 76% ) patients ; the remaining 34 ( 24% ) patients had a negative afb smear test .
the demographic characteristics and the frequency of the 3 nramp1 polymorphisms ( 3utr , d543n , int4 ) between patients and controls are shown in table 1 .
there was no evidence for deviation from hwe regarding the polymorphisms d543n ( p=0.07 ) and int4 ( p=0.08 ) , whereas the 3 utr ( p=29.11 ) was found to deviate significantly from hwe .
the nramp1 allelic frequencies for the d543n and 3utr variants were not found to differ significantly between patients with pulmonary tb and controls .
d543n associated g / g and g / a variants were observed in 138 and 4 samples in the patients group , respectively . in controls , d543n - associated g / g and g / a genotypes
the genotype a / a was absent in both groups . in the patients group ,
the 3utr - associated tgtg / tgtg , tgtg / del and del / del variants were observed in 137 , 4 and 1 sample , respectively . in the control group ,
the numbers of samples with positive 3utr variants were 139 , 4 and 1 sample , respectively .
however , we identified an increased incidence of int4 polymorphism in the patients group compared to the control group , which was close to the borderline of statistical significance ( p=0.058 ) .
three separate logistic regression analyses were performed , after adjustment for age and sex , in order to further investigate the possible association of tb patients with the allelic frequencies of the 3 nramp1 polymorphisms ( table 2 ) .
no significant association was found in patients with pulmonary tb for the d543n and 3utr allele variants .
specifically , when gg allele genotype was used as baseline value , cc homozygotes were found to be at higher risk for developing pulmonary tb ( or=7.31 , 95% ci : 1.3340.17 , p=0.022 ) .
a more detailed analysis was performed by creating 8 categories of genotype combinations : g / tgtg / g , g / tgtg / c , g / del / g , a / del / g , a / tgtg / g , g / del / c , a / tgtg / c and a / del / c ( table 3 ) .
significant differences among the frequencies between patients and controls were found regarding g / tgtg / g and g / tgtg / c genotype combinations
. specifically , lower expression of g / tgtg / g combination ( p=0.005 ) was found in the patients group compared to the control group , while higher expression of the g / tgtg / c combination ( p=0.004 ) was found in the patients group compared to the controls . from a total of 8 genotype combinations , 5 ( g / tgtg / g
, g / tgtg / c , g / del / g , a / del / g , a / tgtg / g ) were included in a multivariate model ( table 4 ) .
the remaining 3 combinations ( g / del / c , a / tgtg / c , a / del / c ) were excluded because of their extremely low frequencies .
g / tgtg / c was found to be associated with a 36% higher risk for developing pulmonary tb ( or=1.36 ; 95% ci : 1.111.66 ; p=0.004 ) compared to the baseline expression of g / tgtg / g .
an association between pulmonary tb and g / del / g , a / del / g and a / tgtg / g genotype combinations , compared to the baseline expression of g / tgtg / g , was not observed .
to our knowledge this is the first study in greece that examines the possible association of nramp1 polymorphisms and the susceptibility to develop culture - positive pulmonary tb .
our study demonstrated that there was not any significant difference in the incidence of nramp1 d543n and 3utr polymorphisms between patients with pulmonary tb and ethnically matched healthy controls having latent m. tuberculosis infection .
an increased incidence of int4- nramp1 polymorphism was found in the patients group , which was close to the borderline of statistical significance .
a positive association of 3utr variant allele and the susceptibility to develop tb was reported in a small caucasian population . however , several studies performed in european populations suggested no statistically significant association between nramp1 polymorphisms and the susceptibility to develop tb .
this was also confirmed in the biggest meta - analysis of published trials to date . in this analysis ,
the odds ratios ( ors ) among 3utr , d543n , int4 locus and tb susceptibility , in the european population , were 1.81 ( 95%ci : 0.664.93 ; p=0.25 ) , 1.88 ( 95%ci : 0.754.67 ; p=0.18 ) and 0.87 ( 95%ci : 0.611.22 ; p=0.41 ) , respectively .
it is believed that the european origin population has a high level of genetical resistance to m. tuberculosis infection , due to natural selection over the last 300 years .
the lower proportion of tb cases in the european population , compared to asian and african populations , could be another reason for the lack of association .
we found a lack of correlation among d543n and 3utr nramp1 polymorphisms and an increased susceptibility to develop pulmonary tb , according to results of the majority of studies in european populations .
although our results are close to statistical significance , there is a trend for a higher incidence of int4-nramp1 polymorphism in patients with pulmonary tb , compared to ethnically matched volunteers with latent m. tuberculosis infection .
this possible association appears for the first time in a european population , and can be possibly attributed to the diverse genetic background among various ethnic populations of the same descent .
specifically , the int4-nramp1 polymorphism can be found in approximately 20% of the chinese population , whereas the frequency in white europeans is about 50% , with significant variation among different european populations .
the int4-cc homozygotes variants were found to have an increased risk for developing pulmonary tb , compared to their corresponding common gg alleles . when a more detailed analysis was performed by creating 8 categories of genotype combinations , higher expression of g / tgtg / c combination ( p=0.004 ) was found in the patient s group as compared to the controls .
this combination was associated with a 36% higher risk for developing pulmonary tb ( or=1.36 ; 95% ci : 1.111.66 ; p=0.004 ) compared to the baseline expression of g / tgtg / g .
however , the limited number of patients and controls found to carry the int4-cc genotype must be taken into consideration in the interpretation of our results . whether the incidence of int4-cc variant or any nramp1 genotype combination is correlated to the development of active disease
most of the studies exploring the possible role of nramp1 polymorpisms in the development of tb did not distinguish between susceptibility to infection with m. tuberculosis and susceptibility to progression to active disease , since the control groups consisted of healthy volunteers without active disease .
our study design distinguishes between susceptibility to infection with m. tuberculosis and susceptibility to progression in an active m. tuberculosis pulmonary disease .
all volunteers in the control group had a latent m. tuberculosis infection without developing the disease .
this fact suggests that int4-nramp1 polymorphisms may have a role in the development of overt pulmonary disease after a latent m. tuberculosis infection . the main limitations of our study are the lack of data in both study groups that can be associated with a higher risk of developing pulmonary tb ( alcohol abuse , chronic corticosteroid therapy , diabetes , poor nutrition / socio - economic status ) , and the relatively small sample size of the population enrolled .
int4-nramp1 polymorphism may be associated with a higher rate of culture - positive pulmonary tb in the greek population compared to ethnically matched healthy controls with latent m. tuberculosis infection .
a lack of association was observed for the other 2 nramp1 polymorphisms ( d543n , 3utr ) .
an increased incidence of g / tgtg / c genotype combination was found in the patients group compared to controls .
g / tgtg / c genotype combination was associated with a 36% higher risk for developing pulmonary tb compared to the baseline expression of g / tgtg / g genotype combination .
the possible role of int4-nramp1 polymorphism in the development of active pulmonary tb , after an initial latent m. tuberculosis infection , needs further investigation . | summarybackgroundample evidence suggests that host genetic factors affect human susceptibility to tuberculosis . the natural resistance associated macrophage protein 1 ( nramp1 ) gene seems to play a role in the pathophysiology of a number of intracellular infections , including mycobacteria .
a case - control study was conducted in the greek population to determine whether nramp1 polymorphisms affect the susceptibility to development of overt pulmonary tuberculosis.material/methodsnramp1 polymorphisms ( 3utr , d543n , int4 ) were evaluated among 142 patients with culture - positive pulmonary tuberculosis and 144 ethnically matched healthy controls having latent m. tuberculosis infection .
patients with human immunodeficiency virus infection were excluded.resultsout of the 3 nramp1 polymorphisms , a trend of increased incidence of int4 polymorphism was found in the patients group compared to the control group .
a lack of association was observed between the 2 groups as far as the other 2 polymorphisms ( d543n , 3utr ) are concerned .
int4-cc homozygotes were found to have a higher risk to develop pulmonary tuberculosis compared to gg homozygotes ( p=0.022 ) .
an increased incidence g / tgtg / c genotype combination was found in the patients group as compared to controls .
g / tgtg / c genotype combination was associated with a 36% higher risk of developing pulmonary tuberculosis ( p=0.004 ) compared to the baseline expression of g / tgtg / g combination.conclusionsint4-nramp1 polymorphism may have a role in the development of culture - positive pulmonary tuberculosis after an initial m. tuberculosis latent infection .
the possible role of int4-nramp1 polymorphism in the development of active pulmonary tuberculosis needs further investigation . |
cardiovascular disease is the leading cause of mortality throughout the world , accounting for 17.3 million deaths / year .
a large number of studies have proven that the epidemiology , clinical manifestations , and clinical prognosis of coronary artery disease ( cad ) have sex - related differences . in the past 30 years
, the mortality of male patients with cad has declined gradually , but an increase had been observed in women .
unfortunately , accurate diagnosis of cad might be more challenging in women than in men because women more frequently present with atypical symptoms .
thus , women are at increased risk of delayed or incorrect diagnosis . according to a report from the american heart association ( aha ) in 2015
, the mortality rate of female patients with cardiovascular disease ( 50.6% ) has exceeded that of men ( 49.4% ) .
conventional coronary angiography ( cag ) is the gold standard diagnostic modality for cad , but it is invasive and associated with several potential complications .
moreover , nearly half of the women who underwent invasive cag were found to have nonobstructive cad .
multislice computed tomography ( msct ) cag is highly accurate for the exclusion of significant coronary artery stenoses ( > 50% luminal narrowing ) , with high negative predictive values ( npvs ) , as compared with conventional cag . at present , the accuracy of msct diagnosis in women is still not clear .
data are mainly based on the male population , with only about 20% of the included patients being female .
many of the studies that have been conducted have small sample sizes and conflicting results .
in addition , whether atypical presentation influences the diagnostic accuracy of msct in women remains unknown .
this study aimed to investigate the effect of sex and atypical symptoms on the diagnostic accuracy of the current 64-slice msct cag technique using conventional cag as the reference standard .
totally , 1246 in - patients who had performed cag from january 2007 to february 2011 in the peking university people 's hospital were included in the study .
demographic information , medical history , and clinical data were obtained from the patients medical records .
the exclusion criteria were nonsinus rhythm ( 145 patients ) , intolerance to iodine contrast ( 11 patients ) , renal dysfunction ( serum creatinine levels 15
mg / l , 87 patients ) , pregnancy ( 0 patients ) , history of percutaneous coronary intervention or coronary artery bypass surgery ( 46 patients ) , uninterpretable images due to poor quality ( 72 patients ) , and other complications not suitable for msct ( 489 patients ) .
finally , a database of 396 patients with suspected or proven cad on admission , who successively underwent both msct and invasive cag , was reviewed .
cad was diagnosed based on invasive cag results , indicating stenoses of 50% in any coronary artery .
the patients were divided into typical and atypical groups based on their symptoms of angina pectoris .
typical angina symptoms were defined as the sensation of chest pain or discomfort that may feel like tightness , heaviness , or burning in the central chest .
the discomfort might move or radiate to the shoulder , arms , jaw , neck , and back .
angina is typically precipitated by exertion or emotional stress , lasting only a few minutes and relieved by rest and nitroglycerin .
this retrospective study was approved by the ethics committee of peking university people 's hospital .
all the patients underwent coronary msct imaging using the general electric lightspeed volume computed tomography ( ge healthcare , milwaukee , wi , usa ) .
patients with a heart rate of > 65 beats / min received additional blockers ( 25 or 50 mg of metoprolol ) 1 h before the ct examination .
then , the patients were injected with 6080 ml of iodinated contrast medium in the antecubital vein at a high flow rate ( 5 ml / s ) , followed by saline flushing and coronary ct angiographic examinations .
the collimation was 64 mm 0.625 mm ; gantry rotation time was 350 ms , tube current was 600750 ma , and voltage was 120 kv .
automated bolus - tracking in the aortic root was used for timing of the scan .
all the images were acquired during a single inspiratory breath hold of about 10 s , with simultaneous electrocardiographic recording .
all images were interpreted in consensus by two experienced radiologists , who were unaware of the clinical presentations of the patients .
the coronary arteries were divided into 13 separate segments according to the modified aha classification .
coronary artery plaque was defined as an area of 1 mm , and a clearly recognizable structure associated with the coronary artery wall in at least two independent image planes .
plaques with a ct density greater than that of the contrast - enhanced coronary lumen were defined as calcified plaques . for patients with suspected or proven cad who needed to undergo a second evaluation of coronary arteries , conventional invasive cag was performed according to the standard protocols .
cags were visually assessed in consensus by two experienced observers who were unaware of the results of the msct cag .
continuous variables were expressed as mean standard deviation ( sd ) and compared between the two groups using the t - test for independent samples . when not normally distributed
, continuous data were expressed as median ( interquartile range ) and compared between the two groups using the nonparametric mann - whitney u - test .
categorical variables were expressed as absolute numbers ( percentages ) and compared between the two groups using the chi - square test .
the diagnostic performance of msct was assessed in comparison with that of cag , which was used as the reference standard .
sensitivity , specificity , positive predictive value ( ppv ) , and npv were calculated .
the youden index was calculated as ( sensitivity + specificity 1 ) to summarize both sensitivity and specificity in a single number between 0 and 1 .
multivariate logistic regression analyses were performed to determine the influencing factors on the diagnostic accuracy of msct .
the diagnostic performance of msct was assessed using the area under the receiver operating characteristic curve ( auc ) of the receiver operating characteristic ( roc ) curves , taking 50% or greater diameter stenoses at conventional invasive cag as the reference standard .
all p values were two - tailed and a significance level of < 0.05 was used .
all statistical analyses were performed using spss version 17.0 ( spss inc . , chicago , il , usa ) .
totally , 1246 in - patients who had performed cag from january 2007 to february 2011 in the peking university people 's hospital were included in the study .
demographic information , medical history , and clinical data were obtained from the patients medical records .
the exclusion criteria were nonsinus rhythm ( 145 patients ) , intolerance to iodine contrast ( 11 patients ) , renal dysfunction ( serum creatinine levels 15
mg / l , 87 patients ) , pregnancy ( 0 patients ) , history of percutaneous coronary intervention or coronary artery bypass surgery ( 46 patients ) , uninterpretable images due to poor quality ( 72 patients ) , and other complications not suitable for msct ( 489 patients ) .
finally , a database of 396 patients with suspected or proven cad on admission , who successively underwent both msct and invasive cag , was reviewed .
cad was diagnosed based on invasive cag results , indicating stenoses of 50% in any coronary artery .
the patients were divided into typical and atypical groups based on their symptoms of angina pectoris .
typical angina symptoms were defined as the sensation of chest pain or discomfort that may feel like tightness , heaviness , or burning in the central chest .
the discomfort might move or radiate to the shoulder , arms , jaw , neck , and back .
angina is typically precipitated by exertion or emotional stress , lasting only a few minutes and relieved by rest and nitroglycerin .
this retrospective study was approved by the ethics committee of peking university people 's hospital .
all the patients underwent coronary msct imaging using the general electric lightspeed volume computed tomography ( ge healthcare , milwaukee , wi , usa ) .
patients with a heart rate of > 65 beats / min received additional blockers ( 25 or 50 mg of metoprolol ) 1 h before the ct examination .
then , the patients were injected with 6080 ml of iodinated contrast medium in the antecubital vein at a high flow rate ( 5 ml / s ) , followed by saline flushing and coronary ct angiographic examinations .
the collimation was 64 mm 0.625 mm ; gantry rotation time was 350 ms , tube current was 600750 ma , and voltage was 120 kv .
automated bolus - tracking in the aortic root was used for timing of the scan .
all the images were acquired during a single inspiratory breath hold of about 10 s , with simultaneous electrocardiographic recording .
all images were interpreted in consensus by two experienced radiologists , who were unaware of the clinical presentations of the patients .
the coronary arteries were divided into 13 separate segments according to the modified aha classification .
coronary artery plaque was defined as an area of 1 mm , and a clearly recognizable structure associated with the coronary artery wall in at least two independent image planes .
plaques with a ct density greater than that of the contrast - enhanced coronary lumen were defined as calcified plaques .
for patients with suspected or proven cad who needed to undergo a second evaluation of coronary arteries , conventional invasive cag was performed according to the standard protocols .
cags were visually assessed in consensus by two experienced observers who were unaware of the results of the msct cag .
continuous variables were expressed as mean standard deviation ( sd ) and compared between the two groups using the t - test for independent samples . when not normally distributed
, continuous data were expressed as median ( interquartile range ) and compared between the two groups using the nonparametric mann - whitney u - test .
categorical variables were expressed as absolute numbers ( percentages ) and compared between the two groups using the chi - square test .
the diagnostic performance of msct was assessed in comparison with that of cag , which was used as the reference standard .
sensitivity , specificity , positive predictive value ( ppv ) , and npv were calculated .
the youden index was calculated as ( sensitivity + specificity 1 ) to summarize both sensitivity and specificity in a single number between 0 and 1 .
multivariate logistic regression analyses were performed to determine the influencing factors on the diagnostic accuracy of msct .
the diagnostic performance of msct was assessed using the area under the receiver operating characteristic curve ( auc ) of the receiver operating characteristic ( roc ) curves , taking 50% or greater diameter stenoses at conventional invasive cag as the reference standard .
all p values were two - tailed and a significance level of < 0.05 was used .
all statistical analyses were performed using spss version 17.0 ( spss inc . , chicago , il , usa ) .
table 1 lists the baseline characteristics of the entire study population . in this study , 396 patients were enrolled , including 141 women and 255 men . according to the results of cag ,
cad diagnosis was established in 346 patients and ruled out in 50 patients . on average , the women were older than the men ( 67 10 vs. 62 11 years , t = 4.416 , p < 0.01 ) .
the number of male smokers was significantly greater than that of female smokers , and more women had no cad .
women showed a lower prevalence of lesions in mid - segments or side branches . with regard to calcified plaque on msct , and the number of diseased and culprit vessels on invasive cag
clinical characteristics of the study group sd : standard deviation ; cad : coronary artery disease ; msct : multislice computed tomography ; nstemi : non - st elevation myocardial infarction ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery .
table 2 lists the diagnostic accuracy of msct in the total population . of the 396 patients , 328 had obstructive cad on conventional invasive cag that were correctly identified by msct , resulting in a sensitivity of 95% ( 95% confidence interval [ ci ] : 9397% ) .
eighteen patients ( 36% , 18/50 ) were identified as false positive . on a per - patient level , the sensitivity , specificity , ppv , npv , total accuracy , and youden index were 95% , 64% , 95% , 64% , 91% , and 0.59 , respectively .
diagnostic accuracy of multislice computed tomography in the total population ( n = 396 ) * p<0.001 , compared with all vessels ; p<0.05 , compared with all vessels ; p<0.001 , compared with all segments ; p<0.05 , compared with all segments .
tn : true negative ; tp : true positive ; fn : false negative ; fp : false positive ; ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery .
the diagnostic performance of msct for detecting significant stenoses in a vessel - based analysis was good .
most of the significantly diseased vessels ( 75% , 570/756 ) were correctly identified using ct .
ninety - six vessels ( 12% , 96/828 ) with nonsignificant stenoses were incorrectly classified as significant stenoses using ct .
the diagnostic accuracy of msct in detecting left main ( lm ) cad is the highest ( 91% vs. 82% , compared with all vessels , = 10.120 , p < 0.05 ) .
msct had a lower diagnostic accuracy ( 74% vs. 82% , compared with all vessels , = 12.432 , p < 0.05 ) in detecting lesions of left circumflex coronary . on a per - segment basis ,
the diagnostic values of msct in detecting lesions in distal segments and side branches were low , and the youden indexes for each segment in comparison with all the segments were 0.22 versus 0.4 ( u = 7.493 , p < 0.05 ) and 0.15 versus 0.4 ( u = 6.767 , p < 0.05 ) , respectively . table 3 shows the diagnostic accuracy of msct in the women compared with that in the men . on a patient - based analysis ,
the ppv ( 91% vs. 97% , = 5.705 , p < 0.05 ) and diagnostic accuracy ( 87% vs. 93% , = 5.093 , p < 0.05 ) of msct in detecting cad were lower in the women .
the women had a lower youden index ( 0.45 vs. 0.70 , u = 2.584 , p < 0.05 ) in msct diagnosis . in the cad population diagnosed using conventional invasive cag , 93% ( 110/118 ) of the female patients and 96% ( 218/228 ) of the male patients were correctly identified using msct .
eleven women ( 48% , 11/23 ) and 7 men ( 26% , 7/27 ) with nonsignificant cad were incorrectly classified as having significant coronary stenoses using ct .
diagnostic accuracy of multislice computed tomography in women versus men * p<0.05 , compared with men .
ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery . on a per - vessel basis (
lm , left anterior descending , left circumflex , and right coronary arteries ) , 564 coronary arteries in women and 1020 vessels in men were included in the study .
the youden index showed no significant difference . using msct , 76% ( 186/244 ) of vessels in the women and 75% ( 384/512 ) of vessels in the men
thirty - eight vessels ( 13% , 38/290 ) in the women and 58 vessels ( 11% , 58/508 ) in the men with nonsignificant stenoses on invasive cag were misdiagnosed using ct .
after exclusion of 104 segments ( 23 for women and 81 for men ) owing to small vessel size , motion artifacts , and undetectable distal segments due to total occlusion of the proximal vessel , 1534 coronary segments in the female population and 2718 segments in the male population were included in this study . on a per - segment basis ,
the npv ( 87% vs. 83% , = 8.563 , p < 0.05 ) and diagnostic accuracy ( 82% vs. 79% , = 5.916 , p < 0.05 ) of msct in detecting cad were higher in the women .
however , the ppv was relatively low in both groups for all the segments ( 60% and 63% , respectively ) and extremely low in distal segments and side branches ( 3050% ) .
thus , the diagnostic values were low for both groups ( youden index : 0.42 and 0.39 , respectively ) . according to the msct results with conventional invasive cag as the reference standard
, patients with true positive and true negative results were defined as the accurate diagnosis group .
table 4 shows that the percentage of men was significantly higher in the accurate diagnosis group .
multivessel diseases were significantly more frequent in the accurate group . in the multivariate logistic regression analysis ,
atypical presentation was an independent influencing factor of the diagnostic accuracy of msct in the women ( odds ratio [ or ] = 4.94 , 95% ci : 1.1620.92 , walds = 4.69 , p < 0.05 ) . multivessel disease ( or = 31.34 , 95% ci : 3.6272.6 , walds = 9.7 , p < 0.01 ) and noncalcified plaque ( or = 4.96 , 95% ci : 1.0124.51 , walds = 3.87 , p < 0.05 ) were also independent influencing factors .
clinical characteristics based on the accuracy of multislice computed tomography cad : coronary artery disease ; msct : multislice computed tomography ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery ; ldl - c ; low - density lipoprotein cholesterol ; hdl - c : high - density lipoprotein cholesterol .
the whole population was divided into two groups , namely , the typical and atypical groups , according to angina pectoris symptoms . table 5 shows that in a patient - based analysis , the ppv ( 98% vs. 74% , = 17.283 , p < 0.001 ) and diagnostic accuracy ( 93% vs. 72% , = 9.571 , p < 0.001 ) of msct in detecting female cad were significantly higher in the typical group than in the atypical group .
the women in the typical group had a higher youden index ( 0.69 vs. 0.29 , u = 2.359 , p < 0.05 ) in msct diagnosis .
not much difference was observed for msct in the diagnosis of male cad between the two groups
. diagnostic accuracy of multislice computed tomography in patients with typical or atypical angina symptoms * p<0.001 ; p<0.05 , compared with atypical group .
ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value the diagnostic performance of msct to detect coronary stenoses of 50% was further investigated with respect to the roc curve .
for all the patients , the auc was 0.84 ( 95% ci : 0.770.90 ) , suggesting a good accuracy .
the auc were 0.90 ( 95% ci : 0.830.97 ) for men and 0.74 ( 95% ci : 0.620.87 ) for women ( z = 2.194 , p < 0.05 ) . in women , patients with typical symptoms had a higher auc ( 0.91 , 95% ci : 0.830.99 ) than those with atypical symptoms ( 0.64 , 95% ci : 0.450.82 , z = 2.690 , p < 0.01 ) .
however , in the men , no significant difference in auc was found between the typical and atypical groups .
table 1 lists the baseline characteristics of the entire study population . in this study , 396 patients were enrolled , including 141 women and 255 men . according to the results of cag ,
cad diagnosis was established in 346 patients and ruled out in 50 patients . on average , the women were older than the men ( 67 10 vs. 62 11 years , t = 4.416 , p < 0.01 ) .
the number of male smokers was significantly greater than that of female smokers , and more women had no cad .
women showed a lower prevalence of lesions in mid - segments or side branches . with regard to calcified plaque on msct , and the number of diseased and culprit vessels on invasive cag
clinical characteristics of the study group sd : standard deviation ; cad : coronary artery disease ; msct : multislice computed tomography ; nstemi : non - st elevation myocardial infarction ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery .
table 2 lists the diagnostic accuracy of msct in the total population . of the 396 patients , 328 had obstructive cad on conventional invasive cag that were correctly identified by msct , resulting in a sensitivity of 95% ( 95% confidence interval [ ci ] : 9397% ) .
eighteen patients ( 36% , 18/50 ) were identified as false positive . on a per - patient level , the sensitivity , specificity , ppv , npv , total accuracy , and youden index were 95% , 64% , 95% , 64% , 91% , and 0.59 , respectively . diagnostic accuracy of multislice computed tomography in the total population ( n = 396 ) * p<0.001 , compared with all vessels ; p<0.05 , compared with all vessels ; p<0.001 , compared with all segments ; p<0.05 , compared with all segments .
tn : true negative ; tp : true positive ; fn : false negative ; fp : false positive ; ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery .
the diagnostic performance of msct for detecting significant stenoses in a vessel - based analysis was good .
most of the significantly diseased vessels ( 75% , 570/756 ) were correctly identified using ct .
ninety - six vessels ( 12% , 96/828 ) with nonsignificant stenoses were incorrectly classified as significant stenoses using ct .
the diagnostic accuracy of msct in detecting left main ( lm ) cad is the highest ( 91% vs. 82% , compared with all vessels , = 10.120 , p < 0.05 ) .
msct had a lower diagnostic accuracy ( 74% vs. 82% , compared with all vessels , = 12.432 , p < 0.05 ) in detecting lesions of left circumflex coronary . on a per - segment basis , the diagnostic values of msct in detecting lesions in distal segments and side branches were low , and the youden indexes for each segment in comparison with all the segments were 0.22 versus 0.4 ( u = 7.493 , p < 0.05 ) and 0.15 versus 0.4 ( u = 6.767 , p < 0.05 ) , respectively .
table 3 shows the diagnostic accuracy of msct in the women compared with that in the men . on a patient - based analysis , the ppv ( 91% vs. 97% , = 5.705 , p < 0.05 ) and diagnostic accuracy ( 87% vs. 93% , = 5.093 , p < 0.05 ) of msct in detecting cad were lower in the women .
the women had a lower youden index ( 0.45 vs. 0.70 , u = 2.584 , p < 0.05 ) in msct diagnosis . in the cad population diagnosed using conventional invasive cag , 93% ( 110/118 ) of the female patients and 96% ( 218/228 ) of the male patients were correctly identified using msct .
eleven women ( 48% , 11/23 ) and 7 men ( 26% , 7/27 ) with nonsignificant cad were incorrectly classified as having significant coronary stenoses using ct .
diagnostic accuracy of multislice computed tomography in women versus men * p<0.05 , compared with men .
ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery . on a per - vessel basis (
lm , left anterior descending , left circumflex , and right coronary arteries ) , 564 coronary arteries in women and 1020 vessels in men were included in the study .
the youden index showed no significant difference . using msct , 76% ( 186/244 ) of vessels in the women and 75% ( 384/512 ) of vessels in the men
thirty - eight vessels ( 13% , 38/290 ) in the women and 58 vessels ( 11% , 58/508 ) in the men with nonsignificant stenoses on invasive cag were misdiagnosed using ct .
after exclusion of 104 segments ( 23 for women and 81 for men ) owing to small vessel size , motion artifacts , and undetectable distal segments due to total occlusion of the proximal vessel , 1534 coronary segments in the female population and 2718 segments in the male population were included in this study . on a per - segment basis ,
the npv ( 87% vs. 83% , = 8.563 , p < 0.05 ) and diagnostic accuracy ( 82% vs. 79% , = 5.916 , p < 0.05 ) of msct in detecting cad were higher in the women .
however , the ppv was relatively low in both groups for all the segments ( 60% and 63% , respectively ) and extremely low in distal segments and side branches ( 3050% ) .
thus , the diagnostic values were low for both groups ( youden index : 0.42 and 0.39 , respectively ) .
according to the msct results with conventional invasive cag as the reference standard , patients with true positive and true negative results were defined as the accurate diagnosis group .
table 4 shows that the percentage of men was significantly higher in the accurate diagnosis group .
multivessel diseases were significantly more frequent in the accurate group . in the multivariate logistic regression analysis ,
atypical presentation was an independent influencing factor of the diagnostic accuracy of msct in the women ( odds ratio [ or ] = 4.94 , 95% ci : 1.1620.92 , walds = 4.69 , p < 0.05 ) .
multivessel disease ( or = 31.34 , 95% ci : 3.6272.6 , walds = 9.7 , p < 0.01 ) and noncalcified plaque ( or = 4.96 , 95% ci : 1.0124.51 , walds = 3.87 , p < 0.05 ) were also independent influencing factors .
clinical characteristics based on the accuracy of multislice computed tomography cad : coronary artery disease ; msct : multislice computed tomography ; lm : left main ; lad : left anterior descending coronary ; lcx : left circumflex coronary ; rca : right coronary artery ; ldl - c ; low - density lipoprotein cholesterol ; hdl - c : high - density lipoprotein cholesterol .
the whole population was divided into two groups , namely , the typical and atypical groups , according to angina pectoris symptoms . table 5 shows that in a patient - based analysis , the ppv ( 98% vs. 74% , = 17.283 , p < 0.001 ) and diagnostic accuracy ( 93% vs. 72% , = 9.571 , p < 0.001 ) of msct in detecting female cad were significantly higher in the typical group than in the atypical group .
the women in the typical group had a higher youden index ( 0.69 vs. 0.29 , u = 2.359 , p < 0.05 ) in msct diagnosis .
not much difference was observed for msct in the diagnosis of male cad between the two groups
. diagnostic accuracy of multislice computed tomography in patients with typical or atypical angina symptoms * p<0.001 ; p<0.05 , compared with atypical group .
ci : confidence interval ; ppv : positive predictive value ; npv : negative predictive value the diagnostic performance of msct to detect coronary stenoses of 50% was further investigated with respect to the roc curve . for all the patients , the auc was 0.84 ( 95% ci : 0.770.90 ) , suggesting a good accuracy .
the auc were 0.90 ( 95% ci : 0.830.97 ) for men and 0.74 ( 95% ci : 0.620.87 ) for women ( z = 2.194 , p < 0.05 ) . in women , patients with typical symptoms had a higher auc ( 0.91 , 95% ci : 0.830.99 ) than those with atypical symptoms ( 0.64 , 95% ci : 0.450.82 , z = 2.690 , p < 0.01 ) .
however , in the men , no significant difference in auc was found between the typical and atypical groups .
our findings suggest that the ppv ( 91% vs. 97% , p < 0.05 ) and diagnostic accuracy ( 87% vs. 93% , p < 0.05 ) of msct in detecting cad were lower in the women than in the men .
a more important finding is that our results suggest that both gender and atypical symptoms may influence the diagnostic accuracy of msct .
numerous studies have proven that significant sex - related differences exist not only in the incidence and prognosis of atherosclerosis disease but also in the accuracy of different clinical examination methods .
anatomic and physiological differences , including body composition , heart rate , coronary calcium level , and coronary diameter between women and men , may affect the diagnostic performance of msct .
our study proved sex - related differences in the performance of msct in detecting obstructive cad . in our study , the sensitivity , specificity , ppv , npv , and accuracy of msct in detecting cad were 95% , 64% , 95% , 64% , and 91% , respectively , on a per - patient level . the per - patient - based specificity and
the sensitivity , specificity , ppv , npv , and diagnostic accuracy of msct on a per - segment level were 50% , 90% , 62% , 85% , and 80% , respectively .
its sensitivity and npv were also relatively low . besides the technical problems , smaller body size and vascular lumen diameter may be possible reasons .
in addition , our study population was almost 5 years older than that of other studies .
the prevalence of calcified plaques was also relatively high in our study . in our study , the women with typical angina pectoris had higher ppv and diagnostic accuracy in msct diagnosis .
thus , the diagnostic accuracy of msct may be different in women with atypical symptoms .
several studies previously investigated the diagnostic accuracy of msct in female coronary heart disease , yielding inconclusive results .
some studies found no significant difference in the performance of msct between women and men . while the study of meijboom et al . suggested a similar sensitivity in diagnosing coronary stenoses between the sexes , the specificity for detection of obstructive cad ( especially in distal and side coronary branches ) was significantly lower in the women than in the men .
another study reported that msct has similar specificity between the sexes but lower sensitivity and accuracy in women .
the inconsistent results between diagnostic studies may be related to the remarkable difference in the number of patients in each study .
however , we should consider that the prevalence of cad is remarkably lower in women than in men .
nonobstruction artery disease and positive remodeling of blood vessels are more common in women . after adjustment for body surface ,
the diameter of the female coronary artery was still smaller than that of the male coronary artery .
studies have proven sex - related differences in plaque morphology on coronary computed tomography angiography .
males tended to have more calcified plaques than females , with a greater tendency to have multiple vessel involvement .
all of these factors might contribute to the significant differences in the diagnostic accuracy of msct between women and men . in our study ,
the diagnostic value of msct in the detection of cad was lower in the women than in the men .
however , it is still a reliable modality for the exclusion of significant coronary artery stenoses in both sexes .
our study suggests that both gender and atypical symptoms might influence the diagnostic accuracy of msct .
our study is limited by its single - center retrospective design and the relatively small number of patients included .
quantitative analysis of calcification and morphologies of plaques were not studied due to technical limitations .
meanwhile , age and the prevalence rates of hypertension and diabetes were significantly higher in the women than that in the men , which might influence the results in our study .
this work was supported by the grant from the beijing science and technology project in china ( no .
this work was supported by the grant from the beijing science and technology project in china ( no .
| background : multislice computed tomography ( msct ) coronary angiography ( cag ) is a noninvasive technique with a reported high diagnostic accuracy for coronary artery disease ( cad ) .
women , more frequently than men , are known to develop atypical angina symptoms .
the purpose of this study was to investigate whether the diagnostic accuracy of msct in women with atypical presentation differs from that in men.methods:we enrolled 396 in - hospital patients ( 141 women and 255 men ) with suspected or proven cad who successively underwent both msct and invasive cag .
cad was defined as any coronary stenosis of 50% on conventional invasive cag , which was used as the reference standard .
the patients were divided into typical and atypical groups based on their symptoms of angina pectoris .
the diagnostic accuracy of msct , including its sensitivity , specificity , negative predictive value , and positive predictive value ( ppv ) , was calculated to determine the usefulness of msct in assessing stenoses .
the diagnostic performance of msct was also assessed by constructing receiver operating characteristic ( roc ) curves.results:the ppv ( 91% vs. 97% , 2 = 5.705 , p < 0.05 ) and diagnostic accuracy ( 87% vs. 93% , 2 = 5.093 , p < 0.05 ) of msct in detecting cad were lower in women than in men .
atypical presentation was an independent influencing factor on the diagnostic accuracy of msct in women ( odds ratio = 4.94 , 95% confidence intervals : 1.1620.92 , walds = 4.69 , p < 0.05 ) . compared with those in the atypical group , women with typical angina pectoris had higher ppv ( 98% vs. 74% , 2 = 17.283 .
p < 0.001 ) , diagnostic accuracy ( 93% vs. 72% , 2 = 9.571 , p < 0.001 ) , and area under the roc curve ( 0.91 vs. 0.64 , z = 2.690 , p < 0.01 ) in msct diagnosis.conclusions:although msct is a reliable diagnostic modality for the exclusion of significant coronary artery stenoses in all patients , gender and atypical symptoms might have some influence on its diagnostic accuracy . |
extracellular matrix ( ecm ) remodeling through cell - ecm interactions is a critical step of many biological and pathological processes such as embryonic development , angiogenesis , wound healing , and cancer cell metastasis .
for instance , when cancer cells move through a dense ecm , they can generate actomyosin forces that deform the collagen fibers to push the cell through the ecm .
reciprocally , the physical properties of the ecm including matrix structure , mechanics , and dimensionality can profoundly influence cellular behavior . in the case of cancer ecm which is usually stiffer than normal tissues ,
the compromised tensional homeostasis affects cell phenotype , rho - dependent cell contractility and oncogene - mediated transformation .
clinically , the increased matrix stiffness and ecm remodeling were observed in premalignant tissue , and this increase was shown to contribute to malignant transformation .
collagen , the most abundant protein in a mammalian body , is the major contributor to tissue mechanical properties .
these mechanical properties have roots in collagen 's microstructure , network organization , and orientation .
collagen has been used in a number of studies that aim to quantify cell - mediated ecm remodeling process and its mechanics .
the measurement scale of ecm remodeling ranges from whole gel contraction assay at centimeter scale [ 811 ] to collagen properties at micrometer scale such as fiber diameter , fiber length , and pore size . also the organization of the collagen matrix in the proximity of the cells has been a subject of several investigations [ 13 , 14 ] . although these methods provide ways to describe the collagen matrix property changes around the cells and at the microscopic level , they lack the information of heterogeneity due to ecm remodeling at the mesoscale that is comparable to cell size . alternatively , stevenson et al
. attempted to quantify the collagen matrix compaction level in the pericellular region by creating an intensity contour map of a confocal collagen reflection image .
this method , however , does not allow an absolute quantitative comparison of different images due to the dependence of intensity range of each image .
nonlinear microscopy techniques such as second harmonic generation ( shg ) provide a noninvasive and label - free tool to image collagen fibers .
collagen has highly crystalline triple - helix structure that is not centrosymmetric , which makes it extremely bright in shg .
shg does not involve the excitation of molecules ; as a result , the molecules do not suffer the effects of phototoxicity or photobleaching . since the first publication of shg on collagen three decades ago , this technique has become a robust tool for imaging tissue structure in both ex vivo and in vivo preparations [ 4 , 1719 ] . in this study
, we aimed to develop a quantitative method based on image spatial correlation of collagen shg images to analyze collagen organization at mesoscale , that is , at the scale of 10100 m , comparable to cellular size .
image spatial correlation provides an unbiased way for estimating the size of spatial organization . by applying image spatial correlation to collagen shg images with proper resolution ,
it is possible to extract the collagen mesoscale organization information . during cell - mediated ecm remodeling
, cells may generate collagen - dense areas through cell - ecm interactions that modify the density of collagen in the regions around the cells .
we applied this method to estimate the mesoscale changes of shg high intensity area size due to the presence of cells in the collagen matrix few days after seeding the cells .
this parameter is referred to as collagen compaction level in this report , the same as stevenson et al .
relevant to our methodology , applying image correlation spectroscopy to shg image has been described by raub et al . , which focused on quantifying collagen matrix pore size and fiber dimension from collagen .
in contrast to the emphasis of the microscopic scale in the previous report , here we applied the spatial correlation technique to analyze the mesoscale structure of collagen and showed that the shg image of collagen combined with image correlation spectroscopy is an effective means to assess the mesoscale properties of an in vitro ecm that involves cellular remodeling .
we also emphasize that the image correlation method used in this work provides unbiased quantitative evaluation of the mesoscale collagen organization .
this is important for comparison of different cell types and for the assessment of the effect of drugs treatment in regard to the large scale organization of the ecm . in this report , we compared the collagen matrix remodeling ability of two human breast cancer cell lines with distinct degrees of invasiveness , mda - mb-231 and mcf-7 , and fibroblast cell line nih/3t3 .
tumor cell invasiveness has been associated with increased contractile force generation , and ecm compaction level may reflect cell contractility .
our results suggested that cancer cells with high invasiveness have significantly more ability to remodel the collagen matrix , consistent with the previous report , while nih/3t3 cell line moderately changed the collagen compaction level .
furthermore , the matrix remodeling ability of invasive cancer cell line can be altered through metalloproteinases ( mmp ) inhibitor or sequestering f - actin , suggesting that mmp and actin cytoskeleton affect collagen remodeling at the mesoscale .
mda - mb-231 , mcf-7 , and nih/3t3 cell lines ( american type culture collection htb-26 , htb-22 , and cl-1658 , resp . ) were obtained from dr .
cells were cultured in dulbecco 's modified eagle 's medium ( dmem ) with high glucose ( sigma , st .
louis , mo ) supplemented with 10% ( v / v ) fetal bovine serum ( fbs ) at 37c in a 5% co2 humid incubator .
type i collagen with original concentration of 3.75 mg / ml was purchased from bd biosciences ( franklin lakes , nj ) .
collagen was diluted with 10x pbs and water to achieve final concentration of 1x pbs and 2 mg / ml collagen .
the final concentration of 5 10 cells / ml cell - collagen mixture was added to 8-well lab - tek chambered coverglass with surface area 0.7 cm per well ( thermo scientific , rochester , ny ) .
collagen was polymerized at 20c for 1 hr and then at 37c for 20 minutes .
medium was changed everyday after collagen polymerization for both collagen - only and cell - collagen matrix to ensure minimum variation caused by evaporation .
images were taken under room temperature after 1 , 2 , and 4 days of collagen polymerization .
mda - mb-231 cells were cultured in type i collagen matrix and applied pharmacological treatment everyday when changing the medium .
the treatment was applied to the cell - collagen matrix for 4 consecutive days before imaging .
the f - actin sequestering drug latrunculin b ( molecular probes , eugene , or ) was used at 1 m final concentration .
the matrix metalloproteinase inhibitor marimastat ( tocris bioscience , bristol , united kingdom ) was used at 10 m final concentration . to assess the degree of collagen matrix remodeling , collagen second harmonic generation ( shg ) images
were collected using lsm 710 ( carl zeiss , maple grove , mn ) with a 40 0.75 n.a .
the mai - tai laser ( newport , irvine , ca ) with emission at 900 nm was used for second harmonic generation .
the collagen shg propagating in backward direction was collected using a bandwidth filter ( 442463 nm ) .
we conducted 4 independent experiments for the samples containing collagen only , collagen with mcf cell line , and collagen with nih cell line .
we did 8 independent experiments for the collagen with mb231 cell line , and 3 independent experiments for each drug treatment .
as shown in figure 1 upper right panel , z - stack shg images with total of 10 slices across 200 m depth were taken ( each slice is 22 m apart along the axial direction ) .
each x - y scan slice of the z - stack image was 1024 1024 pixels with pixel size of 0.8 m and pixel dwell time of 50 s / pixel . the lowest slice was taken right above the glass surface of culture dishes ( slice 1 in the upper left panel of figure 1 ) and went all the way up to 200 m above ( slice 10 in the upper left panel of figure 1 ) .
the spatial correlation function below ( 1 ) was then applied to each x - y plane of shg images :
( 1)gs(,)=i(x , y)i(x+,y+)x , yi(x , y)x , y21 ,
where i is the intensity , and are the spatial increments in the x and y directions , respectively , and the angle bracket indicates the average over all the spatial locations in both x and y directions .
since smaller scale structures are not the subject of this study , the original 1024 1024 pixel images were binned 4 4 , which reduce the images to 256 256 pixels .
the binning and spatial correlation analysis was done by simfcs software developed at the laboratory for fluorescence dynamics ( available at http://www.lfd.uci.edu/ ) . in this study , we have found no specific directionality of collagen fibers and organization , which results in symmetrical spatial correlation images along both x axis and y axis ( see , e.g. , figure 1 , the lower left panel of the correlation image ) .
hence , the cross - sections of x - axis and y - axis were used for fitting .
a script was written in matlab ( mathworks , natick , ma ) to perform a fit of the cross - section of the image spatial correlation function .
figure 1 lower right panel shows that we need at least 2 gaussian functions for the fitting , one very narrow that corresponds to the size of the pixel ( the green dashed curve in figure 1 lower right panel ) and a broad gaussian which corresponds to the low spatial frequency fluctuation of the collagen matrix ( the blue dashed curve in figure 1 lower right panel ) .
the amplitude and standard deviation of the broad gaussian ( a , ) were extracted .
the average peak amplitude of low spatial frequency components is 4.5 ( the average of a ) .
for the images with a smaller than 0.001 , the value of can not be reliably measured . in the following ,
we mainly discuss the value , which reports the mesoscale collagen spatial compaction level .
note that in this repot we used the gaussian fit not as a model of the spatial frequency fluctuations but as an algorithmic approach to estimate the spatial extent of the shg image structure .
data plotting was done using matlab . for each data group , the highest and lowest values
we report the data as significantly different if the p value is smaller than 0.01 .
to evaluate the impact of ecm remodeling by mcf-7 , nih/3t3 , and mda - mb-231 cells , type i collagen matrices with cells embedded were imaged using two - photon microscopy after one , two , and four days of collagen polymerization .
shg z - stack images covered 200 m depth ( 10 z slices in total ) and 819 m of both width and length were collected and analyzed as shown in figure 1 .
collagen matrix with no cells was prepared under the same polymerization and incubation conditions for baseline comparison .
figures 2(a)2(d ) shows the collagen matrix shg images of collagen alone , collagen with mcf-7 , nih/3t3 , and mda - mb-231 cell line , respectively .
contrary to the relatively homogenous collagen fiber distribution from collagen matrix without cell seeding shown in figure 2(a ) , collagen organization heterogeneity can be clearly seen after one day of mda - mb-231 cell seeding , and the collagen compaction level at some local regions increased over time ( figure 2(d ) ) . on the other hand , the less - invasive breast cancer cell line mcf-7 and fibroblast cell line nih/3t3 have more mild effects on collagen compaction level ( figures 2(b ) and 2(c ) ) .
the association of the high collagen matrix density region with the cell locations can be seen in figure 2(e ) , where the collagen fibers ( magenta ) were densely packed around mda - mb-231 cells ( green ) .
our aim is to quantitatively assess the average size of these pericellular collagen compactions due to cell - ecm remodeling . as described in the method section , to capture this mesoscale collagen matrix feature , we calculated image spatial correlation of shg images ( figure 1 ) to statistically estimate the average size of collagen compaction .
the image spatial correlation gives rise to a sharp peak , which corresponds to the pixel size , and a broader distribution , which reflects the actual size of the structure .
the lower right panel in figure 1 represents an example of spatial correlation with this property . to identify the characteristic scale of these two distinct spatial frequency features
the standard deviation value of the sharp gaussian of the shg spatial correlation , as shown in figure 3 , was not affected by cell ecm remodeling .
it coincides with the value of the pixel size and does not have significant change over time .
in contrast , the standard deviation of low frequency component reflects the average size of the collagen compaction , and the amplitude is a function of signal - to - background ratio and of the proportion of the broad gaussian .
here we define as the standard deviation of low spatial frequency component ( i.e. , the broader distribution ) , which in our experiment was in the order of tens of micrometers . within 4 days
, the average of collagen matrix with mda - mb-231 cells increases significantly from 17 m in day 1 , 21 m in day 2 , to 39 m after 4 days of incubation ( figure 3 ) .
the average of collagen matrix without cells shows much smaller change , increasing by only 1 m after 4 days compared to ~20 m increase in the presence of mda - mb-231 cells .
consistent with the hypothesis that less invasive tumor cells may cause less collagen compaction , we found that the collagen shg images with mcf-7 cells embedded showed significantly smaller . the collagen remodeling ability of nih/3t3 cell line lies between mcf-7 and mda - mb-231 cell line .
however , unlike mda - mb-231 cell line , the change of over time was minimal for nih/3t3 embedded collagen matrix .
we exclude the possibility of the value of due to the space occupied by cells based on the following .
( 1 ) the size indicated by is larger than cell body ; ( 2 ) cell size does not change over time , but collagen compaction level increases dramatically over time ; and ( 3 ) the cell size is comparable for the three cell lines examined . to further assess the method 's applicability to evaluate collagen remodeling , we performed latrunculin b treatment and marimastat treatment to mda - mb-231 cells cultured in the type i collagen matrix .
latrunculin b affects the actin polymerization degree , which leads to the altered mechanical properties of the cell , including decreased contractile force .
marimastat is a broad - spectrum matrix metalloproteinase inhibitor that blocks the activity of mmp-1 , 2 , 3 , 7 , 9 , and 12 and has been shown to affect collagen remodeling in vitro . with either latrunculin b or marimastat treatment , the collagen compaction level by mda - mb-231 cells
the treatment with marimastat decreased the magnitude of collagen remodeling from the average = 37 m to = 29 m , while latrunculin b showed even more severe effect that leaded to similar to mcf-7 cell line ( = 21 m ) . for a structure with given geometry in an image , the structure size is linearly correlated to the standard deviation of spatial correlation as obtained from the gaussian fit .
the resulting spatial correlation will have a standard deviation that is half of the original structure size due to the properties of the correlation function .
the amplitude , on the other hand , is proportional to the area of the structure and signal - to - background ratio .
the result from collagen shg images used in this study , thus , represents a weighted average of complex patterns , with two major components : the pixel - size correlation due to the small collagen fiber width and its random orientation ( figure 3 ) and the collagen compaction area created by cell - matrix interaction , which is highly variable ( figures 4 and 5 ) . here
, we demonstrated that by using spatial image correlation it is possible to quantify and differentiate the impact of cells on collagen matrix under different conditions .
the estimated collagen compaction structure size from mda - mb-231 cell after 4 days in collagen is around 70 m .
compared to an average cell body diameter of 2030 m , the collagen structure emerging after cell seeding may be much larger than the single cell .
the interplay between the biophysical properties of the cell and ecm establishes a dynamic mechanical reciprocity between the cell and the ecm in which the cell 's ability to exert contractile stresses against the ecm balances the elastic resistance of the ecm to deformation . in this sense , ecm is effectively a physical extension of the cell and cytoskeleton . in this study , we described a method based on spatial correlation to quantify the degree of matrix remodeling at the tens of micron scale .
we chose cell lines ( mcf-7 , nih/3t3 , and mda - mb-231 ) that presumably could show a difference in regard to collagen compaction at the mesoscale .
we were able to estimate the average collagen compaction area by mcf-7 cells , nih/3t3 cells , and mda - mb-231 cells . with the same initial type i collagen concentration , the more invasive cancer cell line , mda - mb-231 , was able to generate significantly larger collagen compaction areas , while the less invasive cell line ( mcf-7 ) has a modest effect on the collagen matrix .
the nih/3t3 cell line exhibits the collagen remodeling ability between mcf-7 and mda - mb-231 cell lines .
this result is consistent with previous studies showing that tumor cell invasiveness is associated with increased contractile force generation , which affects the collagen remodeling ability .
this notion was further supported by the significant decrease of when mda - mb-231 cells were treated with latrunculin b , which inhibits actin polymerization and decreases cellular contractility . blocking mmps by marimastat , although with smaller impact compared to the effect of actin cytoskeleton disruption , also showed significant alteration on the degree of collagen compaction .
in fact , mmps have been associated to tumor stroma and fibrosis in vivo [ 26 , 27 ] . a recent research conducted by tracking bead displacement to assess
ecm remodeling also showed the treatment of mmp inhibitor reduced the magnitude of ecm deformation , supporting our evaluation by spatial correlation method .
high breast tissue density due to increased type i collagen is one of the single largest risk factors for developing breast cancer .
this higher stiffness correlates with increased mammographic density and has been exploited to detect cancer [ 4 , 3032 ] .
matrix stiffness from type i collagen cross - linking has also been implicated as a contributor to the enhanced invasive behavior in tumor , enhanced cell growth and survival , and promoted migration .
however , what causes the increase in tumor stromal stiffness and how stromal stiffness contributes to tumor progression is still not clear .
in addition , the causality of matrix stiffness and tumor invasiveness has yet to be determined .
our data indicate that tumor cells may be involved in ecm remodeling and contribute to the different degree of collagen compaction based on their invasiveness .
this hypothesis is also supported by in vivo tumor collagen images which showed collagen stretching near invasive cancer cells , which is similar to what we observed in vitro . in this report
, we showed that the image spatial correlation method provides a measure of ecm spatial organization , in this example the collagen compaction by cells , at the scale comparable or larger than the cellular size .
the advantages of the analysis method proposed in this report include ( 1 ) the simple statistical procedure that does not require image segmentation or other predefined parameters , which could be more subjective ; ( 2 ) it does not depend on the intensity range of each image acquired , so the comparison between different images can be achieved without difficulty ; and ( 3 ) the use of shg to image collagen fibers that does not require extra labeling and could be directly applied to in vivo studies . due to its simplicity of implementation , this method has the potential to not only facilitate in vitro ecm remodeling study or ecm assessment for tissue engineering purposes but can be also applied to in vivo research for determining regional collagen organization differences in tissues and tumor sites . by choosing the proper pixel size , it is possible to use this method to detect structures of different scales and to quantify each component 's significance .
provided there is enough axial resolution , image spatial correlation can be used in three - dimensions , making it possible to tell whether collagen exhibits different spatial organization along lateral and axial directions . furthermore ,
although not shown in this report , the directional matrix alignment could be estimated by 2d gaussian fitting to the spatial correlation image , which may increase the application of this method . | extracellular matrix ( ecm ) remodeling is a critical step of many biological and pathological processes .
however , most of the studies to date lack a quantitative method to measure ecm remodeling at a scale comparable to cell size . here , we applied image spatial correlation to collagen second harmonic generation ( shg ) images to quantitatively evaluate the degree of collagen remodeling by cells .
we propose a simple statistical method based on spatial correlation functions to determine the size of high collagen density area around cells .
we applied our method to measure collagen remodeling by two breast cancer cell lines ( mda - mb-231 and mcf-7 ) , which display different degrees of invasiveness , and a fibroblast cell line ( nih/3t3 ) .
we found distinct collagen compaction levels of these three cell lines by applying the spatial correlation method , indicating different collagen remodeling ability .
furthermore , we quantitatively measured the effect of latrunculin b and marimastat on mda - mb-231 cell line collagen remodeling ability and showed that significant collagen compaction level decreases with these treatments . |
oxo clusters of the general composition
tiaob(or)c(oocr)d
are obtained when titanium alkoxides , ti(or)4 , are treated with more than one molar
equivalent of a carboxylic acid.1 the carboxylic acid not only
provides carboxylate ligands but also acts as an in situ water source through esterification with
the eliminated alcohol .
the outcome of the reaction depends , among others , on the groups r and
r as well as the ti(or)4/rcooh ratio .
many oxo clusters have been
isolated with different degrees of condensation ( a : b ratio ) ,
different degrees of substitution ( a : d ratio ) , different
proportions of residual or groups ( a : c ratio ) , and , as a
consequence , different structures .
one of the more prominent structure types is
ti6o4(or)8(oocr)8 , which was obtained for
several r / r combinations.2,3 an example , with r = ipr and oocr = omc ( ti6 ) ( homc
= methacrylic acid ) , is shown in figure1 . molecular structure of
ti6(3-o)2(2-o)2(2-oipr)2(2-omc)8(oipr)6
( ti6 ) : ( top ) ball - and - stick model ; ( bottom ) polyhedral representation .
hydrogen atoms are omitted
for clarity . selected bond lengths and angles : ti1ti2 3.3783(3 ) , ti1ti3 3.1062(3 ) ,
ti2ti3 3.5862(4 ) , ti1o1 1.9047(9 ) , ti1o2 1.7497(9 ) , ti1o3 2.0874(9 ) ,
ti1o4 2.0024(10 ) , ti1o6 2.0465(10 ) , ti1o8 2.0051(10 ) , ti2o2
1.8893(9 ) , ti2o1 1.9061(9 ) , ti2o5 2.1019(10 ) , ti2o7 2.0892(10 ) ,
ti2o10 2.0300(10 ) , ti2o12 1.7598(11 ) , ti3o1 2.0722(9 ) , ti3o3
1.9572(10 ) , ti3o9 2.1627(11 ) , ti3o11 2.0482(11 ) , ti3o13 1.7995(10 ) ,
ti3o14 1.7903(11 ) ; ti1o1ti2 128.00(5 ) , ti1o1ti3
102.63(4 ) , ti2o1ti3 128.65(5). in this article , we describe the structures of mixed - metal oxo clusters that are derived from
that of ti6o4(or)8(oocr)8 .
the variability of
this structural motif shows that this is a robust structure that not only tolerates variations of r
and r , but also that of the metal polyhedra of which it is composed .
mixed - metal clusters have been prepared by addition of carboxylic acids to metal alkoxide
mixtures.4 we have previously shown that use of metal acetates
as one of the components is a good alternative , because they are readily available and easy to
handle.5 the mixed - metal clusters described in this article
were therefore prepared from ti(oipr)4 and metal acetates as precursors
[ equation ( 1 ) ] .
we chose a selection of
divalent metals ( m ) with different ionic radii and different preferred coordination numbers to find
out how these parameters influence the structures of the clusters , keeping in mind that the charges
of the metal as well as the total number of coordination sites must be balanced by the ligands to
get a stable cluster.6(1 ) in previous work on carboxylate - substituted metal oxo clusters , we were initially using
methacrylic acid to get clusters that can be polymerized subsequently to obtain hybrid
materials.7 it turned out that methacrylic acid is
particularly well suited to the production of crystalline clusters .
this may be owing to steric
reasons and/or a suitable balance of substitution versus esterification reaction rates .
we therefore
used methacrylic acid in this work as well , although in this case no subsequent polymerizations were
intended .
although several clusters of the composition
ti6o4(or)8(oocr)8 are known with various
r / r combinations , we prepared the derivative with r = ipr and
oocr = omc ( ti6 ) ( figure1 ) for better comparison of the
structural parameters with that of the mixed - metal clusters reported in this article .
the
centrosymmetric ti6o4 core is formed by two
ti3(3-o ) units , which are connected through two
2-oxygen atoms .
an alternative description of the structure is that of a
ti4o4 ring of four corner - sharing octahedra to which two ti octahedra ( called
outer ti in the following ) are condensed through shared edges .
balancing of charges
and coordination numbers is achieved by two bridging oipr ligands ( connecting the
two edge - sharing octahedra ) , eight bridging carboxylate ligands and six terminal
oipr ligands .
treatment of fe(oac)2 and ti(oipr)4 ( 2 equiv . ) with
methacrylic acid ( 17 equiv . ) resulted in reddish - brown crystals of
feti5o4(oipr)4(omc)10 ( feti5 )
( figure2 ) .
the cluster core of feti5 is isostructural to
that of ti6 . although attachment of the outer
ti octahedra is the same as in ti6 ,
the four ti atoms of the ti4o4 ring are partly replaced by fe atoms owing to
the nearly identical ionic radii of ti ( 0.605 8 ) and low - spin fe in an octahedral coordination ( 0.61
) .
the ti / fe1 site ( corresponding to ti2 in ti6 ) was refined with an occupancy for fe of
34 % and that of ti / fe2 ( corresponding to ti1 in ti6 ) with 16 % .
to
prove incorporation of both metals , the crystals were washed with dry n - heptane and
their metal content was checked with energy - dispersive x - ray spectroscopy ( edx ) , through which both
fe and ti were found in the crystals .
selected bond lengths and angles :
fe / ti1fe / ti2 3.4215(10 ) , fe / ti1ti3 3.4917(10 ) , fe / ti2ti3 3.0466(11 ) ,
fe / ti1o1 1.918(3 ) , fe / ti1o2 1.875(3 ) , fe / ti2o1 1.904(3 ) , fe / ti2o2
1.776(3 ) , ti3o1 1.990(3 ) , fe / ti1o7 2.044(3 ) , fe / ti1o9 2.040(3 ) ,
fe / ti2o11 2.030(3 ) , ti3o8 2.025(4 ) , ti3o10 1.974(4 ) , ti3o12
2.031(4 ) , fe / ti1o3 2.015(3 ) , fe / ti1o5 2.061(3 ) , fe / ti2o4 2.012(3 ) ,
fe / ti2o6 2.047(3 ) , fe / ti2o13 2.137(3 ) , ti3o13 1.902(3 ) , ti3o14
1.773(3 ) ; fe / ti1o1fe / ti2 127.09(15 ) , fe / ti1o1ti3
126.61(14 ) , fe / ti2o1ti3 102.95(12). feti5 needs two negative ligands less than ti6 because of the lower charge of
fe , but the total number of coordination sites to be occupied by the ligands is
the same because all metal atoms in both ti6 and feti5 are octahedrally coordinated .
thus , the two
terminal oipr groups o12 ( on ti2 ) and o14 ( on ti3 ) in ti6 , which are nearly
parallel to each other are replaced by one bridging omc ligand in feti5 ( o9 and o10 ) ( compare
figures1 and 2 ) . as
a consequence of this substitution , the coordination octahedra in feti5 are slightly tilted relative
to ti6 .
this results , among other things , in slightly different distances between the metal centers
( 3.4215 , 3.4917 , and 3.0466 in feti5 compared with 3.3783 , 3.5862 , and 3.1062 in
ti6 for analogous distances ) .
reaction of zn(oac)2 with ti(oipr)4 and methacrylic acid
in different molar ratios afforded the centrosymmetric cluster
zn2ti4o4(oipr)2(omc)10
( zn2ti4 , figure3 ) .
the structure of this cluster is again
structurally related to that of ti6 , with the two outer ti octahedra being replaced by zn
tetrahedra .
contrary to ti6 and feti5 , in which the outer ti octahedra share an edge with one of the
octahedra of the ti4o4 unit , the zn tetrahedra share a corner with two ti
octahedra . the 3-oxygen ( o1 ) is slightly shifted towards the titanium atoms
[ zn
o1 1.871(1 ) and 1.923(1 ) ] , which
results in a widening of the ti1o1ti2 angle to 136.01(7) compared to
128.00(5) in ti6 and 127.1(1) in feti5 .
hydrogen atoms are omitted for clarity . selected bond lengths and angles : zn1ti1
3.1470(4 ) , zn1ti2 3.2701(4 ) , ti1ti2 3.3652(5 ) , zn1o1 1.9747(13 ) ,
ti1o1
1.8712(13 ) , ti1o2 1.7550(13 ) , ti2o1 1.9231(14 ) , ti2o2
1.8784(13 ) , ti2o(13 ) 1.7821(14 ) , zn1o3 1.946(2 ) , zn1o5 1.959(2 ) ,
zn1o7 1.940(2 ) , ti1o4 2.106(2 ) , ti1o6 2.0123(14 ) , ti2o8 2.0435(14 ) ,
ti1o9 2.0752(14 ) , ti1o11 1.9994(14 ) , ti2o10 2.0383(15 ) , ti2o12
2.0936(14 ) ; zn1o1ti1 109.80(7 ) , zn1o1ti2 114.05(6 ) ,
ti1o1ti2 136.01(7). zn2ti4 needs four negative ligands less than ti6 ( and two less than feti5 ) to compensate the
metal charges because two ti are replaced by two zn .
the
total number of coordination sites to be occupied by the ligands is reduced by four relative to ti6
and feti5 ( replacement of two octahedra by two tetrahedra ) .
compared to ti6 , four terminal
oipr ligands are missing ( the ones at ti3 and ti3 * in ti6 ) , and the
oipr ligands bridging ti1 and ti3 in ti6 are replaced by a bridging omc ligand in
zn2ti4 .
in addition to the 3-oxygen atom ( o1 ) , the zn atom is connected to both
neighboring ti atoms through bridging omc ligands , two to ti1 and one to ti2 .
the oxygen atoms of
these three omc ligands , together with the 3-oxygen atom , form a tetrahedron
around zn . the zn
o distances of the omc ligands vary only slightly between 1.940(2 ) and
1.957(2 ) and are not much shorter than the zn
the
o zn o angles vary between 103.24(6 ) and 116.79(6). reaction of cd(oac)2 and an equimolar proportion of
ti(oipr)4 with a tenfold excess amount of methacrylic acid resulted in
centrosymmetric
cd4ti2o2(oac)2(omc)10(hoipr)2
( cd4ti2 , figure4 ) . the structure of this cluster can again
be related to that of ti6 , but there are more profound changes . ( i ) in contrast to the structures
discussed before , the four ti atoms in the center are replaced by cd atoms , ( ii ) acetate groups
[ originating from the cd(oac)2 precursor ] were incorporated in the
structure , ( iii ) two cd atoms are bridged across the cd4o4 ring , and ( iv ) the
structure contains no 2-oxygen atoms , only the 3-o units are
retained .
only hydrogen of the oh groups are shown . selected bond lengths and angles : ti1cd1
3.4499(9 ) , ti1cd2 3.4521(9 ) , cd1cd2 3.8904(8 ) , ti1o1 1.698(2 ) , cd1o1
2.262(2 ) , cd2o1 2.267(2 ) , cd1o2 2.260(2 ) , cd1o2 2.355(2 ) ,
cd1o4 2.232(2 ) , cd2o3 2.267(3 ) , cd2o4 2.336(2 ) , cd2o5 2.368(2 ) ,
ti1o7 2.012(2 ) , ti1o9 1.997(2 ) , ti1o11 1.977(2 ) , ti1o13 1.986(2 ) ,
ti1o14 2.227(2 ) , cd1o6 2.256(2 ) , cd1o8 2.260(2 ) , cd2o10 2.279(3 ) ,
cd2o12 2.228(3 ) ; cd1o1cd2 118.40(9 ) , cd1o1ti1
120.55(11 ) , cd2o1ti1 120.36(11). the titanium atoms are symmetrically connected to the central cd4 unit through four
bridging omc ligands each ( two to each neighboring cd atom ) as well as a 3-o atom
connecting cd1 , cd2 , and ti1 . ti1 is approximately equidistant to both cd [ ti1cd1
3.4499(9 ) , ti1cd2 3.4521(9 ) ] .
the alcoholic proton was identified in the electron
density map , and the long ti1o14 distance [ 2.2186(3 ) ] proves
additionally that this is a coordinated alcohol rather than a oipr group .
o1 bond length of 1.698(2 ) is due to the coordinated roh in the
trans position .
the total positive
charge of the metals , however , is + 24 in ti6 but only + 16 in cd4ti2 .
this means that a
smaller number of ( monoanionic ) ligands must satisfy the coordination requirements of the metals . in
addition to the ligands
discussed before , coordination of the cd4 core must be completed
by two omc and two oac ligands .
thus , one oxygen atom ( o2 ) of the remaining omc ligands bridges cd1 and cd1 , and
the second ( o3 ) is coordinated to cd2 .
the acetate ligands are bridging - chelating [ o4 bridges
cd1 and cd2 ; o4 and o5 chelate cd2 ] .
the coordination octahedron of cd2 is much more
distorted than that of cd1 due to the chelating carboxylate .
whereas the cis
o cd1o angles of cd1 are between 75.0(1 ) and 97.9(1) , those of cd2 are
between 55.50(8 ) and 109.49(9). the 3-omc and the chelating / bridging acetate cause a shorter distance between
the symmetry - related cd1 atoms than the corresponding ti atoms in ti4 or zn2ti4 .
the distance
between cd1 and cd1 [ 3.6618(8 ) ] is comparable to that of
cd1cd2 [ 3.8904(8 ) ] and cd1cd2 [ 3.9525(8 )
] .
however , the symmetry - related cd2 atoms are moved further apart
[ cd2cd2 6.9358(14 ) ] .
another carboxylate - substituted cd / ti oxo cluster reported in the literature ,
cd4ti4o6(ochch2nme2)4(occf3)4(oac)4,9 is also based on four interconnected cd polyhedra . in this case ,
however , the cd4 unit is capped by two condensed ti octahedra on both sides .
reaction of an equimolar mixture of ca(oac)2 and ti(oipr)4
or ti(obu)4 with methacrylic acid ( 4.5 equiv . ) resulted in crystals of
[ ca2ti4o4(oac)2(omc)10]n
( ca2ti4 ) besides much of a colorless precipitate of unknown composition . in ca2ti4
the outer two ti
octahedra of the ti6 structure are replaced by ca distorted pentagonal bipyramids , but the structure
of the ti4o4 core is retained , similar to that in zn2ti4 .
owing to the lower
charge of ca and its higher coordination number , the clusters are condensed to
endless parallel chains of ca2ti4 units ( figure5 ) , contrary to the molecular clusters discussed before .
the
ca2ti4 repeating units are connected through a chelating - bridging acetate
ligand . the acetate ligand is chelating ca1 , while one of its oxygen atoms ( o3 ) is bridging ca1 and
ti1 , and the other ( o4 ) ca1 and ca1. the ca1o4 bond length [ 2.660(2 )
] is much longer than that of ca1o4 [ 2.292(2 )
] .
hydrogen atoms are omitted for clarity . selected bond lengths and angles :
ca1ca1 4.0045(9 ) , ca1ti1 3.5083(6 ) , ca1ti2 3.6524(6 ) ,
ca1o1 2.4549(16 ) , ti1o1 1.965(2 ) , ti1o2 1.730(2 ) , ti2o1 1.760(2 ) ,
ti2o2 1.912(2 ) , ca1o3 2.428(2 ) , ca1o4 2.659(2 ) , ca1o4
2.292(2 ) , ca1o5 2.361(2 ) , ca1o7 2.317(2 ) , ca1o9 2.342(2 ) , ti1o3
2.1116(18 ) , ti1o6 1.9633(18 ) , ti2o8 1.950(2 ) , ti2o10 1.958(2 ) ,
ti1o11 2.021(2 ) , ti1o13 2.004(2 ) , ti2o12 2.026(2 ) , ti2o14 2.159(2 )
; ca1o1ti1 104.55(7 ) , ca1o1ti2 119.20(8 ) ,
ti1o1ti2 135.64(9). in addition to the chelating - bridging acetate ligand and the 3-oxygen , ca1 is
also connected to ti1 through a bridging omc ligand .
the
ca o bond lengths of the omc ligands differ only slightly [ 2.317(2 ) and 2.361(2 )
] , as well as the ti
o bond lengths of these ligands
[ 1.950(2)1.963(2 ) ] . when the molar ratio of ti(oipr)4/ca(oac)2 in the
precursor mixture was increased from 1:1 to 2:1 ,
while keeping the proportion of methacrylic acid
per metal constant , a variation of the structure of ca2ti4 was observed . the ligand sphere and the
linkage of the cluster units in
[ ca2ti4o4(oac)(omc)11(homc)buoh]n
( ca2ti4a ) is slightly different to that of ca2ti4 , and the repeating cluster unit is doubled .
the acetate ligand
linking the cluster units is partly substituted by an omc ligand .
the occupancy of the acetate on
one site is 25 % , and 75 % on the second ( figure6 , indicated by grey dashed lines ) .
selected bond lengths and
angles : ca1ca2 3.7089(8 ) , ca1ti1 3.5606(7 ) , ca1ti2 3.6737(7 ) ,
ca1o1 2.500(2 ) , ti1o1 1.931(2 ) , ti1o2 1.745(2 ) , ti2o1 1.774(2 ) ,
ti2o2 1.875(2 ) , ca1o3 2.560(2 ) , ca1o4 2.510(2 ) , ca1o4
2.360(2 ) , ti1o3 2.101(2 ) , ca1o5 2.362(2 ) , ca1o7 2.417(2 ) , ca1o9
2.358(2 ) , ti1o6 1.935(2 ) , ti2o8 1.967(2 ) , ti2o10 1.957(2 ) , ca1o15
2.489(2 ) , ca2o15 2.812(2 ) , ti1o11 2.025(2 ) , ti1o13 2.034(2 ) , ti2o12
2.045(2 ) , ti2o14 2.127(2 ) ; ca1o1ti1
106.22(8 ) ,
ca1o1ti2 117.52(9 ) , ti1o1ti2 136.12(10). different to ca2ti4 , the structure of ca2ti4a contains a neutral homc ligand , of which one of the
oxygen atoms ( o15 ) is unsymmetrically bridging ca1 and ca2 [ ca2o15 2.812(2 ) ,
ca1o15 2.489(2 ) ] .
this increases the coordination number of ca from seven in
ca2ti4 to eight in ca2ti4a .
the proton of the methacrylic acid is hydrogen bonded to a
non - coordinated butanol , and the proton of the butanol in turn to an oxygen atom ( o7 ) of one of the
bridging omc ligands .
the bridging homc moves the ca atoms closer to each other
[ ca1ca2 3.7089(8 ) compared with 4.0045(9 ) in ca2ti4 ] and also affects
the linking carboxylate group .
o bond lengths of the chelating ligands at ca1 are now
equal [ ca1o3 2.560(2 ) , ca1o4 2.510(2 ) ] , and the ca2o4
distance is slightly lengthened [ 2.3604(19 ) ] . the ca o
bonds of the
chelating carboxylate group at ca2 differ in lengths , but are still shorter than in ca2ti4
[ ca2o19 2.432(2 ) , ca2o20 2.546(2 ) ] .
all other bond lengths
are comparable to those in ca2ti4 . we have previously prepared
sr2ti8o8(oipr)1.73(oac)2.27(omc)16 ,
with a crown ether like structure , from sr(oac)2 , ti(oipr)4 ,
and methacrylic acid.5 a compound with a different structure
was obtained when sr(oac)2 was treated with ti(obu)4 .
the arrangement of the
metal polyhedra of polymeric
[ sr2ti4o4(omc)12(homc)2]n
( sr2ti4 , figure7 ) is the same as that of ca2ti4 and ca2ti4a ,
but there are differences in the ligand sphere , especially in the coordination of sr .
in addition to
3-oxygen atoms , sr1 is connected to ti2 and sr2 to ti4 through two bridging omc
ligands each , similar to ca1 and ti1 in ca2ti4 .
the connection of sr1 and sr2 to ti3 and ti1 by two
omc ligands each is slightly different . in one pair of omc ligands ,
one oxygen atom of the coo group
bridges sr1 and ti1 , whereas the other oxygen atom coordinates to sr2 ( correspondingly , one oxygen
atom bridges sr2 and ti3 , and the other coordinates to sr1 ) . in the second pair of omc ligands , one
oxygen atom of the coo group bridges the two sr atoms and the other oxygen atom binds to ti1 ( or
ti3 , respectively ) .
selected bond lengths and angles : sr1ti1 3.6312(4 ) ,
sr1ti2 3.7280(4 ) , ti1ti2 3.3691(5 ) , sr1o1 2.581(2 ) , ti1o1
1.803(2 ) , ti1o2 1.836(2 ) , ti2o1 1.884(2 ) , ti2o2 1.777(2 ) , sr1o3
2.877(2 ) , sr2o4 2.598(4 ) , sr2o4a 2.438(13 ) , sr1o5 2.595(2 ) , sr2o5
2.705(2 ) , sr1o7 2.632(2 ) , sr1o13 2.946(2 ) , sr1o14 2.558(2 ) , sr1o15
2.558(2 ) , ti1o3 2.017(2 ) , ti1o6 1.962(2 ) , ti1o9 2.048(2 ) , ti1o11
2.087(2 ) , ti2o8 1.953(2 ) , ti2o13 2.026(2 ) , ti2o10 2.013(2 ) , ti2o12
2.071(2 ) ; sr1o1ti1 110.59(7 ) , sr1o1ti2 112.29(7 ) ,
ti1o1ti2 137.04(9). the coordination spheres of the sr atoms are completed by an -homc . the
coordination number for sr is nine with two very long sr o bonds ( > 2.8 ) . the
methacrylic acid bonded to sr1 shows hydrogen bonds to the omc ligand bridging sr1 and ti2
[ sr1o15 2.558(2 ) , o16o7 2.728(2 )
] and that bonded to sr2 hydrogen bonds to o4 of a 3-omc ligand
[ sr2o31 2.556(2 ) , o32o4 2.804(5 )
] . the acid as well as o4 are disordered with 75:25 % occupancy of both
positions .
the minority position shows no hydrogen bonding
[ o32ao(4a ) 3.66(2 ) ] .
the structure of all the clusters described in this article can be derived from that of ti6 .
the
general structure is retained when part of the ti atoms is replaced by two - valent atoms
( fe , zn , cd , ca ,
sr ) , but the lower charge of the second metal renders modification of the ligand
sphere necessary . depending on the preferred coordination number of the two - valent atoms , this
adaptation occurs differently .
in addition to different coordination of the negatively charged
ligands , completion of the coordination sphere is also possible by coordination of neutral ligands
( roh , mcoh ) , as observed in cd4ti2 , ca2ti4a , or sr2ti4 .
fe and ti have similar bonding characteristics with
oxygen and the ionic radii are almost equal ( 0.61 and 0.605 ) , hence the four inner
ti sites are partly replaced by fe atoms ( feti5 ) . in
contrast , the ionic radius of cd is much bigger ( 0.95 ) .
this results in
a different arrangement of the coordination octahedra in cd4ti2 , namely , replacement of the inner ti
atoms by cd .
the lower charge is compensated by a different coordination behavior of the smaller
number of ligands .
although the size of zn is the same as that of fe and
ti ( 0.61 ) , it usually exhibits tetrahedral coordination .
hence a
partial substitution of the ti atoms is not possible . in zn2ti4 , the two outer ti octahedra
ca and sr ions are much bigger , have higher coordination
numbers ( seven - coordinated ca 2.00 , eight - coordinate
ca 2.17 , ten - coordinated sr 2.33 ) , and the
bonds are less directed . in the ca and sr compounds ,
different to feti5 , zn2ti4 , and cd4ti2 where molecular clusters were obtained ,
ca2ti4 , ca2ti4a , and sr2ti4 form chains of condensed clusters in the crystal lattice .
the structures reported in this article impressively demonstrate the subtle balancing of charges
and coordination behavior of both the metals and the ligands to reach a stable cluster.6 in light of the reported results , it is to some extent surprising
that such closely related structures were obtained with a given set of ligands , despite the
considerably different ionic radii and coordination numbers of the metals .
general : all experiments were carried out under an argon atmosphere using standard
schlenk techniques .
ti(obu)4 , fe(oac)2 , and sr(oac)2 were obtained
from aldrich , ti(oipr)4 from abcr ,
ca(oac)2h2o and
zn(oac)2h2o from fluka , and
cd(oac)2h2o from merck .
water - free metal acetates were
obtained by drying under vacuum at 130 c overnight ( verified by ir spectroscopy ) .
all
solvents used for nmr spectroscopy ( eurisotop ) were degassed prior to use and stored over molecular
sieves .
ti6 was prepared analogously to the onpr derivative.2 h and c solution - state nmr spectra were recorded on a bruker avance 250
( 250.13 mhz [ h ] , 62.86 mhz [ c ] ) equipped with a
5 mm inverse - broadband probe head and a z - gradient unit .
general preparative procedure : ti(oipr)4 , the
corresponding water - free metal acetate and an excess amount of methacrylic acid were mixed .
feti5o4(oipr)4(omc)10
( feti6 ) : the red - brown solution of fe(oac)2 ( 1 mmol , 0.174 g ) ,
ti(oipr)4 ( 2 mmol , 0.568 g ) , and methacrylic acid ( 17 mmol , 1.44 g ) was
stirred for one week and then filtered .
reddish - brown crystals were obtained after two weeks , yield
0.120 g ( 41 % rel .
ti ) . zn2ti4o4(oipr)2(omc)10
( zn2ti4 ) : ti(oipr)4 ( 1 mmol , 0.284 g ) , dry zn(oac)2 ( 1
mmol , 0.183 g ) , and methacrylic acid ( 10 mmol , 0.861 g ) were mixed at room temperature .
after 1 week orange crystals were isolated from the mother
liquid , yield ( after washing with dry n - heptane ) : 0.256 g ( 77 % rel .
to ti ) .
h nmr ( cdcl3 , 250 mhz ) : = 1.22 ( m ,
12 h , chme ) , 1.812.11 ( m , 30 h , = cme ) , 4.52 ( sep , 2
h , chme ) , 5.355.68 ( m , 10 h , = ch2 ) , 5.986.43 ( m ,
10 h , = ch2 ) ppm .
ir : $ \tilde { \nu}$
= 611 ( s ) , 686 ( w ) , 790 ( m ) , 825 ( m ) , 859 ( w ) , 942 ( m ) , 988 ( w ) , 1005 ( w ) , 1113 ( w ) , 1162
( w ) , 1241 ( m ) , 1372 ( m ) , 1411 ( s ) , 1552 ( s ) , 1644 ( w ) , 2975 ( w ) cm .
cd4ti2o2(oac)2(omc)10(iproh)2
( cd4ti2 ) : cd(oac)2 ( 1 mmol , 0.231 g ) was mixed with
ti(oipr)4 ( 1 mmol , 0.284 g ) and methacrylic acid ( 10 mmol , 0.861 g ) in
dry ch2cl2 ( 2 ml ) .
the sample was stirred for 3 h and then filtered through a
syringe filter .
after 2 weeks , colorless crystals could be isolated from the mother liquid , yield
( after washing with dry n - heptane ) : 0.198 g ( 48 % rel .
h nmr ( [ d8]toluene , 250 mhz ) :
= 1.36 ( m , 12 h , hochme2 ) , 1.681.78 ( m , 30 h ,
= cme ) , 1.922.01 ( sep , 6 h , cme ) , 4.94 ( m , 2 h ,
hochme ) , 5.165.40 ( m , 10 h , = ch2 ) , 6.036.45 ( m ,
10 h , = ch2 ) ppm .
[ ca2ti4o4(oac)2(omc)10]n
( ca2ti4 ) : dry ca(oac)2 ( 2 mmol , 0.363 g ) and ti(oipr)4
( 2 mmol , 0.568 g ) were treated with methacrylic acid ( 18 mmol , 1.55 g ) . a clear solution was
obtained after 1 h of stirring .
after two weeks colorless crystals formed , in addition to much of a
white insoluble and amorphous precipitate .
the same compound was obtained when dry
ca(oac)2 ( 2 mmol , 0.363 g ) and ti(obu)4 ( 2 mmol , 0.702 g ) were treated with
methacrylic acid ( 18 mmol , 1.55 g ) .
[ ca2ti4o4(oac)(omc)11(homc)buoh]n
( ca2ti4a ) : dry ca(oac)2 ( 1 mmol , 0.182 g ) and
ti(oipr)4 ( 2 mmol , 0.568 g ) were treated with methacrylic acid ( 13.5
mmol , 1.162 g ) .
after two weeks colorless crystals formed , in addition to much of a white insoluble
and amorphous precipitate .
[ sr2ti4o4(omc)12(homc)2]n
( sr2ti4 ) : sr(oac)2 ( 1 mmol , 0.411 g ) and ti(obu)4 ( 1 mmol , 0.351 g )
were treated with methacrylic acid ( 12 mmol , 1.03 g ) .
small amounts of precipitate were formed after
three days in the originally clear solution .
x - ray structure analyses : crystallographic data were collected on a bruker axs smart
apex ii four - circle diffractometer with -geometry at 100 k using
mo - k ( = 0.71073 )
radiation . the data were corrected for polarization and lorentz effects , and an empirical absorption
correction ( sadabs ) was employed .
saint plus software ( bruker analytical x - ray instruments , 2007 ) was used to integrate the frames .
symmetry was then checked with the program platon.10 the structures were solved by charge flipping ( jana2006 ) .
refinement was performed by the
full - matrix least - squares method based on f ( shelxl97 ) with anisotropic
thermal parameters for all non - hydrogen atoms .
hydrogen atoms were inserted in calculated positions
and refined riding with the corresponding atom .
crystal data , data collection parameters , and
refinement details are listed in tables 1 and 2 .
crystal data , data collection parameters , and refinement details.[a ] =
1/[(fo ) +
( xp ) + yp ] , in which p
= ( fo +
2fc)/3 .
crystal data , data collection parameters , and refinement details.[a ] =
1/[(fo ) +
( xp ) + yp ] , in which p
= ( fo +
2fc)/3 .
ccdc-1005662 ( for ti6 ) , -ccdc-1005663 ( for feti5 ) , -ccdc-1005664 ( for zn2ti4 ) , -ccdc-1005665 ( for
cd4ti2 ) , -ccdc-1005666 ( for ca2ti4 ) , -ccdc-1005667 ( for ca2ti4a ) , and -ccdc-1005668 ( for sr2ti4 )
contain the supplementary crystallographic data for this paper .
these data can be obtained free of
charge from the cambridge crystallographic data centre via www.ccdc.cam.ac.uk/data_request/cif . | the mixed - metal oxo clusters
feti5o4(oipr)4(omc)10 ( omc =
methacrylate ) ,
zn2ti4o4(oipr)2(omc)10 ,
cd4ti2o2(oac)2(omc)10(hoipr)2 ,
[ ca2ti4o4(oac)2(omc)10]n ,
and
[ sr2ti4o4(omc)12(homc)2]n
were obtained from the reaction of titanium alkoxides with the corresponding metal acetates and
methacrylic acid .
their structures are derived from ti clusters with the composition
ti6o4(or)8(oocr)8 .
the ca and sr derivatives
consist of chains of condensed clusters . |
bone morphogenetic proteins ( bmps ) are critical for normal intestinal homeostasis , counteracting wnt signaling toward the top of crypts , and allowing differentiation ( scoville et al . , 2008 ) . bmp antagonists , notably grem1 , are expressed in the mesenchyme at the crypt base , restricting bmp activity , potentiating wnt signaling , and maintaining the epithelial stem cell compartment .
germline mutations in bmp pathway members occur in juvenile polyposis and hereditary mixed polyposis ( hmps ) ( hardwick et al . , 2008 ;
hmps is a rare , autosomal - dominant condition in which patients develop multiple types of colorectal polyp and cancer .
hmps results from a 40 kb duplication upstream of grem1 , which causes dramatically increased , ectopic grem1 expression in the normal epithelium ( jaeger et al . , 2012 ) .
in the general population , moreover , single - nucleotide polymorphisms ( snps ) near bmp pathway genes have been found by genome - wide association studies to predispose to colorectal cancer ( crc ) ( jaeger et al .
rs4779584 , near grem1 , was one of the first crc snps to be found .
interestingly , rs4779584 does not show an association with predisposition to benign colorectal tumors ( carvajal - carmona et al . , 2013 ) , suggesting that it may act after tumor initiation . to uncover functional variation tagged by rs4779584
, we had previously refined its association signal using direct genotyping , imputation , and conditional logistic regression ( tomlinson et al . , 2011 ) .
this analysis suggested that rs4779584 captured two independent association signals represented by snps rs16969681 and rs11632715 upstream of grem1 . in this study , we aimed to identify the functional variation tagged by rs16969681 and characterize the mechanism whereby it influenced crc risk .
an updated genetic fine - mapping analysis of snps upstream of grem1 using current snp reference panels confirmed the existence of two independent crc risk snps ( details not shown ) .
other snps in strong linkage disequilibrium ( ld ) with rs16969681 showed an 20-fold lower likelihood of association with disease ( table s1 ) . because rs16969681 tags snps in intergenic dna , its association is most likely caused by modifying a gene regulatory element .
we therefore investigated the epigenetic characteristics of the rs16969681 region ( figure 1 ) .
however , the cell lines used in these studies are not of intestinal origin , so we wished to expand these studies in appropriate cell types .
although grem1 is principally expressed by mesenchymal cells in normal intestine , we found grem1 mrna in 11/32 crc cell lines ( crccls ) , which are all of epithelial origin ( figure s1 ) .
we therefore performed formaldehyde - assisted identification of regulatory elements ( faire ) in four crccls to identify areas of nucleosome - depleted chromatin in the 25 kb region upstream of the grem1 promoter , including rs4779584 and rs16969681 .
the highest signal in grem1-expressing lines was a 1.3 kb peak centered on rs16969681 ( figure 1b ) .
chromatin immunoprecipitation ( chip ) using antibodies against h3k4me2 ( marker of promoters and enhancers ) and panh4ac ( marker of active chromatin ) also showed signals at rs16969681 and the grem1 transcription start site ( tss ) , specifically in grem1-expressing lines ( figures 1c and 1d ) .
the chromatin signature was indicative of a regulatory element , probably an enhancer , located at or near rs16969681 .
in support of this , we demonstrated an interaction between the region containing rs16969681 and grem1 s promoter using chromosome conformation capture ( jaeger et al .
rs16969344 , a snp in perfect ld with rs16969681 , was not associated with total grem1 mrna levels in the cancer genome atlas crc samples .
however , differences in gene - expression levels among cancers may have many different causes , and we therefore searched for allele - specific expression ( ase ) differences .
one crccl in our panel ( colo320 ) was heterozygous at rs16969681 , had two copies of chromosome 15q , expressed grem1 , and was heterozygous for a suitable snp in the grem1 transcript ( rs7162202 in the 3 utr ) .
we found that the normalized allelic ratio ( g : t ) of rs7162202 in colo320 cdna was 1.62 ( p = 0.001 ; figure 2a ) compared with 1.1 ( p = 0.11 ) in a control crccl homozygous for rs16969681 ( sw403 ) , consistent with ase in colo320 .
due to the large distance ( > 30 kb ) between rs16969681 and rs7162202 and no suitable cell line for copy - number - based phasing , we could not determine whether the high - risk allele was associated with higher expression in cis .
we therefore carried out luciferase reporter assays to investigate allele - specific enhancer activity of the rs16969681 region in vitro in sw948 cells ( figure 2b ) .
constructs containing the low - risk allele showed moderate enhancing activity , but this was significantly increased when rs16969681 was mutated to the high - risk allele ( p < 0.001 ) .
a series of deletion constructs around rs16969681 showed increasing enhancer activity ( figure 2b ) .
the smallest construct , with the greatest enhancer activity , showed rs16969681 s high - risk allele to have almost double the activity of the low - risk allele ( p = 0.029 ) .
thus , we found that the region containing rs16969681 carries the hallmarks of an allele - specific transcriptional enhancer .
we wished to test whether modest changes in grem1 levels , such as those resulting from the rs16969681 genotype , could affect intestinal tumorigenesis in vivo .
because the region around rs16969681 is not highly conserved , we used constitutionally heterozygous grem1 mice ( gazzerro et al . , 2007 ) as a model for the effects of rs16969681 .
we confirmed that these animals had 50% reduced grem1 mrna expression relative to wild - type mice and showed no intestinal abnormalities or polyposis phenotype .
grem1;apc animals had a > 50% reduction in adenomas compared with their well - characterized grem1;apc littermates ( p = 0.0162 ; wilcoxon test ; figure 2c ; su et al . , 1992 ) .
we examined tumors from littermates for differences in proliferation ( ki67 immunohistochemistry ) , apoptosis ( casp3 ) , wnt signaling ( -catenin ) , stem cell numbers ( sox9 ) , and canonical bmp pathway activity ( phospho - smad1,5,8 ) .
we observed increased apoptosis in grem1;apc tumors ( p = 0.044 ; n = 12 ; t test ) and no other differences between the mouse genotypes . in order to confirm that the rs16969681 enhancer is functional in vivo , we generated transgenic mice carrying a 2 kb fragment containing rs16969681 upstream of a lacz reporter gene .
-galactosidase expression was found specifically in the intestines and stomach and was absent from other tissues ( figure 2d ) .
interestingly , reporter expression was seen in both mesenchymal and epithelial cells of the intestine ( figure 2diii ) , though grem1 is not normally expressed in the epithelium .
next , we sought to identify the mechanism by which rs16969681 genotype could modify enhancer activity by identifying transcription factors ( tfs ) that bound to the surrounding region .
we used biotin - labeled electrophoretic mobility shift assay probes , a development of butter et al .
( 2012 ) to pull down dna - protein complexes from sw948 and c99 nuclear extracts , followed by quantitative mass - spectrometry ( n.c.h . ,
we combined these data , in silico predictions from biobase / transfac in a 50 bp region around rs16969681 , and mining of public chip sequencing ( chip - seq ) databases ( mokry et al .
we assessed these proteins using chip in grem1-expressing cells ( c99 rs16969681 t / t , ls180 c / c , sw1222 c / t , and sw948 c / c ) and a nonexpressing line ( sw48 c / c ) .
cdx2 and tcf7l2 ( also known as tcf4 ) bound to the rs16969681 region ( figure 3a ) , as did p300 , runx1 , and , less reliably , hnf4a ( figure s3 ) .
tcf7l2 , hnf4a , and p300 are also listed as binding in published encode data . in order to investigate allele - specific tf binding , we quantified rs16969681 alleles in the chip output from the heterozygous cell line sw1222 .
p300 and runx1 showed no evidence of allele - specific binding ( p > 0.5 ) .
however , cdx2 ( odds ratio [ or ] = 4.7 ; p = 0.001 ; n = 81 ; fisher s exact test ) and tcf7l2 ( or = 2.0 ; p = 0.028 ; n = 63 ) showed a significant bias toward the risk allele ( figure 3b ) .
we also found a correlation between grem1 and cdx2 mrna expression in our panel of crc cell lines ( p = 0.0088 ; n = 34 ; spearman s rank correlation ) .
these data again indicate that the enhancer at rs16969681 is important in cells of epithelial origin .
the core consensus cdx2-binding motif , ataaa ( verzi et al . , 2010 ) , occurs both 14 bp upstream and 65 bp downstream of rs16969681 . to confirm that the cdx2-binding region of the rs16969681 enhancer is important for its activity
, we deleted a 71 bp fragment from our luciferase reporter constructs ( figure 2b ) , removing rs16969681 and disrupting the bases adjacent to the core cdx2-binding motifs .
figure 3c shows the resultant significant reduction in enhancer activity and loss of allele - specific expression in sw948 cells . to demonstrate that cdx2 protein can directly regulate grem1 transcription , we overexpressed cdx2 in sw480 , a crccl with low endogenous cdx2 and grem1 .
we achieved consistent cdx2 overexpression using two separate constructs , causing an 3.5-fold increase in grem1 expression ( p = 0.0005 ; figure 3d ) . by comparison , in cell lines with already high levels of grem1 or cdx2 , no effect or less consistent results
several lines of evidence presented above suggest that the rs16969681 genotype might have its effects in dysplastic colorectal epithelium , that is , at a stage after tumor initiation ( carvajal - carmona et al . , 2013 ) .
we noted that cdx2 expression occurs in almost all colorectal adenomas but is lost in some crcs ( qualtrough et al .
, 2002 ) , and we therefore wondered whether grem1 is expressed in adenomas as well as crcs .
we assayed grem1 mrna expression in 15 isolated crypts from three sporadic adenomas and found a 10-fold increase in epithelial expression compared with surrounding normal epithelial tissue ( p = 0.002 ; anova ; figure 3e ) .
we have found that the grem1 locus harbors at least two snps that independently influence the risk of crc .
studies of grem1 expression in the developing limb bud ( zuniga et al . ,
2012 ) have shown that proximal and distal global control regions ( gcrs ) near grem1 regulate gene expression in a complex fashion .
the snp rs16969681 lies upstream of grem1 in a region distinct from the known gcrs and is close to a regulatory element that we have shown to act as an allele - specific grem1 enhancer .
mouse models confirm that relatively modest changes in grem1 expression influence intestinal tumor burden , possibly through variation in apoptosis .
the rs16969681 region differentially binds the intestine - specific tf cdx2 and the wnt effector tcf7l2 .
both these transcription factors play important roles in intestinal function and often co - occupy binding sites ( verzi et al .
cdx2 is described as a master tf that establishes intestinal epithelial identity ( gao et al . , 2009 ; qualtrough et al . , 2002 ) and maintains open chromatin ( verzi et al . , 2013 ) for tfs such as tcf7l2 .
although further investigation is required , we hypothesize ( figure 4 ) that , with the possible exception of the stem cell compartment , cdx2 allows tcf7l2 to act as repressor in the wnt - low environment of the normal epithelium .
however , with high levels of wnt , such as colorectal tumors , tcf7l2 complexes with -catenin and drives grem1 expression , promoting tumorigenesis .
sample sets are those reported in whiffin et al . ( 2013 ) , plus uk2 and scotland2 reported in tomlinson et al .
briefly , 10 cells were crosslinked for 5 min with 1% formaldehyde , neutralized , washed , and scraped .
purified dna was analyzed by sybr green quantitative pcr ( qpcr ) using primers from table s2 .
native chip was carried out as previously described ( umlauf et al . , 2004 ) .
approximately 10 cells were collected , washed in pbs , and lysed , and chromatin was purified using a sucrose cushion .
chromatin was fragmented with mnase1 , and immunoprecipitation ( h3k4me2 , 07 - 030 millipore ; h4ac , 06 - 866 millipore ) was carried out overnight .
antibody complexes were captured with protein a or g dynabeads ( invitrogen ) and washed .
approximately 10 cells were crosslinked for 10 min with 1% formaldehyde , neutralized , washed , and scraped .
cells were lysed and the chromatin sonicated prior to overnight immunoprecipitation ( anti - cdx2 , bethyl laboratories ; anti - tcf7l2 , santa cruz ) .
antibody complexes were incubated with protein a or g beads , washed , and eluted .
for allelic analysis , pcr products were ta cloned using pgem taeasy ( promega ) followed by colony pcr and sequencing . for ase analysis , genomic dna and rna
paired cdna and genomic dna samples , in triplicate , were processed using the iplex assay and detected by sequenom maldi - tof mass spectrometry .
the products were run using the sequenom genotyping protocol , visualized using the massarray typer 4.0 , and the areas under the curves used to calculate the allelic ratios of each sample .
luciferase reporter assays were carried out as described ( jaeger et al . , 2012 ) .
briefly , putative enhancer fragments in the pgl3 promoter vector ( promega ) were cotransfected into sw948 cells with 1/10 concentration of pgl4 renilla vector .
luciferase and renilla activity was measured using the dual luciferase assay system ( promega ) and the avent luminometer .
all work was performed according to spanish regulations for animal experimentation and approved by the consejo superior de investigaciones cientificas ethics committee .
two - kilobase constructs containing rs16969681 and rs16969862 , the minimal beta - globin promoter , lacz , and a sv40 polyadenylation signal were linearized , diluted to 5 ng / ml in microinjection buffer , and used to inject fertilized mouse eggs from cba / ca c57bl/6 crosses .
f0 embryos of 9.513 days postcoitum ( dpc ) stages and tissues from f0 animals at postnatal day 1 ( p1 ) and p35 were harvested .
embryos and dissected tissues were fixed overnight , washed and placed in 10 ml of x - gal solution for 220 hr at 37c , and then postfixed .
cdx2 chip - seq data from caco2 cells ( gene expression omnibus series gse23436 ; verzi et al . ,
2010 ) and tcf7l2 data from ls147 t cells ( series gse18481 ; mokry et al . , 2010 )
sw480 cells were plated out and transfected with cmvcdx2 ( addgene ) and cmvflag - cdx2 ( barros et al . , 2011 ) .
cells were lysed , rna and cdna generated , and cdx2 and grem1 mrna levels measured by quantitative rt - pcr ( qrt - pcr ) using the taqman system ( applied biosystems ) . individual crypts from normal and adenomatous patient epithelium were isolated as described previously ( jaeger et al . , 2012 ) to ensure exclusion of any pericryptal myofibroblasts .
grem1 mice , a kind gift of e. canalis , were crossed with pgk - cre mice to inactivate one copy of grem1 in the germline .
these mice were crossed with apc mice to generate grem1 ; apc and grem1 ; apc animals .
mice were aged for 5 to 6 months and litters sacrificed when one member showed symptoms of intestinal tumors .
all procedures were carried out in accordance with home office uk regulations and the animals ( scientific procedures ) act 1986 .
mice were housed at the functional genomics facility , wellcome trust centre for human genetics , oxford .
| summarya rare germline duplication upstream of the bone morphogenetic protein antagonist grem1 causes a mendelian - dominant predisposition to colorectal cancer ( crc ) .
the underlying disease mechanism is strong , ectopic grem1 overexpression in the intestinal epithelium . here
, we confirm that a common grem1 polymorphism , rs16969681 , is also associated with crc susceptibility , conferring 20% differential risk in the general population .
we hypothesized the underlying cause to be moderate differences in grem1 expression .
we showed that rs16969681 lies in a region of active chromatin with allele- and tissue - specific enhancer activity .
the crc high - risk allele was associated with stronger gene expression , and higher grem1 mrna levels increased the intestinal tumor burden in apcmin mice .
the intestine - specific transcription factor cdx2 and wnt effector tcf7l2 bound near rs16969681 , with significantly higher affinity for the risk allele , and cdx2 overexpression in cdx2/grem1-negative cells caused re - expression of grem1 .
rs16969681 influences crc risk through effects on wnt - driven grem1 expression in colorectal tumors . |
congenital adrenal hyperplasia ( cah ) is an autosomal recessively inherited disorder of adrenal steroid synthesis , in which glucocorticoid and mineralocorticoid deficiencies lead to a compensatory increase in adrenocorticotropic hormone ( acth ) secretion , which in turn causes adrenocortical hyperplasia and overproduction of intermediate metabolites and adrenal androgens1 ) .
testicular adrenal rest tumors ( tarts ) are important complications of cah , resulting in infertility , and were first described in 19402 ) .
tarts have been observed in 0 - 94% of cah patients , dependent on patient selection and the method of tumor detection3 ) .
tarts are thought to arise from aberrant adrenal cells that migrate to the scrotum during the descent of the testes or from pluripotent stem cells in the testes4 ) .
the growth of these cells in patients with cah may be caused by the chronically elevated levels of acth or other , as yet unknown , growth promoting factors5 ) . in patients with cah ,
although tarts are usually benign , they may obstruct the seminiferous tubule , leading to possible gonadal dysfunction5 ) .
intensive glucocorticoid therapy , which suppresses acth secretion , leads to regression of the tumors .
we describe here a patient with untreated cah who presented with huge tarts , which regressed with dexamethasone therapy .
a 2-month old boy was admitted to our primary clinic because of poor weight gain , hyponatremia , and hyperkalemia .
initially , he was thought to have electrolyte abnormalities caused by dehydration and discharged without an exact diagnosis . when he was 6 years old , he manifested voice change , acne , testicular enlargement , and premature pubarche .
his height and weight were 124 cm ( 2.31 sds ) and 27 kg ( 2.63 sds ) , respectively .
the volume of each testis was 7 ml , the stretched penile length was 7 cm , and his bone age was 13 years by greulich and pyle 's method , which was remarkably advanced for his chronological age .
his serum 17-hydroxypregnenolone ( 17-ohp ) and testosterone levels were elevated to 480 ng / ml ( normal range : 0.32 - 3 ng / ml ) and 2.0 ng / ml ( normal range : 0.03 - 0.1 ng / ml ) , respectively .
he was treated with hydrocortisone ( 15 mg / m / day ) and 9-fludrocortisone ( 0.1 mg / day ) .
17 years , he visited our outpatient clinic because of bilateral testicular enlargement and short stature .
he was scheduled to be treated by surgical resection of testicular tumors because testicular biopsy demonstrated large , polygonal , and eosinophilic cells with round nuclei and prominent nucleoli , which are consistent findings with leydig cell tumors ( fig .
his height and weight were 155.1 cm ( -2.90 sds ) and 68 kg ( 0.49 sds ) , respectively , and body mass index ( bmi ) was 30.2 ( > 97th percentile ) .
his right and left testicles measured 106 cm , 7.54.5 cm , respectively ( fig .
1 ) , with both being of firm consistency . his pubic hair tanner stage was grade v. gynecomastia and skin hyperpigmentation were noted .
his serum 17-ohp concentration was 506.20 ng / ml ( normal range : 0.32 - 3 ng / ml ) , acth level was 187.6 pg / ml ( normal range : 2 - 49 pg / ml ) , plasma renin activity was 20.0 ng / ml / h ( normal range : 0.4 - 8.8 ng / ml / h ) , and dehydroepiandrosterone sulfate ( dhea - s ) level was 270.6 g / dl ( normal range : 30 - 555 g / dl ) .
he had an androstenedione level of 18.0 ng / ml ( normal range : 0.57 - 1.5 ng / ml ) , a luteinizing hormone ( lh ) concentration of 0.23 miu / ml ( normal range : 1.54 - 7.0 miu / ml ) , a follicle - stimulating hormone ( fsh ) level of 1.6 miu / ml ( normal range : 1.54 - 7.0 miu / ml ) and a testosterone level of 6.13 ng / ml ( normal range : 3.5 - 9.7 ng / ml ) . his serum sodium and potassium concentrations were 141 mmol / l and 4.1 mmol / l , respectively , and serum -fetoprotein and -hcg levels were 1.4 ng / ml ( normal range < 20 ng / ml ) and 1.0 miu / ml ( normal range < 5 miu / ml ) , respectively .
abdominal computerized tomography revealed a 1.5 cm - sized adrenal adenoma on the left side and focal adrenal hyperplasia on the right side .
mutation analysis of cyp21a2 revealed a compound heterozygote , c.293 - 13a > g and c.1066c > t ( p.r356w ) . to reduce the size of his tumors and to improve his hormonal profile , he was prescribed acth suppressive therapy with dexamethasone ( 0.25 mg tid ) .
the volume of each testis estimated using a prader orchidometer was 25 ml ( fig .
his serum 17-ohp and acth concentrations had decreased to 1.0 ng / ml and 9.4 pg / ml , respectively , and serum testosterone concentration was reduced to 0.05 ng / ml ( normal range : 3.5 - 9.7 ng / ml ) .
he was switched from dexamethasone to prednisolone ( 5 mg twice a day ) for maintenance , which had no effect on the volume of his testes . following the switch ,
17-ohp concentration was 19.8 ng / ml , acth concentration was 45.3 pg / ml , plasma renin activity was 20.0 ng / ml / h , and testosterone level was 3.0 ng / ml .
tarts can be the first manifestation of cah and have been reported in 18% of undiagnosed cah patients2 ) . tarts usually develop in untreated or inadequately treated patients with cah because of chronically elevated acth levels .
in addition , increased levels of lh during the pubertal period has been found to stimulate tumor growth by binding of lh to receptors located in tumor tissues .
therefore , tarts are usually detected in adolescence and young adulthood , even in adequately treated patients .
tarts are usually nodular , firm , well delineated , and separated by fibrous bands . because of their histopathologic resemblance on microscopy , it is difficult to differentiate tarts from leydig cell tumors6 ) .
both tumors contain polygonal cells with abundant eosinophilic or granular cytoplasm in cord - like arrangements separated by dense fibrotic tissue4 ) .
tarts , however , never contain the cytoplasmic rod - like crystalloids termed reinke crystals , a pathognomonic finding of leydig cell tumors found in 20 - 40% of patients with the latter condition7 ) .
for example , leydig cell tumors can appear at any age , and only 3% are bilateral8 ) .
in contrast , tarts are more common during adolescence and young adulthood , and are usually bilateral9 ) .
thus , bilateral testicular masses in adolescents should be suspected of having tarts with cah although the histologic findings are consistent with leydig cell tumors .
tarts are benign and reversible without surgical intervention if the tumors are found before stage iv by classification proposed by claahsen - van der grinten hl et al.3 ) .
no cases of metastatic tarts have been reported and tumors shrink after high - dose glucocorticoid therapy2 ) . in our patient
, tarts could not be clearly differentiated from leydig cell tumors histopathologically , but the marked reduction in testicular size following high - dose glucocorticoid therapy suggested that the tumors were tarts , not leydig cell tumors .
although tarts have no malignant features , they can cause severe testicular damage by compressing the seminiferous tubules .
chronic obstruction of the tubules has been found to cause peritubular fibrosis and tubular hyalinization , leading to irreversible testicular damage10 ) .
in addition to their mechanical effects , adrenal androgens produced by such tumors may be toxic to leydig cells and germ cells11 ) .
therefore , early recognition and treatment is required for better prognosis . because tarts are located adjacent to the mediastinum testis , only tumors of > 2 cm are detectable by palpation . to find small - sized tumors ,
ultrasonography has been found to be as sensitive as magnetic resonance imaging in detecting small tarts12 ) .
several treatment modalities have been described that reduce tumor size and improve gonadal function . because these tumors result from elevated acth concentration ,
intensive glucocorticoid therapy , which suppresses acth secretion and leads to regression of tumors , has been found effective .
dexamethasone has a longer half - life than has hydrocortisone ( 36 to 54 hours vs. 8 to 12 hours ) and more effectively suppresses the hypothalamic - pituitary axis13 ) .
testis - sparing surgery is preferred for prepubertal tumors , with psychological and cosmetic advantages .
however , in the case of longstanding tumors in patients with poorly controlled cah , irreversible testicular damage may persist after surgery10 ) .
molecular analysis of cyp21a2 in our patient revealed mutations common in cah of the salt - wasting type , but our patient had not shown any symptoms of adrenal crisis during childhood .
this may have been because his tarts had some features of adrenal cells because of the presence of cyp11b1 and cyp11b2 in tumor tissue14 ) .
it is unusual to find a cah patient of the salt - wasting type who in the absence of treatment , did not present with salt - wasting symptoms . because tarts may cause deterioration in semen production as well as testosterone secretion ,
semen analysis may help to evaluate gonadal function in adults . as poor compliance with medication can aggravate tarts , patients should be regularly followed - up and controlled with appropriate medication , to prevent residual tumor regrowth . in conclusion , early detection of tarts is important in preventing irreversible testicular damage .
patients with cah should be regularly examined by digital palpation and radiological methods such as ultrasonography , to detect small - sized tumors . | testicular adrenal rest tumors ( tarts ) are considered to be formed from aberrant adrenal tissue that has become hyperplastic because of elevated adrenocorticotropic hormone ( acth ) in male patients with congenital adrenal hyperplasia ( cah ) . a 6-year - old boy presented with testicular enlargement and pubic hair .
he was diagnosed with cah complicated by precocious puberty .
however , he was not followed - up . at the age of 17
, he visited the outpatient clinic because of testicular enlargement and short stature .
his right and left testicles were 106 cm and 7.54.5 cm , respectively .
his height was 155.1 cm ( standard deviation score [ sds ] , -2.90 ) .
the diagnosis of cah due to 21 hydroxylase deficiency was confirmed by mutation analysis of cyp21a2 .
histological examination of the testes showed large , polygonal , eosinophilic cells with round nuclei and prominent nucleoli , which were suggestive of tarts .
he was treated with dexamethasone for 3 weeks and tumors regressed .
subsequently , dexamethasone was replaced by prednisolone and 9-fludrocortisone ; thereafter , the reduced testis size has been maintained . |
sepsis is systemic inflammatory response syndrome ( sirs ) resulting from a wide spectrum of infectious agents .
this condition can further lead to severe sepsis and septic shock , and now it is the focus and challenge of critical care medicine ( 1,2 ) .
recent data have shown that 18 million of new sepsis cases occur each year worldwide , with a high fatality rate of 30% ( 3 , 4 ) .
they also have the highest fatality rate , especially those with severe sepsis ( 6 , 7 ) .
recent data suggests that sepsis and its induced septic shock and multiple organ dysfunctions ( mods ) are among the leading causes of admission to intensive care units ( icus ) and it leads to an extremely high fatality rate in pediatric intensive care unit ( picu ) ( 8,9 ) .
the key to decrease the fatality rate of pediatric sepsis , especially severe sepsis , is early diagnosis , accurate assessment and timely treatment .
however , the diagnosis of sepsis and evaluation of its severity only based on clinical signs and symptoms such as temperature , heart rate and respiratory rate is quite difficult , due to its untypical and diverse clinical signs as well as the complex and dynamic pathophysiologic process ( 10 ) .
failure to recognize the severity of illness in the early course of disease may lead to inappropriate disposition or treatment .
therefore , it is of utmost importance to develop a clinical tool that is more accurate and objective than clinicians experience to stratify severity of sepsis ( 11 ) . in 2001
, the north american and european sepsis definitions conference proposed the piro ( predisposition , infection , response , organ dysfunction / failure ) concept with the suggestion that sepsis could be stratified on the basis of easily measured biological indicators in a way similar to cancer , with the tnm ( tumor node metastasis ) staging system ( 12 ) . in recent years
, severity stratification scoring systems such as acute physiology and chronic health evaluation ii ( apacheii ) , sequential organ failure assessment ( sofa ) , pediatric risk of mortality ( prism ) score have been applied to various septic populations ( 13,14 ) .
these scoring systems indeed played a part in assessing sepsis in children with relatively comprehensive parameters .
however , they were computationally complex and devised either for adult patients or for general critical disease , and mainly focus on the prediction of survival or death ( 15,16 ) , thus not suit for pediatric sepsis .
moreover , because of important differences between adults and children with respect to comorbidities , organ failure , and baseline mortality rate , efficacy from adults may not be generalized to children(5 ) . to our knowledge ,
scoring model established specifically for pediatric septic patients with high clinical application value has not yet been reported .
the aim of this study was to develop a scoring model based on selected objective variables with high specificity and sensitivity , to aid severity assessment in children with sepsis .
patients presenting with sepsis were retrospectively recruited from the picu in children s hospital of hunan province between mar 2011 and mar 2013 .
this hospital is the only comprehensive children s hospital in this province , with 80 picu beds and over 200 two - way referral hospitals .
more than 80% of the serious children patients in hunan province were admitted in this hospital .
so , the patients in this hospital are highly representative of all serious children patients in hunan province .
electronic management was implemented for all medical records in this hospital with complete and reliable information , which guaranteed the quality of data .
all selected patients met the pediatric - specific diagnostic criteria for sepsis established by the international pediatric sepsis consensus conference ( 17 ) . in this study
patients who had been admitted for less than 24 h , and patients with important information such as age , gender and prognosis missing were excluded . to develop the scoring model , 75% of the patients
were randomly assigned to the training group and the remaining 25% to the validation group , according to their admission number .
we used the training group to establish the model , and the validation group to evaluate the model .
the hospital electronic medical records for all patients were reviewed by specially trained doctors and eligible patients were selected according to the unified diagnostic criteria and exclusion criteria .
the following information was collected : demographics , clinical and physiologic data , diagnosis data and prognosis data . in order to standardize data collection ,
clinical and physiologic data were taken as the worst value recorded during the first 24 h after admission .
the independent variables considered for inclusion into the sepsis scoring model included demographic variables ( age , gender ) and vital signs ( temperature , heart rate , systolic blood pressure etc . ) ; infection related indicators ( leukocyte platelet proealcitonin , c - reactive protein etc . ) ; organ dysfunction related indicators ( bilirubin , creatinine , total bilirubin , d - dimer , brain natriuretic peptide etc . ) .
this study was approved by xiangya medical school research ethics committee , changsha , china .
we did nt get written or verbal informed consent from participate or the next of kin , caretakers , or guardians on behalf of the minors / children since we have not access the patients .
no any potential harm to the participants was apparent . to ensure anonymity , every participant
regression estimation technique was used to impute the missing values ( the proportion of data missing for individual variables was less than 5% ) .
results are presented as numbers with percentages in parentheses for categorical variables and mean ( sd ) or median ( quartiles ) for continuous variables .
univariate analysis was used to select a subset of predictors associated with sepsis severity at p<0.1 level to be considered for inclusion in the multivariable scoring model .
continuous variables were categorized to binary or categorical indicators required for a clinically useful scoring system in sepsis .
multivariate analysis was performed by stepwise discriminant analysis to identify variables independently associated with the severity of sepsis ( =0.05 , =0.1 ) .
we established the accurate discriminate model based on the discriminant coefficients resulting from the stepwise discriminant analysis .
the simplified scoring model was developed based on standardized canonical discriminant coefficients obtained from discriminant analysis , with the general rule of multiplying the coefficients by 10 and round off to the nearest integer . to assess the accuracy of the models , we calculated the sensitivity ( sn ) and specificity ( sp ) , and then constructed receiver operating characteristic ( roc ) curves by plotting the sn against ( 1-sp ) at different cutoff value .
the discriminant performance of the scoring systems was assessed by the area under the roc curve ( aroc ) , with values close to 1.0 indicating high diagnostic accuracy .
patients presenting with sepsis were retrospectively recruited from the picu in children s hospital of hunan province between mar 2011 and mar 2013 .
this hospital is the only comprehensive children s hospital in this province , with 80 picu beds and over 200 two - way referral hospitals .
more than 80% of the serious children patients in hunan province were admitted in this hospital .
so , the patients in this hospital are highly representative of all serious children patients in hunan province .
electronic management was implemented for all medical records in this hospital with complete and reliable information , which guaranteed the quality of data .
all selected patients met the pediatric - specific diagnostic criteria for sepsis established by the international pediatric sepsis consensus conference ( 17 ) . in this study
patients who had been admitted for less than 24 h , and patients with important information such as age , gender and prognosis missing were excluded . to develop the scoring model , 75% of the patients
were randomly assigned to the training group and the remaining 25% to the validation group , according to their admission number .
we used the training group to establish the model , and the validation group to evaluate the model .
the hospital electronic medical records for all patients were reviewed by specially trained doctors and eligible patients were selected according to the unified diagnostic criteria and exclusion criteria .
the following information was collected : demographics , clinical and physiologic data , diagnosis data and prognosis data . in order to standardize data collection ,
clinical and physiologic data were taken as the worst value recorded during the first 24 h after admission .
the independent variables considered for inclusion into the sepsis scoring model included demographic variables ( age , gender ) and vital signs ( temperature , heart rate , systolic blood pressure etc . ) ; infection related indicators ( leukocyte platelet proealcitonin , c - reactive protein etc . ) ; organ dysfunction related indicators ( bilirubin , creatinine , total bilirubin , d - dimer , brain natriuretic peptide etc . ) .
this study was approved by xiangya medical school research ethics committee , changsha , china .
we did nt get written or verbal informed consent from participate or the next of kin , caretakers , or guardians on behalf of the minors / children since we have not access the patients .
no any potential harm to the participants was apparent . to ensure anonymity , every participant
regression estimation technique was used to impute the missing values ( the proportion of data missing for individual variables was less than 5% ) .
results are presented as numbers with percentages in parentheses for categorical variables and mean ( sd ) or median ( quartiles ) for continuous variables .
univariate analysis was used to select a subset of predictors associated with sepsis severity at p<0.1 level to be considered for inclusion in the multivariable scoring model .
continuous variables were categorized to binary or categorical indicators required for a clinically useful scoring system in sepsis .
multivariate analysis was performed by stepwise discriminant analysis to identify variables independently associated with the severity of sepsis ( =0.05 , =0.1 ) .
we established the accurate discriminate model based on the discriminant coefficients resulting from the stepwise discriminant analysis .
the simplified scoring model was developed based on standardized canonical discriminant coefficients obtained from discriminant analysis , with the general rule of multiplying the coefficients by 10 and round off to the nearest integer . to assess the accuracy of the models , we calculated the sensitivity ( sn ) and specificity ( sp ) , and then constructed receiver operating characteristic ( roc ) curves by plotting the sn against ( 1-sp ) at different cutoff value .
the discriminant performance of the scoring systems was assessed by the area under the roc curve ( aroc ) , with values close to 1.0 indicating high diagnostic accuracy .
during the 2-year study period , 687 patients admitted to picu met the criteria of sepsis or severe sepsis , of whom 43 ( 6.3% ) were excluded for less than 24 h of hospital stay .
634 children were included in the final analysis , with a response rate of 92.3% .
424 ( 66.9% ) patients improved or recovered , 49 ( 7.7% ) gave up treatment and were discharged , and 161(25.4% ) patients died .
the average age was 16.9 24.4 months ( range 1 month to 14 yr ) and 65.3% were male .
the two groups had no statistical difference in gender , age , length of hospital stay , mechanical ventilation , blood culture ; cause of sepsis and the proportion of patients with severe sepsis ( table 1 ) .
a total of 585 patients were included in the prognostic analysis due to 49 cases ( 7.7% ) were abandoned or left hospital voluntarily with no available prognostic information .
we compared the initial characteristic between the included 585 patients and the excluded 49 patients .
table 2 reveals that there were no statistic difference in sepsis severity and mean score between the two group ( p>0.05 ) , which indicated that our results resulted from the remained sample may be reliable .
clinical characteristics of patients in the training and validation groups results are presented as numbers with percentages in parenthesis characteristic of the included 585 patients and excluded 49 patients group a : 585 patients included in the prognostic analysis ; group b : 49 patients excluded in the prognostic analysis due to lack of prognostic information table 3 compares the characteristics and clinical variables of mild sepsis versus severe sepsis in the training group .
several clinical variables were strongly associated with severe sepsis in the univariate analysis , including d - dimer , heart rate , respiratory rate , platelets , potassium , capillary refill time , prothrombin time , pao2/fio2 ratio , base excess , blood lactate , total bilirubin , serum total protein , alanine aminotransferase , urea nitrogen , creatinine , uric acid , myoglobin , proealcitonin , brain natriuretic peptide , and troponin .
univariate analysis of suspected factors of severe sepsis in training group ( 476 patients ) binary and categorical data are presented as n and percentages of totals , using the pearson s chi - square test/ normally distributed data are presented as mean ( sd ) , using student s t - test/ other nonsymmetrical distributed continuous data are presented as medians and 25th to 75th percentile ranges , using mann - whitney test / hr : heart rate ; alt : alanine aminotransferase ; pt : prothrombin time ; pfg : fasting plasma glucose ( fpg ) ; bun : urea nitrogen ; cr : creatinine ; pct : proealcitonin ; bnp : brain natriuretic peptide ; crp : c - reactive protein the nineteen statistically significant factors identified by the univariate analysis were taken to fisher stepwise discriminant analysis to construct a discriminant function ( table 4 ) . as a result , seven variables were retained in the final discriminant model , including prothrombin time , d - dimer , pao2/fio2 ratio , total bilirubin , serum total protein , uric acid and myoglobin .
these results can be presented in the form of the following equation to establish the accurate discriminant model for severe sepsis :
z=7.312 + 0.702*x11 ( total bilirubin)+0.634*x15(uric acid)+0.680*x6 ( ddimer)+0.934*x12 ( serumtotal protein)+0.795*x5 ( prothrombin time)+0.616*x16 ( myoglobin)+0.551*x7 ( pao2/fio2 ratio )
the average discriminant function value for severe sepsis group was za=0.45 , and for mild sepsis group was zb=0.92 , distinguishing value zc= ( za + zb)/2= ( 0.45 + 0.92 ) / 2 = 0.24 . thus , zi 0.24 was discriminated as severe sepsis , and zi < 0.24 was discriminated as mild sepsis . for convenience in clinical use
, we generated a simplified score using weightings defined by the standardized discriminant coefficients of the model ( multiplied by a constant and rounded to the nearest integer ) ( table 5 ) .
simplified scoring model developed based on the accurate discriminate model coefficient : standardized canonical discriminant coefficients the score is 0 if the variable is normal ; for abnormal variables , the score equals to the coefficients multiplied by 10 and rounded to the nearest integer .
the performance of the discriminant model in discriminating mild sepsis and severe sepsis was estimated both in the training sample and validation sample using roc curves .
the arocs ( fig.1a ) of accurate discriminate model were 0.816 ( 95% ci 0.771 to 0.861 ) in the training group and 0.836 ( 95%ci 0.765 to 0.907 ) in the validation group .
for simplified scoring model , the aroc remained relatively high with aroc 0.800 ( 95%ci : 0.7530.846 ) in the training set and 0.825 ( 95%ci : 0.7500.899 ) in the validation set ( fig.1b ) .
receiver operator characteristic ( roc ) curves for the accurate discriminate model and simplified scoring model in the training ( a ) and validation ( b ) groups .
these scores ranged from 0 to 23 , with a mean ( sd ) value of 11.6 ( 6.2 ) .
table 6 shows the prevalence of severe sepsis and fatality rate with each level of scores . as the score rises , the proportion of severe sepsis and the fatality rate increases significantly , and the percentage of improved or recovered decreases significantly ( trend chi - square tests p<0.05 ) .
prevalence of severe sepsis and prognosis in total patients according to summed score a total of 585 patients were included in the prognostic analysis due to 49 cases ( 7.7% ) were abandoned or left hospital voluntarily with no available prognostic information .
during the 2-year study period , 687 patients admitted to picu met the criteria of sepsis or severe sepsis , of whom 43 ( 6.3% ) were excluded for less than 24 h of hospital stay .
634 children were included in the final analysis , with a response rate of 92.3% .
424 ( 66.9% ) patients improved or recovered , 49 ( 7.7% ) gave up treatment and were discharged , and 161(25.4% ) patients died .
the average age was 16.9 24.4 months ( range 1 month to 14 yr ) and 65.3% were male .
the two groups had no statistical difference in gender , age , length of hospital stay , mechanical ventilation , blood culture ; cause of sepsis and the proportion of patients with severe sepsis ( table 1 ) .
a total of 585 patients were included in the prognostic analysis due to 49 cases ( 7.7% ) were abandoned or left hospital voluntarily with no available prognostic information .
we compared the initial characteristic between the included 585 patients and the excluded 49 patients .
table 2 reveals that there were no statistic difference in sepsis severity and mean score between the two group ( p>0.05 ) , which indicated that our results resulted from the remained sample may be reliable .
clinical characteristics of patients in the training and validation groups results are presented as numbers with percentages in parenthesis characteristic of the included 585 patients and excluded 49 patients group a : 585 patients included in the prognostic analysis ; group b : 49 patients excluded in the prognostic analysis due to lack of prognostic information
table 3 compares the characteristics and clinical variables of mild sepsis versus severe sepsis in the training group .
several clinical variables were strongly associated with severe sepsis in the univariate analysis , including d - dimer , heart rate , respiratory rate , platelets , potassium , capillary refill time , prothrombin time , pao2/fio2 ratio , base excess , blood lactate , total bilirubin , serum total protein , alanine aminotransferase , urea nitrogen , creatinine , uric acid , myoglobin , proealcitonin , brain natriuretic peptide , and troponin .
univariate analysis of suspected factors of severe sepsis in training group ( 476 patients ) binary and categorical data are presented as n and percentages of totals , using the pearson s chi - square test/ normally distributed data are presented as mean ( sd ) , using student s t - test/ other nonsymmetrical distributed continuous data are presented as medians and 25th to 75th percentile ranges , using mann - whitney test / hr : heart rate ; alt : alanine aminotransferase ; pt : prothrombin time ; pfg : fasting plasma glucose ( fpg ) ; bun : urea nitrogen ; cr : creatinine ; pct : proealcitonin ; bnp : brain natriuretic peptide ; crp : c - reactive protein
the nineteen statistically significant factors identified by the univariate analysis were taken to fisher stepwise discriminant analysis to construct a discriminant function ( table 4 ) . as a result
, seven variables were retained in the final discriminant model , including prothrombin time , d - dimer , pao2/fio2 ratio , total bilirubin , serum total protein , uric acid and myoglobin .
these results can be presented in the form of the following equation to establish the accurate discriminant model for severe sepsis :
z=7.312 + 0.702*x11 ( total bilirubin)+0.634*x15(uric acid)+0.680*x6 ( ddimer)+0.934*x12 ( serumtotal protein)+0.795*x5 ( prothrombin time)+0.616*x16 ( myoglobin)+0.551*x7 ( pao2/fio2 ratio )
the average discriminant function value for severe sepsis group was za=0.45 , and for mild sepsis group was zb=0.92 , distinguishing value zc= ( za + zb)/2= ( 0.45 + 0.92 ) / 2 = 0.24 .
thus , zi 0.24 was discriminated as severe sepsis , and zi < 0.24 was discriminated as mild sepsis . for convenience in clinical use , we generated a simplified score using weightings defined by the standardized discriminant coefficients of the model ( multiplied by a constant and rounded to the nearest integer ) ( table 5 ) .
simplified scoring model developed based on the accurate discriminate model coefficient : standardized canonical discriminant coefficients the score is 0 if the variable is normal ; for abnormal variables , the score equals to the coefficients multiplied by 10 and rounded to the nearest integer .
the performance of the discriminant model in discriminating mild sepsis and severe sepsis was estimated both in the training sample and validation sample using roc curves .
the arocs ( fig.1a ) of accurate discriminate model were 0.816 ( 95% ci 0.771 to 0.861 ) in the training group and 0.836 ( 95%ci 0.765 to 0.907 ) in the validation group .
for simplified scoring model , the aroc remained relatively high with aroc 0.800 ( 95%ci : 0.7530.846 ) in the training set and 0.825 ( 95%ci : 0.7500.899 ) in the validation set ( fig.1b ) .
receiver operator characteristic ( roc ) curves for the accurate discriminate model and simplified scoring model in the training ( a ) and validation ( b ) groups .
we calculate the severity scores for each of the study subjects . these scores ranged from 0 to 23 , with a mean ( sd ) value of 11.6 ( 6.2 ) .
table 6 shows the prevalence of severe sepsis and fatality rate with each level of scores . as the score rises , the proportion of severe sepsis and the fatality rate increases significantly , and the percentage of improved or recovered decreases significantly ( trend chi - square tests p<0.05 ) .
prevalence of severe sepsis and prognosis in total patients according to summed score a total of 585 patients were included in the prognostic analysis due to 49 cases ( 7.7% ) were abandoned or left hospital voluntarily with no available prognostic information .
sepsis is a severe condition triggered by systemic inflammation in response to infection ( 18 ) , which remains one of the common critical illnesses encountered in the picu .
it is very complicated for clinicians to diagnose and assess pediatric sepsis due to its untypical symptoms , rapidly development and dynamic pathophysiological process ( 19,20 ) .
thus , practical clinical scoring models with few indicators are urgently required in pediatric critical medical care to assess patients condition ( 21 ) .
this study specially designed to stratify severity of sepsis for pediatric patients , using a score based on seven variables drawn from patient clinical findings and laboratory examinations .
advances in statistical methods have supplied the tools necessary to model complex relationships among many variables relevant to outcomes . in this study
other alternatives would have been the use of synthetic evaluation model , for example hierarchy method and delphi method ( 22 ) .
however , those methods always select indicators subjectively or need other auxiliary methods to select indicators objectively . besides
, specialists were required to participate in and reach a high agreement coefficient to obtain reliable results .
therefore , the availability and comprehensiveness of discriminant analysis and the simplicity of presenting the results as simple scores have conducted us to this election .
discriminant analysis is a classification statistical method using the known categories of sample to establish the discriminant model .
the stepwise discriminant analysis is the ideal statistical method for our analysis by using fewer indicators to achieve stable discriminant effect ( 23 ) .
receiver - operating curve ( roc ) analysis has been used to determine the performance of a model in many studies ( 24 ) .
based on 634 patients , we selected 27 variables , which are readily available in most of the institutions and closely related with various systems of the body .
nineteen of them entered into discriminant analysis by univariate analysis , and 7 were retained in the final model .
most of the severity indicators found in our study are in concordance with previous study ( 2527 ) .
other indicators , such as c - reactive protein , tumor necrosis factor and procalcitonin , have been reported as potential biochemical markers of infection ( 2830 ) , however have not been shown to be associated with severity of pediatric sepsis in our study .
one explanation may be that only children diagnosed with sepsis were included in this study , so the biochemical markers of infection can not be used to distinguish the severity of infected children .
these variables and their coefficients are different from the widely used but complicated scoring systems such as apacheii , sofa , and prism score ( 15,16 ) .
this difference may be related to our specific research objects , which fully shows the rationality and uniqueness of our model in assessing severity of sepsis in children .
previous studies have attempted to derive clinical scores as a tool to identify seriously ill sepsis patients ; however , most of them looked at factors which may be predictive of fatality ( 4,31 ) .
shapiro et al . ( 32 ) firstly derived and validated the mortality in emergency department sepsis ( meds ) score to address the need for an early risk stratification tool for sepsis in adult patients .
the score was based on nine variables drawn from patient profile , clinical findings , and initial laboratory examination .
the performance of the meds score has been validated in various populations to predict the 28-d fatality rate , with aroc 0.76 to 0.82 ( 33,34 ) .
then corinne alberti et al . developed a risk of infection to severe sepsis and shock score ( rissc ) to estimate the risk of worsening sepsis in critically ill patients with infection ( 35 ) .
the score included 12 variables such as temperature , heart rate , platelets , which are subsequently simplified into four subclasses of risk .
unfortunately , scores in the above studies were devised based on adult groups whose average age were over 60 yr old , which were not suitable for assessment of disease severity in pediatric population .
yet there are some scoring systems for mortality prediction in pediatric sepsis have been developed in recent past .
( 36 ) constructed and validated a prediction - scoring model for late - onset neonatal sepsis from clinical , laboratory , and management variables in a retrospective cohort .
the validity of this score was good with arocs from 0.80 to 0.85 , but its practical application only suits for neonates .
wong et al . ( 37 ) derived a pediatric sepsis biomarker risk model using 12 gene probes associated with outcome in children with septic shock with an aroc 0.811 .
however , it only suits for sepsis shock children and has technical limitation for widely clinical application . in the present study
, we evaluated the accurate discriminate model with training sample and validation sample , which showed a good discriminatory performance in assessing the severity of sepsis ( aroc 0.815 to 0.836 ) .
it was similar to the clinical value of prediction - scoring model for late - onset neonatal sepsis and pediatric sepsis biomarker risk model ( 36 ) , better than the prism reported by de araujo costa et al ( 38 ) and apache score in assessing critical illness of children reported by ana lilia et al .
it can be applied easily by clinicians with simple calculation procedures installed on their personal computers . for those inconvenient to use a computer ,
, we provide a more convenient simplified scoring model with similar clinical discriminant effect ( auroc:0.8000.825).the simplified scoring model was superior to the mews , scs and rems in predicting septic fatality reported by ghanem - zoub ( 39 ) , and also better than the sofa and cis score reported by shigeto oda ( 13 ) .
in addition , we provided the risk estimation of severe sepsis and fatality according to the summed score , which is not only of diagnosis value but also of clinical predictive value .
although we used scientific statistical methods to develop and verify the model , some limitations should be noted .
firstly , the performance of the model can be optimized with more predictable factors included . in our analysis ,
not all variables that might be considered for inclusion into a severity score of sepsis were obtained .
for example , we had no information on nervous system ( data regarding gsw were only available in 67 patients . ) , cytokines levels or patients genetic predisposition in our population .
however , examination of such indicators is costly and currently limited to few centers throughout the world .
different etiologies may confer different severity and prognoses but the underlying etiology is often unknown until relatively late in the course of hospitalization .
factors that account for the accuracy of the model also contribute to its complexity , and sophisticated parameters are not uniformly obtained in resource - limited settings .
accordingly , we intended to create a clinical tool based on commonly available clinical variables .
all of these additional diagnostic tests merit further evaluation and incorporating such variables into a sepsis risk score might be feasible in a not - too - distant future .
secondly , our study was conducted at single center in chinese population and may not be representative of all the picus worldwide .
thirdly , 49 patients exclusion from the prognostic analysis might have introduced a bias in this study .
an accurate discriminate model and simplified scoring model proposed in this study are new , applicable tools for severity assessment in pediatric sepsis patients . using generally available indicators
, clinicians can easily stratify the disease severity and predict the risk of severe sepsis in septic children , which are very important in guiding treatment and improving outcomes .
we propose that this severity - scoring model should be further evaluated for severity stratification and mortality prediction in larger prospective study as well as in other ethnic groups .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication , and/ or falsification , double publication and/ or submission , redundancy , etc . ) have been completely observed by the authors . | background : multiple severity scoring systems have been devised and evaluated in adult sepsis , but a simplified scoring model for pediatric sepsis has not yet been developed .
this study aimed to develop and validate a new scoring model to stratify the severity of pediatric sepsis , thus assisting the treatment of sepsis in children.methods:data from 634 consecutive patients who presented with sepsis at children s hospital of hunan province in china in 20112013 were analyzed , with 476 patients placed in training group and 158 patients in validation group .
stepwise discriminant analysis was used to develop the accurate discriminate model .
a simplified scoring model was generated using weightings defined by the discriminate coefficients .
the discriminant ability of the model was tested by receiver operating characteristic curves ( roc).results : the discriminant analysis showed that prothrombin time , d - dimer , total bilirubin , serum total protein , uric acid , pao2/fio2 ratio , myoglobin were associated with severity of sepsis .
these seven variables were assigned with values of 4 , 3 , 3 , 4 , 3 , 3 , 3 respectively based on the standardized discriminant coefficients .
patients with higher scores had higher risk of severe sepsis .
the areas under roc ( aroc ) were 0.836 for accurate discriminate model , and 0.825 for simplified scoring model in validation group.conclusions:the proposed disease severity scoring model for pediatric sepsis showed adequate discriminatory capacity and sufficient accuracy , which has important clinical significance in evaluating the severity of pediatric sepsis and predicting its progress . |
control of the fouling
properties of biomaterials , e.g. , protein
adsorption and cell adhesion , is essential for ultimate functioning
of these materials as implants and prostheses .
functional
antifouling properties are most frequently added through physisorption
or chemical tethering of nonfouling moieties .
covalently attached
layers are more durable than physisorbed adlayers , but making stable
coatings on more complex surfaces as efficient in reducing biofouling
as self - assembling monolayers ( sams ) on gold remains a challenge .
initial investigation on properties of
antifouling sams revealed
that moieties that resist the adsorption of proteins and cells typically
are hydrophilic , include hydrogen - bond acceptors , but do not include
hydrogen - bond donors , and have an overall neutral charge .
since then , hydrophilic poly(ethylene glycol ) ( peg)-based materials
have been widely used to develop antifouling surfaces using varying
surface preparation techniques .
similar
to hydrophilic polysaccharide containing materials and coatings based
on zwitterionic molecules , the antifouling properties of peg - based
materials have been attributed to the presence of an hydration layer
near the surface , which acts as a barrier . to design antifouling materials , ethylene glycol - based materials
recently ,
the functionalization of substrates with polymer brushes has proven
to increase surface coverage and thereby increase effectivity of the
applied coating .
brush - like coatings based on peg ,
often in the form of a poly[oligo(ethylene glycol ) methyl methacrylate ]
( polyoegma ) , have for instance been successfully applied via surface - initiated
atom transfer radical polymerization ( si - atrp ) on 2d substrates and on electrospun fibers .
cell adhesion
is presumed to be preceded by nonspecific protein
adsorption , which agrees to the fact that peg - functionalized materials
often show a decrease in protein adsorption when a reduction in cell
adhesion is also observed .
the first proteins to adsorb upon contact with the biomaterial are
high mobility proteins , such as albumin .
subsequently , displacement
by less abundant proteins with higher affinities occurs , starting
with globulin and fibrinogen , and ending with high molecular weight
kininogen .
this process is called the vroman effect . to quantitatively measure the adsorption of proteins
on biomaterial
surfaces in vitro ,
several techniques have been used ,
such as fluorescence microscopy , ellipsometry , surface plasmon resonance
( spr ) , and quartz crystal microbalance with dissipation ( qcm - d ) .
qcm - d offers label - free frequency and real - time monitoring of the
hydrated mass adsorption using the change in resonance frequency and
dissipation energy of a piezoelectric quartz crystal with a resolution
of 1 ng / cm . recently , protein
adsorption has been investigated on spin - coated layers of different
thermoplastic polyurethanes and on varying
antifouling coatings containing chondroitin sulfate , peg , carboxymethylated
dextran , or zwitterions .
using noncovalent supramolecular interactions , based on the self - complementary
2-ureido-4[1h]-pyrimidinone ( upy ) moiety , we were able to develop modular biomaterials
with upy - modified polycaprolactone ( pcldiupy ) ( scheme 1 ) as the structural base material .
more recently , this system has been further expanded by the incorporation
of peg2kdiupy ( additive 1 , scheme 1 ) to prevent cell adhesion .
previously , we have shown that the addition
of peg2kdiupy reduces the hydrophobicity of the surface ,
leads to decreased cell adhesion on electrospun meshes in
vitro , and reduces cell infiltration in vivo in a chain - extended upy - modified pcl material . here
, our supramolecular toolbox is extended with bifunctional
peg10kdiupy ( additive 2 , scheme 1 ) and monofunctional meopeg5kupy ( additive 3 , scheme 1 ) , previously used as hydrogelators .
these additives are of interest because they
contain larger peg chains and hence could lead to more hydrophilic
surfaces and thus less cell adhesion .
furthermore , both compounds
contain an extra aliphatic dodecyl spacer between the upy and the
peg , which can shield the urea groups from interactions with water
of the peg and can lead to enhanced anchoring in the pcldiupy base
material .
the meopeg5kupy mimics half of the peg10kdiupy and therefore allows investigation of the effect of the number
of anchoring points . with our previous results , and the presumption
that cell adhesion is preceded by nonspecific protein adsorption ,
investigation of protein adsorption on our materials provides more
insight into the non - cell - adhesive properties of the peg - containing
materials .
the surface morphology of the spin - coated supramolecular
polymer
films was studied using atomic force microscopy ( afm ) and water contact
angle ( wca ) measurements .
the adhesion of epithelial and endothelial
cells on the polymer film was investigated , and the adsorption of
the first three proteins of the vroman series ( albumin , globulin , and fibrinogen ) was quantified using qcm - d .
pcldiupy
and peg10kdiupy were
synthesized by symo - chem bv ( eindhoven , the netherlands ) .
pbs tablets , 1,1,1,3,3,3-hexafluoroisopropanol ( hfip ) , edta ,
non - idet np-40 , gelatin from porcine skin , bovine serum albumin ( bsa ) ,
fibrinogen from bovine plasma , -globulins from bovine blood ,
mowiol 4 - 88 , anti - human vinculin mouse igg1 antibody ( v9131 ) , atto
488 conjugated phalloidin , and 4,6-diamidino-2-phenylindole
( dapi ) were purchased from sigma - aldrich ( zwijndrecht , the netherlands ) .
egm-2 bulletkit , penicillin / streptomycin , and trypsin / edta were purchased
from lonza ( breda , netherlands ) .
tris , nacl , and triton x-100
were purchased from merck millipore , fetal bovine serum ( fbs ) from
greiner bio , and dulbecco s modified eagle medium ( # 41966 )
from gibco life sciences .
bsa , fibrinogen , and -globulins were
obtained as powders and used without further purification .
solutions of all polymers
were prepared at a concentration of 20 mg / ml in hfip . for the mixtures
,
5 vol % of the additive polymer solution was added to 95 vol % solution
of pcldiupy .
this results in 5:95 wt % additive : pcldiupy mixtures ,
corresponding to 5 , 1.24 , and 2.45 mol % mixtures for additive 1 , 2 , and 3 , respectively ( table s7 ; calculation is provided in the supporting information ) .
samples were prepared
by spin - coating 100 l of a 20 mg / ml solution of the polymers
in hfip at 5000 rpm for 30 s on either glass coverslips with a diameter
of 15 mm ( for afm ) or 13 mm ( for cell culture ) or on the gold - coated
sensor ( for qcm - d ) .
the samples were left to dry for 1 h at room temperature
before continuing the experiments .
afm images were recorded at
room temperature using a digital instrument multimode nanoscope iv
operating in the tapping regime mode using silicon cantilever tips
( ppp - nch-50 , 204497 khz , 10130 n / m ) .
the root - mean - square
( rms ) for an area of 100 m was used to compare
the different materials .
water contact angles
were measured at room temperature on an oca30 ( dataphysics ) .
water
droplets ( 5 l ) were applied on the polymer films on glass ,
and the angle at the polymer air water interface was
determined after 5 s using an automatic fitting routine ( sca20 software ) .
the mean and the standard deviation of three samples are reported .
human umbilical vein endothelial cells
( huvec , lonza ) were cultured in egm - bulletkit medium ( endothelial
basal medium supplemented with 2% fbs and growth factors , including
vegf ) supplemented with 1% penicillin / streptomycin and expanded in
t-75 culture flasks that were coated with a 0.1% gelatin solution
for 15 min at 37 c .
proximal epithelial cells from a human kidney
( hk-2 , atcc ) were cultured in dmem , supplemented with 10% fbs and
1% penicillin / streptomycin , and expanded in t-75 culture flasks .
cells
were subcultured at a 8090% confluency by washing with pbs
and incubating in 2 ml of trypsin / edta for 12 min ( huvec )
and 35 min ( hk-2 ) at 37 c and diluted in 8 ml of culture
medium before centrifugation for 5 min at 180 g .
cells
were resuspended and counted using a nucleocounter ( nc-100 , chemometec
copenhagen , denmark ) and diluted to 8104 cells / ml culture medium
and reseeded at 106 cells per t-75 flask .
huvecs were used for experiments up to p5 and
hk-2 cells up to p16 .
glass coverslips with
a diameter of 13
mm ( # 0 , vwr ) , either covered with spin - coated polymer , coated with
gelatin , or bare , were placed in minusheet tissue carriers with 13
mm o.d .
( minucells and minutissue - vertriebs gmbh ) to prevent films
from detaching during culture and were then sterilized under uv for
1 h. next , the secured coverslips were placed in 24 wells culture
plates , and 250 l culture medium was pipetted under the coverslip .
cells were harvested as in subculture and resuspended in culture medium
and diluted to 2.6 10 cells / ml .
62 l of
cell suspension was pipetted onto the exposed surface of the coverslips ,
and cells were left to adhere for 1.5 h at 37 c and 5% co2 , before adding 500 l of culture medium on top of the
cells .
cells grown on coverslips
were fixated and permeabilized simultaneously by incubation in 3.7%
formaldehyde and 0.5% triton x-100 in pbs for 15 min at room temperature .
samples were then washed with pbs twice and subsequently blocked in
2% bsa at least overnight at 4 c .
next , slips were washed in
net - gel ( 50 mm tris , 150 mm nacl , 5 mm edta , 0.05% np-40 and 0.25%
gelatin ) , prior to overnight incubation with primary mouse igg1 anti - human
vinculin antibody 1:250 in 80% net - gel and 1% bsa in pbs at 4 c .
samples were then washed in net - gel three times and subsequently incubated
for 1.5 h at room temperature with 1:250 goat anti - mouse igg1 alexa
555 conjugated secondary antibody and 1:500 atto 488 conjugated phalloidin
in net - gel , followed by a double 5 min wash in net - gel and a nuclear
counterstain with 0.1 g / ml dapi in pbs .
after three washes
in pbs , coverslips were mounted on microscopy slides with mowiol and
stored in the dark at 4 c until visualization with a zeiss axiovert
200 m microscope .
qcm - d was performed on the q - sense e4 instrument
( biolinscientific
ab ) using gold - coated at - cut quarts discs with a fundamental frequency
of 4.95 mhz ( qsx 301 gold , biolinscientific ab ) .
sensors were rinsed
with piranha solution and subsequently heated for 10 min at 70 c
in a 5:1:1 mixture of ultrapure water , ammonia , and 30% hydrogen peroxide
( base piranha ) .
sensors were rinsed with water , acetone , and isopropanol
and dried with nitrogen .
clean crystals were mounted to record their
fundamental frequency in air and subsequently removed for spin - coating .
after mounting the sensors
with the spin - coated material , sensors were measured in air .
afterward ,
pbs was passed over the surface at 0.1 ml / min until the signal equilibrated .
subsequently , the protein solution was passed over the surface at
0.1 ml / min .
frequency and dissipation changes were recorded until
both signal equilibration , and the sensors were rinsed with pbs .
each
experiment was repeated 3-fold , and means and standard errors of the
mean are reported .
after each experiment , the system was cleaned
by rinsing with 50 ml of 2 wt % solution of hellmanex iii ( hellma )
in ultrapure water , followed by rinsing with 100 ml of ultrapure water .
shifts in frequency
and dissipation for overtones 3 , 5 , 7 , 9 , and 11 were analyzed using
the voigt
voinova model using the qtools software ( q - sense ) .
a protein density of 1250 kg / m was used for all protein
layers .
as became evident from the measurements , the viscosity of
the protein solutions was higher than the viscosity of pbs and hence
was accounted for in the model , together with one homogeneous viscoelastic
layer . for pbs at 37 c ,
the measured viscosity in the qcm - d
was 0.0071 pa s , and a density of 1000 kg / m was used .
the use of qcm - d to
quantify protein adsorption requires a homogeneous and thin polymer
film .
initial experiments showed that drop - casting leads to thick
films in the micrometer range at higher concentrations or to inhomogeneous
films caused by dewetting at lower concentrations .
therefore , spin - coating
was selected as the technique to reproducibly prepare thin polymer
films .
the surface roughness of the polymer films prepared by spin - coating
was assessed using afm ( table s1 ) .
the
addition of upy - peg did slightly increase the rms roughness for spin - coated
samples , but no large differences are observed .
afm phase images show
a fibrous morphology on the nanometer length scale for pristine pcldiupy
films , similar to nanofibers obtained previously in drop - cast samples ,
attributed to the highly directional self - assembly of the combined
upy and urea moieties .
when 5 wt % of
additive 1 was added , a similar nanofibrillar morphology
is observed ( figure 1 ) .
for the samples with additive 3 , domains of peg can
be observed on the larger scale , which were also visible in the height
images ( figure s1 ) .
details of the microphase - separated
regions show pcldiupy - like fibers in the darker regions while the
lighter regions are hypothesized to represent the peg phase .
a homogeneous
polymer film was obtained for mixtures with additive 2 , with well - defined fibrillar morphology .
we hypothesize that the
speed of spin - coating inhibits both nanophase and microphase separation ,
leading to suboptimal fiber formation , but intimate mixing of the
components .
afm phase micrographs of spin - coated films of pcldiupy with 5 wt
% of the upypeg additives on glass .
the water contact angle is 74.7 0.2.
a sharp decrease in contact angles is observed with the addition of
upypeg , with contact angles of 59.8 1.3 for pcldiupy
with additive 1 , 56.7 0.9 with additive 2 , and 44 14 with additive 3 .
the
presence of the hydrophilic components in the films prepared from
the polymer mixtures is clearly reflected in the water contact angles .
the adhesive behavior
of cells on our materials with the different peg - containing additives
was evaluated based on the staining of f - actin , an important part
of the cytoskeleton , and vinculin , a focal adhesion protein involved
in anchoring these microfilaments to the extracellular matrix .
on pcldiupy and the mixture with additive 1 , hk-2 cells show a well - spread morphology and clear actin
fibers , terminating in defined vinculin spots , indicating good adhesion
( figure 2 , and single
channel greyscale images in figure s2 ) .
less huvecs adhered to pcldiupy and the mixture with additive 1 than hk-2 cells and show a decrease in cell spreading ( table s6 ) . moreover fewer vinculin spots are
observed , indicating less firm adhesion to the material .
cells and huvecs show a clear decrease in adhesion and spreading on
the mixtures with additives 2 and 3 ( table s6 ) .
cells show a rounded morphology and
have a lack of actin fibers and vinculin spots .
previous studies show
a significant decrease in cell adhesion materials functionalized with
additive 1 . here , the reduction
in cell adhesion is not so obvious , which could be explained by the
lower additive 1 to base material ratio of 5:95 compared
to 10:90 , as previously shown by van almen et al . for huvecs and 3t3 fibroblast cells cultured on drop - cast
films up to 7 days .
furthermore , mollet et al . showed a reduction of cell adhesion for additive 1 to base material ratio of 30:70 for hk-2 cells on electrospun
meshes , cultured for 14 h.
interestingly , additives 2 and 3 do reduce the cell adhesion clearly even though
the absolute amount of peg mixed in does not vary much : 1:1.2:1.2
for additive 1:2:3 ( table s7 ; calculation is provided in supporting information ) , suggesting that the
length of the peg chain and improved anchoring in the pcldiupy base
polymer due to the additional alkyl spacer are of importance for non - cell - adhesive
properties , whereas the different number of anchoring points between
additives 2 and 3 does not appear to have
an effect .
fluorescence microscopy images of hk-2 cells and huvecs cultured
on spin - coated polymer films for 1 day , stained for f - actin ( green ) ,
vinculin ( red ) , and nuclei ( blue ) .
the
thickness of the polymer films on the qcm - d sensors was estimated
by comparing the frequency shift of the empty sensors with that of
the spin - coated sensors .
the frequency shift has been converted to
layer thickness using the sauerbrey equation and a polymer density
of 1145 kg / m .
for all mixtures , films of approximately
100 nm thick were obtained ( figure s3 )
showing that spin - coating indeed leads to reproducible , thin films .
qcm - d measurements were performed to quantify the amount of protein
adsorbed under physiologically relevant conditions .
representative
data for one experiment , in which the adsorption of albumin is measured ,
is shown in figure 3a .
after equilibration with pbs at 37 c , adsorption of mass
can be observed upon introduction of a solution of 30 mg / ml albumin
in pbs .
exposure of the pcldiupy - coated sensors to the albumin solution
resulted in a frequency shift ( f ) for the
fifth overtone of 42.6 3.6 hz .
this frequency shift was accompanied
by a dissipation shift ( d ) of ( 10.8
0.3 ) 10
. the large d/f ratio of 0.25 indicates the nonrigid coupling
of mass to the sensor , which can be largely attributed to the viscosity
of the albumin solution .
this
indicates that the remaining adsorbed mass is more rigidly coupled
to the sensor after rinsing .
( a )
example of frequency
and dissipation changes as a function of time during the adsorption
of albumin . for clarity ,
every tenth data point for the fifth , seventh ,
and ninth overtone is shown .
( b ) overview of the adsorption of albumin
( 30 mg / ml ) , -globulin ( 10 mg / ml ) , fibrinogen ( 3 mg / ml ) , and
a mixture of these three proteins under physiological conditions ( 37
c ) on spin - coated polymer films .
adsorption is represented as
mean sd ( n 3 ) . * p 0.01 . because of the large
dissipation values , the sauerbrey equation
for rigid coupled mass is not valid , and the voight voinova
model was used . in this study ,
one viscoelastic
layer was used to model the protein adsorption and thus obtain parameters
for the shear modulus , the shear viscosity , and the thickness ( or
mass ) of the adsorbed protein layer .
the protein density was set at 1250 kg / m , which accounts for approximately 50% of surface coverage .
varying the protein density between 1000 kg / m ( density of water ) and 1400 kg / m ( density of
proteins ) is known to have limited effect of the modeled mass and
hence on the results obtained .
using
this model , good fits to the data can be obtained ( figure 3a ) . as expected , the
viscosity of the albumin solution is similar for
the different films ( table s2 ) .
the comparable
values obtained for the voigt mass while the sensor is immersed in
the albumin solution and the sauerbrey mass after rinsing with pbs
further supports the hypothesis that the spread in overtones and large
dissipation values are largely due to the viscosity of the albumin
solution for the adsorption of albumin on pristine pcldiupy and indicate
that hardly any albumin is removed from the surface after rinsing .
the voigt mass has been converted to the thickness of the adsorbed
protein layer using the protein density . for mixtures with additives 2 and 3 a trend can be observed toward less adsorption
of albumin on the films , but the differences are not significant ( figure 3b ) .
the protein layers
are approximately 2 nm thick for all samples , which indicates the
formation of a monolayer , consistent with results found previously
for albumin .
therefore , the samples containing
additive 2 or 3 are probably not fully covered
by albumin .
next , the adsorption of -globulin from a
10 mg / ml solution
was studied as general model for globulins .
the modeled bulk viscosity
indicates that the solution is less viscous ( table s3 ) .
similarly to the albumin adsorption , the voigt mass is
comparable to the sauerbrey mass after rinsing with pbs .
in contrast to the adsorption of albumin , a significant decrease
in adsorption can be observed when the surfaces are pegylated with
additive 2 or 3 ( figure 3b ) .
the mixture with additive 2 reduces the adsorption by 40% , and the mixture with additive 3 shows a reduction of 60% in adsorption compared to pristine
pcldiupy .
values between 100 and 800 ng / cm have been reported
before for the adsorption of -globulin . for the third protein in the vroman series , fibrinogen ,
the viscosity of the solution is similar to the
viscosity of the -globulin solution ( table s4 ) . in this case
, adsorption decreases with increasing molecular
weight of the peg , but the differences are not significant ( figure 3b ) .
this is partially
as a result of the large variation in adsorbed values measured using
qcm - d ( table s4 ) .
the layer thickness of
around 15 nm is lower than the long axis of fibrinogen of 47 nm , but
twice the short axis of 57 nm .
a likely explanation is that
the proteins adsorb not end - on but orientate sidewards as suggested
before for softer layers . although
above results give insights into the adsorption of the
individual components in blood plasma on biomaterials , in
vivo the proteins are always mixtures .
therefore , all tested
proteins were added in a single solution at the same concentrations
as used above ( albumin : 30 mg / ml ; -globulin : 10 mg / ml ; fibrinogen :
3 mg / ml ) and introduced in the qcm - d . even though the increased viscosity
of the solution leads to large dissipation values , viscoelastic modeling
allows for the separation of the contribution from the solvent and
the adsorbed protein layer ( table s5 ) .
here
, a large discrepancy between the voigt and the sauerbrey mass
after rinsing with pbs is observed , which might be a result of either
soft , flexible protein layers or an effect of the shear stresses due
to the more viscous solution .
the decreased modeled viscosity for
polymer films containing additive 3 shows that the model
obtained for this sample needs to be interpreted with caution , but
the trends match the values obtained for the sauerbrey mass after
rinsing with pbs .
the protein layers formed on the polymer films
consisting of the
pristine pcldiupy , the mixture with additive 1 , and the
mixture with additive 3 were slightly thicker than the
layers formed from the solution of albumin , suggesting that mainly
albumin adsorbs , which subsequently prevents the adsorption of the
other proteins in these mixtures . for the sample with additive 2
the thickness of the protein layer is larger than the thickness
of the albumin layer , suggesting that on this sample not only albumin
but also the other proteins adsorb .
experiments using optical
waveguide light mode spectroscopy on
block copolymers containing peg have also shown that albumin can block
the adsorption of other proteins when studied sequentially .
although the hydrophilic upypegs can reduce
the adsorption of -globulin and fibrinogen , this effect appears
to be canceled by the fast adsorption of albumin on these films .
even though the reduction in protein adsorption is limited after
mixing in 5 wt % of the upypeg additives , the adhesion of epithelial
and endothelial cells is strongly impeded on films of polymer mixtures
with additives 2 and 3 .
the results on cell
adhesion are in accordance with a slight reduction in adsorption of
-globulin on mixtures with additives 2 and 3 but do not match the adsorption of the other protein solutions
studied here .
importantly , in previous studies , when a reduction in
cell adhesion is observed on materials that also resist the adsorption
of proteins , protein fouling is commonly measured prior to cell adhesion .
with these three additives , we have created a toolbox to tune non - cell - adhesive
properties of our biomaterials in a modular fashion .
incorporation
of peg10kdiupy and meopeg5kdiupy clearly reduced
cell adhesion and spreading compared to the pristine material . using
qcm - d in combination with viscoelastic modeling , and thereby separating
the contribution of solvent viscosity and protein adsorption
, we showed
that we are able to quantify the adsorption of proteins on pcldiupy
surfaces modified in a supramolecular fashion with upypeg .
the more
hydrophilic surfaces created by incorporation of peg2kdiupy ,
meopeg5kupy , and peg10kdiupy had a limited effect
on protein adsorption , which shows that non - cell - adhesive behavior
can not per definition be correlated with a possible reduction in protein
adsorption . | fouling properties
of new biomaterials are important for the performance
of a material in a biological environment . here , a set of three supramolecular
polymeric additives consisting of ureidopyrimidinone ( upy)-functionalized
poly(ethylene glycol ) ( upypeg ) were formulated with upy - modified polycaprolactone
into thin supramolecular material films .
the antifouling properties
of these material films were determined by investigation of the relation
of cell adhesion and protein adsorption on these materials films .
the presence of the upypeg additives at the surface of the films was
evident by an increased hydrophilicity .
adhesion of human epithelial
and endothelial cells was strongly reduced for two of the upypeg - containing
films .
analysis of adsorption of the first three proteins from the
vroman series , albumin , -globulin , and fibrinogen , using quartz
crystal microbalance with dissipation in combination with viscoelastic
modeling , revealed that the surfaces containing the upypeg additives
had a limited effect on adsorption of these proteins . despite a limited
reduction of protein adsorption ,
upypeg - containing mixtures were non - cell - adhesive ,
which shows that non - cell - adhesive properties of supramolecular polymer
surfaces are not always directly correlated to protein adsorption . |
the incidence of obesity defined as body mass index ( bmi ) 30 kg / m has increased drastically worldwide during recent decades .
obesity is associated with a cluster of metabolic disorders , including increased risk of insulin resistance , type 2 diabetes ( t2 dm ) , hypertension , dyslipidemia , and cardiovascular disease ; these disorders and obesity per se constitute a serious threat , known as the metabolic syndrome ( ms ) .
it is well known that chronic inflammation is a key feature of obesity ; this obesity - induced inflammation builds the common soil for the pathogenesis of ms . therefore
, resetting the immunological balance in obesity is a crucial approach for the management of ms .
white adipose tissue ( at ) has been regarded as the primary goal of pharmacological intervention since obesity - induced inflammation is mainly initiated and exacerbated in this organ [ 35 ] .
although several prominent molecular mechanisms have been proposed to trigger inflammation in white at , including hypoxia , endoplasmic reticulum stress , lipotoxicity , and metabolic endotoxemia , these factors can not fully explain the origin of inflammation .
on the other hand , current anti - inflammatory strategies are not sufficient enough in the treatment of ms .
these facts suggest that there are lots of unknowns responsible for at inflammation in obesity . in recent years
, a growing body of evidence has highlighted the modulating roles of mirnas in immune / inflammatory system [ 6 , 7 ] and their involvement in the obesity - related metabolic disorders including t2 dm and atherosclerosis [ 8 , 9 ] .
as yet , whether these mirnas could be another mechanism for mediating at inflammation and whether these tiny molecules could serve as potent therapeutic anti - inflammatory targets for ms are still not fully settled .
during the past decade , it became clear that inflammation is a key feature of obesity .
the inflammatory response triggered by obesity involves many components of the classical inflammatory response to pathogens and includes the increases in circulating inflammatory cytokines and acute - phase proteins ( e.g. , c - reactive protein ) , recruitment of leukocytes to inflamed tissues , activation of tissue leukocytes , and generation of reparative tissue responses ( e.g. , fibrosis ) .
however , the nature of obesity - induced inflammation , referred to as metainflammation ( metabolically triggered inflammation ) , is unique compared with other inflammatory paradigms ( e.g. , infection and autoimmune disease ) in several key aspects .
first , the chronic nature of obesity produces a low - grade activation of the innate immune system that affects metabolic homeostasis over time .
second , childhood obesity may place individuals at risk for lifelong inflammation , since inflammatory markers are elevated in obese children as young as 3 years old .
third , this chronic inflammation is composed of recurrent acute episodes of nutrition - related immune activation induced by nutrient availability ( fasting or high - fat meals ) .
this fluctuation may be associated with the induction of pro- or anti - inflammatory mediators .
the metabolic syndrome ( ms ) refers to the clustering of cardiovascular risk factors that include central obesity , hyperglycemia , dyslipidemia , and hypertension .
the ultimate importance of this cluster is to identify individuals at high risk of both t2 dm and cardiovascular disease .
growing evidence implicates obesity - induced inflammation as an important mechanism linking obesity to the ms in metabolically active organs .
assessment of gene expression networks in obese at has identified a robust pattern of overexpressed inflammatory genes associated with metabolic disease [ 12 , 13 ] .
multiple inflammatory mediators abnormally secreted by at and the crosstalk between immune and metabolic cells can impair insulin signaling and induce oxidative stress and endothelial dysfunction , leading to systemic insulin resistance and cardiovascular disease .
deregulated macrophage - myocyte and macrophage - hepatocyte signaling can impair insulin sensitivity as well [ 14 , 15 ] .
hypothalamic inflammation , which is induced very rapidly by a high - fat diet may provoke hyperphagia and has been documented to impair insulin release from cells , peripheral insulin action , and potentiate hypertension [ 1719 ] .
thus , chronic excess of nutrients , such as lipids and glucose may simultaneously trigger inflammatory responses , which further disrupt metabolic function , enhancing stress , and inflammation .
therefore , breaking this vicious circle by resetting the immunological balance in obesity is a crucial approach for the management of ms .
obesity is characterized by excessive expansion of white adipose tissue ( at ) , which has been thought to be the primary site for the initiation of obesity - associated inflammation .
although at 's principal function deals with energy storage , it serves as an active secretory organ as well . a number of bioactive peptides or proteins , collectively named adipokines , are produced and secreted by fat and/or nonfat cells of white at .
they act in an autocrine / paracrine manner to regulate local at function and also act in an endocrine manner to influence the functions of distant tissues such as liver , skeletal muscle , and cardiovascular and central nervous systems [ 5 , 20 ] . in obesity ,
white at is remodeled dynamically by adipocyte hypertrophy ( increased size ) , hyperplasia ( increased number ) , immune cell infiltration , endothelial cell overactivation , and extracellular matrix overproduction [ 2124 ] .
this remodeling may trigger metabolic and hypoxic stress , resulting in activation of multiple inflammatory signaling pathways , ultimately leading to dysregulation of numerous adipokines including proinflammatory cytokines , chemokines , growth factors , acute - phase proteins , and complement - like factors .
such a deregulation is the main feature of at low - grade inflammation and contributes to the pathogenesis of ms .
the growing at is submitted to hypoxia induced by hypoperfusion at the earliest stages of expansion .
observations have proved that at is poorly oxygenated in the obese state in humans and rodents due to a reduction in adipose tissue blood flow [ 25 , 26 ] .
this hypoxia could be a determinant mediator of obesity - induced inflammation in at by activating multiple signaling pathways such as hypoxia inducible factor-1 ( hif-1 ) and nuclear factor kappa b ( nf-b ) in adipocytes and macrophages , thereby altering the expression of many proinflammatory adipokines [ 2729 ] .
in addition , some studies reported that hypoxia also induces at inflammatory responses indirectly by causing adipocyte death ( e.g. , apoptosis and necrosis ) and lipolysis [ 3032 ] .
the endoplasmic reticulum ( er ) is responsible for much of a cell 's protein synthesis and folding , but it has also a role in sensing cellular stress .
obesity results in conditions that increase the demand on the er , such as hypoxia and excess of cytokines , lipids , or glucose .
subsequently , the er stress induces a complex response known as the unfolded protein response ( upr ) , which alters a cell 's transcriptional and translational programs to cope with these stressful conditions and resolve the protein - folding defect .
er stress and the upr lead to obesity - induced inflammation and metabolic abnormalities by several distinct mechanisms , including the activation of jnk - ap1 ( jun n - terminal kinase - activator protein 1 ) and ikk ( ib kinase)-nfb pathways , the induction of the acute - phase response , and the production of reactive oxygen species ( ros ) [ 34 , 35 ] .
obesity is characterized by a positive energy balance , and the classical response to this nutrient oversupply is at hypertrophy .
this limited storage capacity , coupled with the overstimulation of hormone - sensitive lipase , leads to massive increase in free fatty acid ( ffa ) release and accumulation ectopically .
tlrs are a family of pattern - recognition receptors that play a critical role in the innate immune system by activating proinflammatory signaling pathways in response to microbial pathogens . at
tlr expression in at is inducible by inflammatory stimulation and linked to downstream activation of nf-b or jnk and subsequent release of proinflammatory adipokines [ 37 , 38 , 40 ] .
tlr4-deficient mice and c3h / hej mice ( which have a loss - of - function mutation in tlr4 ) are partially protected from fat - induced inflammation and insulin resistance in their visceral at [ 41 , 42 ] .
ffas also increase the infiltration and activation of macrophages , especially the cd11c+ subset , thereby exacerbating their proinflammatory activity .
high - fat diet given to mice chronically increased plasma lipopolysaccharide ( lps ) concentration two to three times ; a threshold that has been defined as metabolic endotoxemia .
obese subjects with type 2 diabetes have 76% higher circulating lps than healthy controls and this high level of lps decreased significantly after surgical weight loss .
elevated plasma lps levels result from increased absorption of lps across the intestinal barrier triggered by high - fat diet .
recently , three underlying mechanisms have been proposed , including the changes in gut microbiota environment , the increased availability of chylomicrons , and the permeability of the gut epithelium [ 4749 ] . in murine adipocytes
, lps initiates inflammation via tlr4 and induces the secretion of proinflammatory cytokines via downstream activation of nf-b or mitogen - activated protein kinases ( mapk ) signaling pathways . in human adipocytes , stimulation by lps increases release of tumor necrosis factor - alpha ( tnf- ) , interleukin-6 ( il-6 ) , and monocyte chemoattractant protein-1 ( mcp-1 ) by nf-b activation and upregulates tlr2 expression .
micrornas ( mirnas ) are endogenous ~22 nt rnas that can bind to the 3-untranslated region ( 3-utr ) of target mrnas to repress mrna expression at the posttranscriptional level . as a group
, mirnas may directly regulate expression of over 30% of human and mouse genes and more than 60% of human protein - coding genes have been under selective pressure to maintain pairing to mirnas .
specific mirnas have been implicated in adipocyte differentiation and mature adipocyte function , including lipolysis , glucose - uptake , and insulin sensitivity [ 8 , 10 ] . on the other hand
, mirnas have been defined as important immunomodulators by regulating the differentiation , induction , and function of immune cells and the expression of multiple cytokines in the immune system .
these findings could shed light on the possible links between mirnas and adipose tissue immunity / inflammation .
studies conducted by some groups have demonstrated that mirna regulation is indispensable for stable immune process .
ablation of whole mirnas by deletion of argonaute 2 ( ago2 ) or dicer impairs pre - b - cell differentiation and the succeeding peripheral b - cell generation .
several individual mirnas have also been reported to be involved in the b - cell biology .
for example , overexpression of mir-17~92 clusters enhances b - cell proliferation and survival , while mir-150 profoundly impairs early b - cell differentiation and mature b - cell responses .
mirnas have been shown to be key regulators of the t - cell lineage induction pathways .
deletion of dicer at an early stage of t - cell development compromised the survival of t - cell lineage .
the best evidence for mirnas playing a role in specific developmental stages of t - cell differentiation is from mir-181 , which reduces the number of t - cells in haematopoietic overexpression systems and also increases the sensitivity of t - cell receptor signaling .
in addition , mirnas also play pivotal roles in the induction , function , and maintenance of the regulatory t - cell ( treg ) lineage .
mirnas can enhance thymic and peripheral induction of treg cells [ 55 , 58 ] .
several mirnas ( including mir-155 , mir-146a , mir-21 , and mir-9 ) have been consistently found to be rapidly induced by innate immune activation ( e.g. , toll - like receptor ) , indicating that they may regulate the innate immune response .
target prediction analyses indicate that up to a half of innate immune genes could be under the direct regulation of mirnas .
a study on 613 genes , which regulate immunity utilizing a computational approach , identified 285 genes as mirna targets .
major targets include transcription factors , cofactors , and chromatin modifiers whereas upstream factors , such as ligands and receptors ( cytokines and chemokines ) were , in general , poor or nontargets .
the mechanisms by which mirnas regulate cytokine expression include direct regulation by binding to seeding sites in mrna 3-utr and indirect regulation .
for example , of the interleukin genes ( il1 - 29 ) studied , 9 had predicted mirna binding sites . out of 20 interleukin receptor genes examined , only 2 had high probability mirna target sites .
these findings suggest that the regulation of cytokine genes by mirnas occurs often via indirect mechanism .
recent studies indicate indeed that mirnas could indirectly regulate cytokine genes via au - rich elements ( are ) located in the mrna 3-utr by targeting are - binding proteins . are are the cis - acting structural rna motifs that are important determinants of cytokine message stability .
are - mediated mrna degradation is regulated by a number of trans - acting factors , called the are - binding proteins ( which include tristetraprolin ( ttp ) , au rich binding factor 1 ( auf1 ) , and members of the hu protein r ( hur ) family ) .
repression of several are components by mirnas may alter the levels of inflammatory cytokines as well as of other immune genes [ 60 , 62 ] .
obesity - induced inflammation is associated with dysregulated expression of mirnas in white adipose tissue .
a number of mirnas have been found dysregulated in white at during obesity and closely associated with obesity - related metabolic disorders .
a recent study identified 21 mirnas , which were differentially expressed in epididymal at between lean mice fed a standard diet and mice rendered obese by a high - fat diet .
ortega et al . performed mirna array on human subcutaneous at : 50 of the 799 mirnas tested ( 6.2% ) significantly differed between obese ( n = 9 ) and lean ( n = 6 ) subjects . among these 50 mirnas , 17
these data are concordant with those obtained in overweight or obese subjects by klting et al .
they showed significant correlations between the expression of selected mirnas and both at morphology and key metabolic parameters , including visceral fat area , hba1c , fasting plasma glucose , and circulating leptin , adiponectin , and il-6 .
another two mirnas ( mir-175p and mir-132 ) were significantly decreased in the omental fat and the circulation of obese subjects .
a very recent study defined a set of eleven adipocyte - specific mirnas downregulated in obese subjects as all of them were concomitantly altered in both the white at and the isolated adipocytes .
xie et al . showed that tnf- treatment of 3t3-l1 adipocytes mimicked the changes of mirna expression observed in at of obese mice . with obesity and inflammation being so intrinsically associated , the dysregulated expression of mirnas in inflammatory adipocytes highly suggests the particular importance of mirnas in obesity - induced inflammation .
indeed , a few individual mirnas have been reported as playing a crucial role in the inflammatory state of at so far ( figure 1 ) .
expression of mir-221 and mir-222 has been positively correlated to tnf- and negatively correlated to apn expression in white at of mice . yet , the direct effects of both mirnas on these pro- or anti - inflammatory adipokines are still unraveled .
mir-132 , which was downregulated in human obese omental fat , has been reported to activate nf-b and the transcription of il-8 and mcp-1 in primary human preadipocytes and in in vitro differentiated adipocytes .
zhuang et al . demonstrated that mir-223 played a crucial role in modulating macrophage polarization in a pattern that protects mice from diet - induced at inflammation and systemic insulin resistance .
macrophages by targeting pknox1 in vitro , while mir-223 deficient mice fed a high - fat diet exhibited increased at inflammation characterized by enhanced proinflammatory activation of macrophages .
the group of brichard has identified several mirnas , which were regulated by adiponectin ( a promising anti - inflammatory adipokine ) in white at in vivo .
the mir883b-5p , which was upregulated by adiponectin and downregulated in human obesity repressed the lps - binding protein ( lbp ) and tlr4 signaling , acting therefore as a major mediator of the anti - inflammatory action of apn .
moreover , mir883b - silencing in the de novo at formed from in vivo differentiation of preadipocytes also induced lbp production and tissue inflammation .
very recently , arner et al . overexpressed individual mirnas ( which have been defined downregulated in at in human obesity ) in human adipocytes differentiated in vitro .
they found that a set of nine mirnas significantly reduced the secretion of chemokine ( c - c motif ) ligand 2 ( ccl2 ) which is an initiator of at inflammation by attracting the migration of inflammatory cells into the tissue . among these mirnas ,
only mir-126 was predicted by in silico analysis and confirmed by luciferase reporter assay in 3t3-l1 cells to bind directly to the 3-utr of ccl2 , whereas mir-193b affected indirectly the ccl2 production through downregulating the transcription factors ( tfs ) of ccl2 ( relb , stat6 , and ets1 ) .
thus , mirnas may mediate at inflammation by regulating either the activation of macrophages or the production of adipokines .
the mechanism by which mirnas regulate adipokines in at seems via direct and indirect mechanisms .
mirnas may act directly on the target inflammatory adipokines or indirectly by first regulating the intermediate machinery components like the tfs , which , in turn , control the expression of adipokines .
in addition to the innate immunity , recent studies have disclosed the importance of the adaptive immunity in at inflammation .
the subsets of the lymphocyte lineage , including cd8 + and cd4 + t - cells , tregs , natural killer t - cells ( nkt ) , and b - cells have been revealed to infiltrate into at which may orchestrate at inflammation .
yet , the mechanisms underlying the infiltration of lymphocytes into at remain largely unknown ( figure 1 ) .
whether mirnas could play roles in modulating the infiltration or activation of the lymphocytes in at may arouse research interest and requires to be investigated in the future .
more recently , adipocytes have been reported to secrete mirnas in the form of adipocyte - derived microvesicles ( adms ) , which may regulate the function of distant or neighboring cells .
one study found that microvesicles released from cultured 3t3-l1 adipocytes harbored 143 mirnas , most of them being adipocyte - specific and reflecting the abundance of their expression levels in the donor cells .
interestingly , the mirnas - containing microvesicles were transported into cultured macrophages which were incubated with adms containing - medium for 24 h ; they were also present in rat serum in vivo . yet , whether the mirnas inside the vesicles were functional in their new location remains to be determined .
furthermore , another group discovered that mirnas - microvesicle complexes derived from the larger primary rat adipocytes were transferred into and expressed in the smaller adipocytes and were involved in the transcription of multiple genes for lipid synthesis and cell growth .
hypertrophied adipocytes are the main sources of at inflammation , the mirna - mediated interactions ( adipocytes - macrophages and larger adipocytes - smaller adipocytes ) may be novel mechanisms for mirnas contributing to at inflammation ( figure 1 ) .
the facts that mirnas are largely dysregulated in obesity and that specific mirnas regulate obesity - associated inflammation suggests the potential of these molecules as targets for therapeutic intervention in obesity and obesity - related metabolic disorders .
chen et al . screened serum mirnas of healthy chinese subjects and found over 100 and 91 serum mirnas in male and female subjects , respectively . yet , how these mirnas make their way into the circulation is still mysterious .
new studies have revealed that circulating mirnas can reside in microvesicles ( exosomes , microparticles , and apoptotic bodies ) and in protein / lipoprotein complexes ( high density lipoprotein ( hdl ) and argonaute 2 ) ; these formations make the circulating mirnas protected and resistant to rnase activity and degradation .
therefore , the levels of mirnas in serum are remarkable , stable , reproducible , and tissue - specific among individuals .
multiple reports have noted the potential use of blood mirnas as biomarkers for the obesity , cardiovascular diseases , atherosclerosis , and t2 dm .
heneghan et al . showed that circulating levels of mir-17 - 5p and mir-132 were significantly decreased in obese individuals compared to nonobese individuals and reflected mirna expression in omental fat .
the mirna expression in blood and omental fat from obese patients correlated significantly with bmi , fasting blood glucose , and glycated hemoglobin .
cardiac specific mir-1 , mir-208 , and mir-499 were induced consistently in blood of patients with myocardial infarction and strongly correlated to markers of cardiac damage including troponin t and creatine kinase - mb ( ck - mb ) activity [ 7881 ] .
circulating mirnas have also been proposed as highly sensitive biomarkers for t2 dm . in the bruneck cohort of 822 subjects , decreased serum levels of mir-126 in t2 dm emerged as a significant predictor of t2 dm and correlated negatively with increasing glucose intolerance . silencing of key mirna and replacement of certain tissue - specific mirna whose expression is known to be decreased are potential therapeutic interventions .
techniques .
mirnas can be deactivated and silenced by anti - mirna oligonucleotides ( amos ) , mirna sponges , and mirna masking .
amos are synthetic antisense oligonucleotides that competitively inhibit the interaction between mirnas and their mrna targets .
the most widely employed types of amos are 2-o - methyl amo , 2-o - methoxyethyl amos , and locked nucleic acids ( lnas ) . given that mirnas have been observed to function often in clusters in pathological processes , the
this technique requires a vector encoding large quantities of transcripts ( anti - mirnas ) which display numerous and tandem binding sites for a cluster of mirnas of interest .
the mirna masking is an alternative mirna - knockdown strategy to the amo approach , with the advantage of targeting mirnas in a gene - specific manner . unlike amo , which binds to the endogenous mirna directly
, a mirna mask binds to the mirna 's binding site located in the 3-utr of its target mrna , thereby avoiding off - target effects .
conversely , with regard to those mirnas with decreased expression in disease , the fundamental principle strategy is to restore their expression
. this can be achieved through mirna mimicry or plasmid / viral vector - encoded mirna replacement .
mirna mimics are small chemically altered double - stranded rna molecules that imitate endogenous mirnas .
plasmid / viral vectors encoding mirnas are encouraging strategies to replace mirna in vivo , with good transduction efficiency and minimal toxicity [ 85 , 86 ] .
micrornas and therapeutics . since the obesity - induced inflammation is considered to build the common soil for the pathogenesis of ms , some potential anti - inflammatory therapies have been investigated .
several drugs in current clinical practice show the anti - inflammatory properties : the peroxisome proliferator - activated receptor ( ppar ) family and nonacetylated salicylates .
other strategies have been explored by targeting the cytokines and chemokines or their receptors ( e.g. , the anti - tnf or anti - ccr2 ( chemokine ( c c motif ) receptor 2 ) therapies ) .
although there have been some encouraging results , it is likely that the benefits of these approaches are limited [ 87 , 88 ] .
therefore , the development of mirna - based therapeutics has raised interest in this field . to date
, the greatest efforts have been made in exploring the potential application of mirnas therapeutics in cancer and liver disorders .
gain or loss of function of individual mirnas have been reported in almost every solid and hematological cancer , with therapeutic suppressing effect in tumor cell proliferation , progression of tumors , and the metastatic process .
the liver specific mir-122 has been proved to regulate cholesterol biosynthesis and hepatitis c virus replication .
silencing of mir-122 by intraperitoneal administration of high affinity lna anti - mir-122 has resulted in dose - dependent lowering of plasma cholesterol in mice and nonhuman primates ( monkeys ) .
this was achieved without significant adverse reactions or hepatic toxicity [ 89 , 90 ] . likewise , induction or reduction of mirna expression that is known to be under / upper - expressed and involved in at inflammation using viral or liposomal delivery of tissue - specific mirnas to affected at
yet , the application of mirnas therapeutics in at in vivo is poorly investigated because fat cells are insusceptible to selective transfer of exogenous nucleotide due to the containing lipid droplets of adipocytes .
encouragingly , the discovery that mirnas could be secreted by adipocytes and accepted by neighboring adipocytes or macrophages in the form of adipocyte - derived microvesicles [ 75 , 76 ] raises the hope that it could be possible to import the targeting mirnas into at in vivo by the manner of microvesicles .
this manner could construct the microvesicles containing specific mirnas and inject the constructed microvesicles into white at .
although significant advances have been made in mirna - based therapy , various challenges remain to be overcome before clinical use .
first , one individual mirna may have multiple potential targets , which may coordinate or antagonize each other 's functions .
in addition , mirna - target interactions depend not only on the sequence of the target site but also on the cellular context , in which the interactions occur .
this complexity explains the difficulties in predicting the spectrum of side effects and toxicity profiles , which may be associated with mirna - based therapeutics .
high levels of exogenous mirna can compete with the endogenous mirna biogenesis processing , leading to cell toxicity [ 91 , 92 ] .
another critical issue is the site - specific , safe , effective , and repeated delivery of mirna in vivo .
the systemic intravenous delivery of viral or liposomal - mediated mirna oligonucleotides have been well achieved in cancer and liver disease .
however , targeting certain mirnas in other specific tissues is poorly achieved due to the invasive nature of access for repeat delivery and lack of cell - specific up - take .
restoration of tissue - specific mirna expression could be a more rational approach for managing local abnormalities with more efficiency and precision and with less systemic side effects [ 83 , 92 ] .
evidence has provided that mirnas are largely dysregulated in white at in human obesity ; specific mirnas regulate the polarization of macrophages and the expression and secretion of adipokines in white at ; a subset of mirnas could be packaged into adipocyte - derived microvesicles and delivered into blood or neighboring cells , mediating intercellular communication .
furthermore , with the recent discovery that mirnas are secreted into circulation in a stable manner and correlated closely with crucial metabolic parameters , the plasma mirna profiles could therefore be used as attractive biomarkers for the ms .
finally , the establishment of mirnas - based techniques allows exploring further fascinating roles of mirnas and their applications in the management of ms . despite huge challenges lying in mirna - targeting therapeutics , especially in developing safe and effective delivery of targeting mirnas in specific tissues in vivo ,
the development of mirna - based therapeutics for ms is warranted because of the severe problem that metabolic disease poses to society . | obesity is associated closely with the metabolic syndrome ( ms ) .
it is well known that obesity - induced chronic inflammation plays a fundamental role in the pathogenesis of ms .
white adipose tissue ( at ) is the primary site for the initiation and exacerbation of obesity - associated inflammation .
exploring the mechanisms of white at inflammation and resetting the immunological balance in white at could be crucial for the management of ms .
several prominent molecular mechanisms have been proposed to mediate inflammation in white at , including hypoxia , endoplasmic reticulum stress , lipotoxicity , and metabolic endotoxemia .
recently , a growing body of evidence supports the role of mirnas as a new important inflammatory mediator by regulating both the adaptive and innate immunity .
this review will focus on the implication of mirnas in white at inflammation in obesity , and will also highlight the potential of mirnas as targets for therapeutic intervention in ms as well as the challenges lying in mirna - targeting therapeutics . |
cortistatin a is a marine steroid with highly selective and perhaps mechanistically unique antiangiogenic activity .
herein we report a synthesis of this natural product by way of cortistatinone , an intermediate ideally suited for investigating the key pharmacophore of the cortistatin family .
the synthesis begins with a terrestrial steroid and traverses a route to cortistatin a through the discovery of unique chemical reactivity .
specifically , we demonstrate the first example of a directed , geminal ch bisoxidation , a new fragmentation cascade to access expanded b - ring steroid systems , a chemoselective cyclization to install the hallmark oxabicycle of the cortistatin family , and a remarkably selective hydrogenation reaction , which should find extensive use in future syntheses of the cortistatins and designed analogues .
the synthesis displays a level of brevity , efficiency , and practicality that will be crucial in evaluating the medicinal potential of this fascinating class of marine steroids . | |
a 43-year - old male with a history of uncontrolled hypertension was admitted to our neuroscience intensive care unit form an outside institution with a diagnosis of a hunt & hess grade 4/fisher grade 3 sah ( fig .
an evd was placed on admission and a digital subtraction angiography ( dsa ) was performed which demonstrated presence of the right vertebral artery fusiform aneurysm with the posterior inferior cerebellar artery ( pica ) arising from the base of a focal dilatation from the fusiform aneurysm segment ( fig .
after consideration of all the treatment options , endovascular treatment was elected in view of poor grade sah and patients ' cardiopulmonary status .
a microcatheter was subsequently placed into the vertebral artery with coiling of the focal dilatation arising off the aneurysm along with deployment of a 3.7518 mm ped completely covering the fusiform vertebral aneurysm for vessel remodeling .
a raymond class ii occlusion of the focal dilatation arising from the fusiform aneurysm was achieved ( fig .
the evd was converted to a ventriculo - peritoneal shunt before the patient was discharged home in a good neurological condition .
he was alert and oriented with no focal def icits at discharge except presence of right sided sixth cranial nerve palsy .
routine follow up angiography done at 6 months as per our institutional protocol showed coil compaction and enlargement and regrowth of both the focal dilatation and the fusiform aneurysm without migration of the pipeline stent ( fig .
even though the patient was asymptomatic , considering significant enlargement of the aneurysm with a prior presentation with sah , he was taken back to the neuro - interventional suite with placement of three overlapping ped 's ( 3.5 mm182 and 3.514 mm ) to increase the overall coverage area and further reduce the shear stress which possibly caused the aneurysm to continue growing despite treatment with a single ped on presentation .
the post procedure course was uneventful and he was discharged home with no new neurological deficits .
a follow up angiography was performed at 3 months which showed no residual aneurysm ( fig .
fusiform aneurysms represent a challenging subgroup of intracranial aneurysms which generally presents with ischemic stroke , cranial nerve palsies , brainstem compression , hydrocephalus and intracranial hemorrhage .
sah is generally uncommon in patients with fusiform aneurysm ; but when present increases the complexity associated with management of these aneurysms .
given the complex morphology of fusiform aneurysms , both endovascular and surgical treatment options remain limited and technically challenging .
poor grade sah along with associated co - morbid medical conditions often results in pursuing the endovascular route for treatment of intracranial aneurysms even for cases for which there is potential for increased complications .
use of stents in ruptured sah is such an indication as they often require administration of dual antiplatelet agents which associated risks of hemorrhagic complications , either spontaneous or associated with interventions such as placement or revision of evds and vps .
flow diverters induce disruption of flow near the aneurysm neck , inducing thrombosis into the aneurysm sac while preserving physiological flow in the parent vessel and adjacent branches and have been the most recent advancement in endovascular treatment of intracranial aneurysms
. the term " flow diversion " refers to the concept of hemodynamic influence on an aneurysm by reducing both inflow and outflow with flow diverters being stent - like endovascular implants designed to alter the blood flow in an artery in a way that favors the flow along the longitudinal axis of the target vessel and reduces inflow and outflow of an aneurysm covered by the device .
various flow diverters commercially available includes ce mark device ( silk , balt extrusion , montmorency , france and the pipeline embolization device ( ped , ev3 , irvine , ca , usa )
. a tight mesh of small cell sizes and a high longitudinal flexibility of these newer flow diverters offer the possibility to cover an aneurysm orif ice and in carefully selected cases , may successfully be used to reconstruct intracranial vessel dissections . while approved for large or giant necked aneurysms involving the ica from the petrous to the superior hypophyseal segments , a number of studies have shown the effectiveness of stent - assisted coiling in sah with good overall clinical outcome ; albeit at higher rates of complications [ 6 , 7 ] .
most of these complications have been hemorrhagic secondary to the use of antiplatelet and anticoagulant medications .
however , once the acute period is over with good aneurysm occlusion , delayed thrombosis of the stent remains the foremost concern along with other device - related complications such as creeping of the stent or migration / shortening of the ped .
overall , the use of ped has been shown to result in excellent aneurysm occlusion in various series . while immediate angiographic exclusion of the aneurysm at the end of the original procedure is observed only in up to 10 - 20% of patients treated with ped ,
total aneurysm occlusion rates of more than 90% at 1 year have been reported in various studies even in large and giant aneurysms and even fusiform and dissecting type aneurysms through remodeling of the parent vessel over time .
the use of ped in wide neck aneurysms makes it an ideal choice to be used in conjunction with coil placement as conventional stent porosity would not provide significant protection against coil migration as compared to ped in some cases where smaller coils needs to deployed . while the lack of immediate aneurysm occlusion with flow diverters necessitates obliteration of aneurysm sac using simultaneous coil placement in patients with sah , as the occlusion rates with use of flow diverting stents such as ped increases up to 90% at one year follow up , any residual aneurysm neck after securing of the aneurysm dome should not be concerning as the flow diverter stent should actually lead to complete aneurysm occlusion due to its mechanism of action . the case described in this report
was treated with this hypothesis in mind . to avoid the possibility of coil migration / herniation without a scaffold , a ped was used to address this and also to treat the fusiform vertebral artery aneurysm .
the use of conventional low coverage stent assisted coiling was not considered optimal due to the origin of the pica from the fusiform aneurysm segment of the artery .
placement of flow diverter ped was considered the best option as it would allow coiling of the focal dilatation of the aneurysm and leave the pica patent due to maintenance of the flow secondary to associated pressure gradient . hence once positioned and prior to coil detachment into the focal dilatation from the aneurysm , a 3.75 mm18 mm ped device
was partially deployed to cover the pica and the fusiform aneurysm while temporarily jailing the microcatheter .
the coil was then detached , and the remainder of the stent was deployed , preventing migration of the coil mass .
the combined use of both modalities , the coil and the ped , provided immediate protection from re - hemorrhage , while counting on the long term protection through remodeling of the parent vessel by the ped .
unfortunately , in our case , the follow up angiogram showed aneurysm regrowth with coil compaction despite well positioned ped which required another procedure . the plausible hypothesis for aneurysm expansion in our case
may be related to the low coverage of the fusiform segment of the aneurysm which allowed progression of the aneurysm in the diseased fusiform segment due to persistent shear stress in the vessel and led to recurrence of the aneurysm .
we hypothesize that in such cases it may be important to reduce the shear stress in the vessel wall to allow vessel remodeling ; failure of which may result in persistent growth of the fusiform aneurysm as was seen in the present case .
unfortunately , the number of ped to be employed and what is constituted as adequate coverage is considered controversial with no consensus .
technically , a single ped is considered adequate if the target lesion is fully covered and in case of immediate contrast medium stagnation . however , placement of multiple devices may be of value in cases with an unchanged aneurysm perfusion after implantation of ped indicating an insufficient effect of flow diversion and cases of widespread longitudinal vessel dilatation where a telescoping implantation of multiple devices was necessary to cover the entire lesion .
while the use of multiple peds appears intuitive in cases with fusiform aneurysms to decrease the shear stress on the diseased vessel wall , it may not obviate the chances of incomplete occlusion in these subgroup as seen in the study by jabbour et al . and may unnecessarily increase procedural time , procedural cost , and the risk of thromboembolic complications . on the other hand , a dense packing of the device during the implantation process does raise the degree of coverage resulting in larger flow diversion and a larger protective effect arguing in favor of implantation of more than one flow diverter across the orifice in order to increase the density with an improved flow diversion towards the parent artery . the benefit of increased density needs to be balanced with risk of side branch or parent vessel occlusions due to an in - stent thrombosis and other thromboembolic complications .
we believe that in the absence of definitive evidence for either and balancing the risk benefits ratio , concept of a staged therapy with initial rather defensive implantation of devices followed by additional flow diverters dependent on the follow - up results appears intuitive .
while this approach may be useful for patients who does not presents with sah , poor response with aneurysm regrowth in patients with sah may be concerning due to risk of re - rupture as could have happened in our case . in these cases of fusiform aneurysm presenting with sah , it may be prudent to utilize multiple peds to increase the coverage area ; what is considered adequate is debatable and is up to the discretion of the treating neurointerventionlist as even with more than one ped , jabbour et al . reported with poor outcome in patients with fusiform aneurysm .
hence considering an earlier follow up angiography in patients with fusiform aneurysms treated with ped especially in patients with sah may be of value to detect increase in size of the aneurysm earlier and tailor the number of devices necessary to achieve the desired outcome or pursue alternative treatment modality to prevent aneurysm rupture .
fischer , et al recently reported complete aneurysm occlusion or decrease in size of the aneurysm in about 96% of patients with fusiform or dissecting aneurysm at a follow up of 1 year .
interestingly , in the same study ; only 24% of cases showed complete cure and 30% showed partial occlusion at 3 months follow - up angiography .
there was no aneurysm growth in the study at three or one year follow - up angiogram demonstrating the safety and eff icacy of ped in fusiform and dissecting aneurysms .
none of the patients in the study had acute sah though , and a number of patients were treated with more than one peds .
in contrast to this study , siddiqui et al reported less favorable results in a series of 7 large or giant vertebrobasilar fusiform aneurysms treated with flow diverters with significant morbidity and mortality ; the poor outcome being secondary to post - treatment aneurysm ruptures in two and lack of neurological improvement caused by brainstem infarcts in another 2 patients .
suboptimal outcome with the use of ped in fusiform aneurysms was also echoed in the study by jabbour , et al in which they found fusiform aneurysm shape as a negative predictor for aneurysm obliteration despite a significantly higher number of peds ( 1.8 ) used in this subset of patients .the variable results from different studies highlights the complexity associated with use of ped in vertebrobasilar fusiform aneurysms .
as the data on the use of ped in vertebrobasilar aneurysms presenting with sah is not robust , clinicians should be careful in extrapolating the excellent outcome following the use of flow diverters in well studied conditions .
nevertheless , with the increasing use of ped outside more common fda approval indications , apart from demonstrating the efficacy , the clinical relevance of understanding the patterns of failure can not be overemphasized as highlighted in the present case . in conclusion ,
an unusual case of persistent aneurysmal growth following treatment with ped assisted coiling of a fusiform vertebral artery aneurysm is reported . as the use of ped is becoming increasingly common for a number of clinical indications , the mechanism and instances of failure needs to be understood to prevent complications and optimize the use of this newer device .
we suggest considering an earlier follow up angiography in patients with fusiform aneurysms treated with ped to detect inadequate response to treatment with possible need of subsequent multiple / overlapping ped to increase the chances of successful outcome . | the complex morphology of vertebrobasilar fusiform aneurysms makes them one of the most challenging lesions treated by neurointerventionists .
different management strategies in the past included parent vessel occlusion with or without extra - intracranial bypass surgery and endovascular reconstruction by conventional stents .
use of flow diversion has emerged as a promising alternative option with various studies documenting its efficacy and safety . however , there are various caveats associated with use of flow diversion in patients with fusiform vertibrobasilar aneurysms especially in patients presenting with acute subarachnoid hemorrhage ( sah ) .
we report a rare case of persistent aneurysmal growth after coiling and placement of the pipeline embolization device ( ped ; ev3 , irvine , california , usa ) for sah from a fusiform vertebral artery aneurysm . as consequences of aneurysm rupture
can be devastating especially in patients with a prior sah , the clinical relevance of recognizing and understanding such patterns of failure can not be overemphasized as highlighted in the present case . |
lynch syndrome ( also known as hereditary nonpolyposis colorectal cancer [ hnpcc ] ) results from mutations in mismatch repair genes ( mlh1 , msh2 , msh6 , or pms2 ) and is associated with a variety of extra - colonic sites , like endometrium , stomach , ovaries , urinary tract and kidneys , biliary tract , pancreas , small intestine , brain and skin.1 endometrial cancer is the most common malignant disorder observed after hnpcc . considering that lynch syndrome is an autosomal - dominant inherited cancer syndrome , identifying a family history using genetic counseling can lead to further tests based on the current guidelines ( revised amsterdam2 or bethesda3 criteria ) . and identification of individuals who have increased risk of occurring hereditary cancers would enable screening and early detection of cancer , which may reduce disease - specific mortality . recording
a patient 's family history is the first step in the work - up of lynch syndrome patients .
however , recording an accurate family history is often neglected or inconsistent.4,5 it can be challenging to advise patients to proceed with additional testing when they do not have a family history of cancer . screening for colorectal cancer in hnpcc
has been proven to improve survival and the cost - effectiveness of treatments , but the benefits of screening for gynecological cancers in lynch syndrome patients have not yet been proven . additionally , there is no consensus regarding the optimal screening tests to perform based on country or patient ethnicity .
here , we present a case of endometrial cancer in a lynch syndrome patient that did not have a family history of cancer .
a 55-year - old korean woman was admitted to a local clinic for vaginal bleeding .
an endometrial biopsy revealed the presence of adenocarcinoma ( endometrioid type , grade 1 ) .
the patient received pelvic magnetic resonance imaging , computed tomography scan , and other diagnostic tests .
the patient had a cancer antigen 125 ( ca 125 ) level of 9.06 u / ml and no other abnormal masses were visible in imaging tests .
complete surgical staging including total hysterectomy , bilateral salpingo - oophorectomy , washing cytology , and pelvic lymph node dissection was performed .
the operation was concluded after confirming a frozen biopsy of the pelvic lymph node was not cancerous . although this patient had no family history of cancer ( her three sisters and parents were alive without any cancer diagnoses at the time of counseling ) , screening immunohistochemistry ( ihc ; mlh1 , msh2 ) was performed due to the genetic risk of lynch syndrome .
the results indicated that mlh1 was positive in 90% f the sample and msh2 was negative ( fig .
1 ) . the tissue pathology reported the tumor stage was ia and adjuvant treatment was not recommended .
according to diagnostic algorithms and after genetic counseling , microsatellite instability ( msi ) and gene sequencing were performed .
msi was analyzed for five markers ( bat25 , bat26 , d2s123 , d5s346 , and d17s250 ) .
the results indicate there were two unstable markers ( bat25 and bat26 ) , and the patient was scored msi - high ( unstable ) .
further gene sequencing analyses revealed one missense mutation ( c.23c > t [ p.thr8met ] ) ( fig .
this case demonstrates that lynch syndrome - related cancer can occur in a patient despite the lack of any family history .
this is the first case report of an endometrial cancer patient with a lynch syndrome germline mutation without family history in korea .
the risk of developing a second cancer is approximately 25% after 10 years and 50% after 15 years following the initial diagnosis of lynch syndrome.6 therefore , the patient and her family were offered cancer screening and genetic counseling .
approximately 2% to 5% of endometrial cancers may be caused by an inherited susceptibility.7 in lynch syndrome carriers , the lifetime risk of endometrial cancer has been estimated to be 42 to 54% , and may equal or exceed the risk of colorectal cancer.8 lynch syndrome is caused by a germline mutation in one of the dna mismatch repair genes ( mlh1 , msh2 , msh6 , and pms2 ) , and accounts for the majority of inherited cases.7 in younger patients , the incidence of endometrial cancer increases to 9% and better survival rates.9,10 information on family history is important for identifying individuals that could benefit from genetic counseling and predictive genetic testing
after counseling and describing the risk and benefits obtained from these results , further confirmative tests for detection of a pathogenic mutation in one of the dna mismatch repair genes were performed with the patient 's permission . with the advances in molecular biology now available for the diagnosis of genetic cancers , clinicians can help prevent these cancers by counseling patients and prescribing adequate and appropriate tests .
in known mutation carriers without any colorectal adenomas , screening colonoscopy appears to be the most reasonable choice.11 however , prophylactic subtotal colectomy remains an option for patients who have significant anxiety about cancer risk or concern about the safety of repeated colonoscopies .
additionally , colectomy can be used if the patient is unable to receive periodic surveillance colonoscopies.12 more information is required to determine the appropriate genetic tests for counseling and prevention according to the country and ethnicity of the patient .
it is also important to identify patients with lynch syndrome because families of mutation carriers may benefit from genetic counseling , testing , and intensified cancer surveillance . in conclusion
, gynecological oncologists should provide information of the genetic cancer risk to endometrial cancer patients in an effort to prevent second cancers in patients and family with lynch syndrome .
patients should undergo the correct genetic tests and should be counseled appropriately , which is important for reducing cancer mortality and costs . | lynch syndrome is a genetic malignancy syndrome affecting the colon , endometrium , and other organs .
it is difficult to find a lynch syndrome patient without any family history of cancer .
we have recently examined an endometrial cancer patient with a msh2 gene mutation without a family history of cancer .
a 55-year old korean woman was admitted to a local clinic for vaginal bleeding .
an endometrial biopsy revealed the presence of adenocarcinoma ( endometrioid type , grade 1 ) .
after surgical staging , no further adjuvant therapy was required .
analysis of the tissue using immunohistochemistry ( ihc ) showed the endometrium stained negatively for msh2 .
microsatellite instability ( msi ) was analyzed for five markers .
the patient was scored as unstable .
further , additional gene sequencing revealed one missense mutation in c.23c > t ( p.thr8met ) .
this is the first case of lynch syndrome endometrial cancer in korea in which the patient does not have any family history of cancer . |
this concise review presents an expanded summary of discussions held during a symposium entitled maintenance of an efficient and equilibrated immune system through the novel use of natural health products and that was the object of a recent meeting report ( 1 ) .
the symposium brought together between 20 and 30 scientists in disciplines ranging from herbalism , ethnobotany and phytochemistry , through to basic and clinical research in fields such as immunology , cancer and nutrition .
it was held in montreal , canada , on september 23 and 24 , 2004 under the auspices of the lucie and andr chagnon foundation , canada 's largest philanthropic organization .
participants were asked to discuss the relationship between the modulation of immune system functions and the prevention of chronic diseases , and to explore natural health products ( nhps ) that may find novel and judicious use in this context .
other symposium details , including the list of participants , can be found in the published meeting report ( 1 ) .
the complexity of the immune system is secondary only to that of the central nervous system although the immune system is older in evolutionary terms ( 2 ) . our immune system is often conceptualized as possessing two principal components : innate and adaptive immunity , which work in concert to defend the body against infection .
it is neither anticipatory ( no memory ) nor clonal and does not respond to environmental changes .
the innate system reacts directly to a wide variety of microorganisms through receptors for highly conserved and essential microbial molecules , such as lipopolysaccharides present on the cell wall of gram - negative bacteria and lipoteichoic acids present in gram - positive bacteria .
indeed , the cells of the innate immune system represent the first line of defence in the immunosurveillance network .
the primary cells involved in the identification and spontaneous lysis of offensive targets ( virus - infected cells , tumor cells , bone marrow stem cells and embryonic cells ) are natural killer ( nk ) cells , which have been the object of several ground breaking studies in the later part of the last century ( 38 ) .
these cells originate from the bone marrow , from which they travel in a unidirectional and almost exclusive way to the spleen ; very few nk cells are found in lymph nodes or other secondary sites such as the gut - associated and bronchi - associated lymphatic tissue ( 3,915 ) . nonetheless , nk cells are lymphoid in morphology and need cells of the macrophage / monocyte lineage for the full expression of nk cell production or function .
nk cells are numerically and functionally low in youth and old age , and maximal during the reproductive periods in mammals .
moreover , an inverse relationship exists between the rise and fall of nk cells and the incidence of tumor growth .
the studies of dussault and miller ( 1618 ) have elucidated some of the factors underlying this relationship . in elderly mice , bone marrow nk cell production and their capacity to bind onto offensive target cells
are significantly reduced relative to that of the young adult mammal , thus attenuating any potential for tumor cell lysis .
in contrast , very young mammals produce nk cells at a level comparable to that of young adults , but there exist nk - suppressive factors in the microenvironment of the spleen that prevent nk cells from lysing offensive targets . in humans
this demonstrates , in part , the importance of nk cells and of the adaptive system in general for the maintenance of optimal immune function .
in contrast , the adaptive system , also termed specific immunity , is acquired through interactions with the environment .
adaptive immune responses are central to the body 's ability to eliminate bacterial , viral and parasitic infections .
understanding these responses is the key to understanding the phenomena of allergy , autoimmunity , vaccination , cancer and organ graft rejection / acceptance .
adaptive immunity has been known for over a century and an abundant literature exists , which is fully reviewed in classical works such as those of roitt and delves ( 19 ) and paul ( 20 ) .
the cells involved in adaptive responses are thymus ( t)-derived cytotoxic t lymphocytes and helper t lymphocytes ( the latter existing in two subsets : thl and th2 ) , bone ( b ) marrow - derived lymphocytes and assorted accessory cells ( dendritic cells , macrophages and stromal cells ) .
lymphocytes ( along with several of their accessory cells ) circulate to and through the spleen , lymph nodes , specialized lymphoid tissue in the gut ( peyer 's patches and appendix ) , bronchi and oropharynx ( adenoids and tonsils ) .
secondary sites serve as clonal generating stations for t and b lymphocytes , once the appropriate antigen ( from bacterial , viral or parasitic sources ) has been encountered .
thus , an army of identical and antigen - specific lymphocytes is uniquely directed at the offensive antigen , ensuring its elimination in normal animals , including humans .
cytotoxic t lymphocytes destroy virus - infected cells by making direct contact with the latter in order to induce cytolysis .
th1 cells , on the other hand , activate the appropriate accessory cell , and it is the latter that then delivers the cytolytic blow .
th1 cells are the target of the aids virus , and when the vital th1 cells are crippled throughout the body ( advanced aids ) , opportunist infections ultimately kill the infected host ( human or animal ) .
th2 lymphocytes activate b lymphocytes whose function is to produce an appropriate antibody against the offensive antigen , leading to its destruction and elimination .
in addition to being a requisite for adaptive immunity , the innate system is responsible for the early killing and clearance of infectious pathogens and the resolution of the inflammatory response .
the most important means by which the immune system communicates internally and externally is through the use of cytokines , small soluble signaling molecules .
cytokines play a crucial role in the selection , initiation and modulation of an appropriate immune response .
the signaling network of cytokines is characterized by its complexity ( synergy and antagonism ) and by its redundancy ( parallelism ) that ensure the speed and flexibility required for an effective immune response
. the immune system can give rise to several disorders when it is weakened or overactivated ( table 1 ) .
for instance , a defective adaptive immune response can lead to recurrent infection despite previous encounters with antigens , to lowered tolerance to self - organs and tissues leading to organ - specific autoimmunity , and to faulty recognition and elimination of transformed cells leading to cancer .
however , an overactivated innate immune response can cause chronic infection because of inefficient clearing of pathogen or chronic inflammation due to an inefficient regulation or resolution of the inflammatory response . chronic inflammation can lead to extensive non - specific destruction of neighboring tissue .
in addition , inflammation is increasingly found to be involved in the development of several chronic diseases such as arteriosclerosis , diabetes , neurodegenerative diseases and even cancer .
immune dysfunction has also been linked to conditions such as chronic pain , anxiety and depression , albeit sometimes as a consequence rather than a cause , and may be involved in other disease processes in a manner that is currently not fully understood ( 22 ) .
indeed , its diverse influence upon health may be in part due to its evolution as a defence mechanism and in part due to its origins , possibly as a sensory organ ( 23 ) .
a first question to address is whether the immune system is a good target to select in the fight to prevent chronic diseases .
in fact , it is accepted that the immune system represents our arsenal to combat infection and disease .
as mentioned , an underactivated immune system increases risks of opportunistic infection and uncontrolled neoplastic tissue growth , while an overactivated immune system leads to many afflictions , including inflammation , allergy and autoimmunity .
hence , an unchecked and unbalanced immune system hampers good health and quality of life .
yes given as an answer by the experts regarding the pertinence of targeting the immune system to prevent chronic disease .
however , it must be noted that the immune system carries out its important defensive function in intimate and coordinated interaction with the nervous and endocrine systems ( 24,25 ) .
the innate complexity of the immune system and of its communication tool , the cytokines , is thus further complicated by these interactions with the nervous and endocrine systems .
some concern also arises when considering that targeting the immune system may be too reductionist an approach , particularly when dealing with complex pathologies like cancer .
these considerations form the basis of the but part of the answer given in the title to this subsection .
it is important to consider that invertebrate species lack adaptive immunity but are clearly able to deal effectively with infections .
it is also interesting to note that these organisms do not appear to develop cancer or autoimmune disease ( at least not in any readily recognizable form ) , but they do demonstrate senescence ( 2628 ) . therefore , the apparent absence of these age - related diseases in invertebrates could support an evolutionary argument to focus disease prevention upon maintenance of efficient and equilibrated innate immunity .
if this line of reasoning is correct , then the immune cells of greatest interest are ones of the nk cell lineage described above .
these immune cells are present in invertebrate and vertebrate species and serve as a link between innate and adaptive immunity in vertebrates , as mentioned ( 25 ) .
indeed , nk cell function has been identified as a reliable measure of immunotoxicity for use in pharmaceutical safety testing ( 29 ) and could perhaps provide a means to monitor immune system equilibrium . another important point to consider relates to the problem of markers for immune system function .
it is difficult to reach a consensus on the appropriate markers to use in order to monitor the maintenance of a healthy immune system , primarily because biological heterogeneity precludes readily identifiable thresholds for concern in the prevention of chronic disease .
however , there exist potential novel avenues to explore , including the use of nk cell activity , as mentioned above , or the use of a constellation of markers , of which cytokines and cytokine receptors would be a major part .
much has been written about the validity and validation of biomarkers for health monitoring ( 30 ) . while it is beyond the scope of this paper to discuss these issues at length ,
firstly , biomarkers must be relevant to the question being asked and practical to use in the desired setting since advances in analytical sensitivity can now provide detection limits at concentrations often below those that have biological importance ( 31 ) .
secondly , it is generally easier to define thresholds for intervention ( i.e. when a disease requires treatment ) than for early detection or prevention ( i.e. before onset of the disease ) .
indeed , thresholds for disease prevention require a good definition of what constitutes the normal condition .
however , biological heterogeneity and the redundancy present within the immune system makes the establishment of precise
although nk cell activity and cytokine assays can be used as reliable measures for treatment intervention ( 32,33 ) they have yet to be shown to be practical biomarkers for defining a threshold for the early detection of chronic illnesses . finally , one interesting alternative to conventional immune system biomarkers is the presence and quality of microparticles present in the blood .
such microparticles , including exosomes , originate from several cells , including some prominent immune cells , and may represent a novel type of intercellular signaling mechanism ( 34,35 ) .
hence , research into the development of novel and pertinent biomarkers will certainly contribute to finding ways to modulate the immune system and prevent certain chronic diseases .
in this section we briefly review several experimental models and approaches that have been used to study immunomodulatory nhps . table 2 summarizes the advantages and limitations of each modality .
human clinical studies remain the gold standard upon which scientists and health care professionals can rely to determine the usefulness and efficacy of a given nhp .
nonetheless , animal models , in vitro bioassays , and modern proteomic and genomic technologies can be most helpful in delineating the mode of action of nhps .
finally , epidemiological studies bring a wealth of information on the influence of genetic ( germ line ) and environmental factors on health outcomes in the general population and pertinent subgroups . in addition
, it is generally recognized that a great effort should be placed on the standardization of methods used to study immune modulation by nhps . in terms of herbals ,
as mentioned in table 2 above , the greatest inconsistencies of clinical research related to immunomodulatory nhps are caused in major part by the lack of quality control of the source materials used ( 36 ) .
the us agency nccam came to a similar conclusion after its first 5 years of functioning and is now focusing on more preclinical studies to prepare better clinical studies , notably with herbal products ( ) .
during the last century , modern medicine has contributed major advancements to sanitation and public health initiatives and discoveries like antibiotics and vaccination .
it is in the realm of chronic diseases that modern medicine and drugs have been more disappointing and that nhps have elicited resurgent interest . in this section ,
we review the evidence for the immunomodulatory action of several nhps , including probiotics , products of animal origin ( principally milk and thymic extracts ) , nutritional factors ( especially vitamins , minerals and fatty acids ) and herbal products .
table 3 summarizes the strongest evidence brought forward for some of the most promising nhps .
the gastrointestinal ( gi ) tract and the skin epidermis are the major barriers humans possess to confront the external environment .
both are responsible for maintaining systemic integrity and use lymph nodes as efficient immune checkpoints .
the gi tract distinguishes itself by holding the greatest number of lymph nodes , and also by being the most intricate and complex immune organ .
they have received a lot of research attention , particularly in clinical studies in children and the elderly ( 3942 ) .
indeed , they positively affect general health going from the development of the immune system in infants right after birth to the enhancement of general immunity in the elderly ( critically exposed to immunosenescence ) .
probiotics offer much promise because they appear not only to help with the maturation of the visceral immune system but also to equilibrate the body 's defence mechanisms . in that sense
, they act as true immunomodulating agents , enhancing immunity when it is weakened or developing , and buffering exaggerated reactions ( e.g. allergies and inflammatory bowel disease ) .
they also represent the only nhp category that research has shown to have well established curing as well as preventive properties .
probiotics exert their actions principally by
improving the intestinal immunologic barrier function through spatial exclusion of other pathogenic microbial that could otherwise colonize the gut ( 43,44).restoring normal intestinal permeability ( 45 ) in the face of inflammatory or allergic diseases by still ill - defined mechanisms that include cell wall and dna components playing the role of immunomodulatory products ( 46 ) and by lowering the production of pro - inflammatory cytokines ( 47,48).synthesizing vitamins ( folic acid , biotin and other group b vitamins , vitamin k and so on ) ( 116 ) .
suggestions have been made that probiotics may absorb heavy metals ( 4951 ) and thus help their elimination through the feces .
probiotics may also promote the production of enzymes that help decrease the severity of certain food intolerances / allergies ( 52 ) .
improving the intestinal immunologic barrier function through spatial exclusion of other pathogenic microbial that could otherwise colonize the gut ( 43,44 ) . restoring normal intestinal permeability ( 45 ) in the face of inflammatory or allergic diseases by still ill - defined mechanisms that include cell wall and dna components playing the role of immunomodulatory products ( 46 ) and by lowering the production of pro - inflammatory cytokines ( 47,48 ) . synthesizing vitamins ( folic acid , biotin and other group b vitamins , vitamin k and so on ) ( 116 ) .
data on milk peptides , bovine colostrum and thymic extracts are less convincing in terms of immune modulation , most of the research having been done in cellular models in vitro and showing contradicting results ( 5364 ) .
several peptides released from enzymatic hydrolysis of milk proteins were reported to modulate immune functions such as phagocytosis , lymphocyte proliferation and cytokine production .
the type of enzymatic treatment has an influence on the immunomodulatory functions of peptides generated from milk proteins and , without standardized procedures , it is difficult to drawn conclusions from those studies .
further research , especially clinical trials , will be required to identify the impact of those products on the immune system in humans .
however , stronger evidence was presented to support the usefulness of a number of micronutrients to prevent certain chronic illnesses . a new term coined
nutraprevention is applied to the use of empirical combinations of foods , spices and other food - grade herbals in the fight against cancer ( 65 ) .
so far , this approach is based on epidemiological studies clearly showing relationships between cancer prevalence and food consumption in different populations around the world .
moreover , research is now showing potent antiangiogenic activity for polyphenolic components of green tea ( 6668 ) ; angiogenesis being the process by which growing tumors promote the development of new blood vessels to ensure their survival .
several plant constituents , including soy isoflavones ( 69,70 ) and compounds in cruciferous vegetables ( 71,72 ) , are also known to modulate enzymes involved in xenobiotic biotransformation , namely , the activation or detoxification of foreign , pro - carcinogenic chemicals .
vitamin c has been clearly linked to the modulation of immune function , particularly in the context of novel research showing improved cognitive status in patients with neurodegenerative disorders ( 73 ) , which are increasingly shown to involve an immune dysfunction component .
similarly , polyunsaturated fatty acids , in particular the now famous -3 fatty acids , have been used successfully in the management and prevention of several inflammatory and allergic diseases ( 7476 ) . however
, clinical research on lipids has yielded inconsistent results concerning their potential to modulate immune system function .
this state of affairs is related to poor study design and to the great variability in immune cells selected and methods used to assess immune function .
intake of green tea , brassica vegetables ( e.g. broccoli , cabbage and others ) and soy foods has been shown to be associated with lower risk of several cancers in epidemiologic studies [ reviewed in ( 7782 ) ] .
furthermore , data from experimental studies suggest that green tea and its constituents , such as isoflavones , may modulate markers of inflammation and immune function ( 8387 ) . a limited number of herbal products also show promise in the realm of immune modulation .
it is notably the case with echinacea that has received significant scientific attention in the past two decades , particularly in studies on upper respiratory tract infections ( 36,88,89 ) . among its many immune modulating activities
however , novel mechanisms of action include effects on b and t lymphocytes as well as nk cells ( 90,91 ) .
in particular , currier and miller ( 90 ) observed that upon incorporating echinacea in the daily diet of elderly mice for only 14 days nk cell production in the bone marrow , as well as the number of mature functional nk cells reaching the spleen , increased to levels found in young adults ( 92 ) .
brousseau and miller ( 93 ) have also revealed that daily dietary intake of echinacea throughout life , beginning in youth , increased the number of individuals reaching old age to 74% as opposed to 46% in control animals .
thus , any herbal treatment that would augment nk cells would clearly be worthy of investigation for its therapeutic / prophylactic potential , especially in the realm of immune modulation .
indeed , it appears to have both immunostimulant and immunosuppressant activities , which would lend credence to the view that ginseng is an
immunostimulant activities have been attributed to polysaccharide fractions , notably acting on both macrophages and b lymphocytes ( 94,95 ) .
however , ginsenosides display immunosuppressant activity in vitro by inhibiting cytokine release in activated macrophages ( i. lemaire , unpublished results ) .
hence , further research is warranted on ginseng and other adaptogenic herbals to define their potential use in preventing chronic diseases related to immune dysfunction .
dysfunction of the immune system is clearly implicated in the development of several chronic diseases . hence , attempting to maintain an efficient and equilibrated immune system is a valid approach to prevent certain chronic illnesses . however , this approach is limited by the great complexity of the immune system and its close interactions with the nervous and endocrine functions .
this is further hampered by the difficulty in identifying reliable and convenient analytical tools to assess immune function and its modulation . in that context ,
interesting areas for future research include nk cells , constellation of cytokine markers and receptors and assessment of blood - borne microparticles . despite these limitations , research on nhps
has shown that certain products offer novel avenues to modulate the immune system and help prevent chronic diseases .
the most promising ones are probiotics , nutraprevention ( combinations of biologically - active fruits , vegetables and spices ) and so - called adaptogenic plants ( e.g. echinacea and ginseng ) . notwithstanding these encouraging results , future research efforts with nhps will need to address the important issues of standardization in both product quality and research methodology . among areas to favor in the research agenda on nhps and immune modulation , this review highlights the following needs : ( i ) studies on the interaction between nhps and immune cells , particularly the influence of nhps on the innate immune system ; ( ii ) epidemiological studies and clinical studies with nhps in chronic illnesses where immune dysfunction may play a role .
finally , social / economic impact of nhps in health care must also be considered .
health effects of imbalances of the immune system tools to study nhps as related to the immune system promising nhps exhibiting modulatory effects on the immune system | the immune system is increasingly found to be involved in the development of several chronic illnesses , for which allopathic medicine has provided limited tools for treatment and especially prevention . in that context , it appears worthwhile to target the immune system in order to modulate the risk of certain chronic illnesses .
meanwhile , natural health products ( nhps ) are generating renewed interest , particularly in the prevention and treatment of several chronic diseases .
over 20 scientists from fields related to immune function and nhps were thus convened to establish the state of knowledge on these subjects and to explore future research directions .
this review summarizes the result of discussions held during the symposium .
it thus seeks to be thought provoking rather than to comprehensively cover such broad areas of research .
notably , a brief overview of the immune system is presented , including potentially useful targets and strategies to keep it in an equilibrated state , in order to prevent certain disorders .
the pertinence and limitations of targeting the immune system to prevent chronic diseases is also discussed .
the paper then discusses the usefulness and limitations of current experimental tools available to study the immune modulating effects of nhps .
finally , a concise review of some of the most studied nhps showing promising immunomodulatory activity is given , and avenues for future research are described . |
renewable energy from biomass has the potential to reduce dependency on fossil fuels , in addition to combat the environmental issues . the energy policy act of 2005 mandates blending of 7.5 billion gallons of alternative ( biofuel ) fuels by 2012 . the biofuels such as ethanol and biodiesel available in the market are predominantly produced from corn , sugar cane , and soybean oil .
it has been reported that biomass and bioenergy provides only about 4% of the total primary energy used in the us .
lignocellulosic materials are the most abundant renewable resources on earth . according to kadam and mcmillan , about 80100 dry tons of corn stover / year
corn stover was the most researched biomass and it alone might not be able to fulfill the fuel requirement of the us .
warm season grasses such as switchgrass , big bluestem , and prairie cord grass are grown in most part of the nation . in general , the warm season grasses have higher sugar content than cool season grasses .
several studies have indicated that these warm season grasses have greater potential as feedstock to produce biofuels [ 68 ] .
the sugars in lignocellulosic materials mostly exist as polysaccharides such as cellulose and hemicellulose , which are not readily available for hydrolytic enzymes .
pretreatment is an inevitable first step in the conversion of biomass to biofuels and the most expensive step too .
the focus of pretreatment research is to develop processes that would reduce the bioconversion time , lower the cellulase enzyme usage , and increase ethanol yields .
several pretreatment methods such as dilute acid , alkali , organic solvents , wet oxidation , ammonia fiber explosion , hydrothermal ( water , steam ) , milling , and irradiation have been investigated with varying degrees of success for the past three decades .
no perfect pretreatment method has been established to produce biofuels from biomass on commercial scale .
extrusion might be a viable continuous pretreatment method , which has many advantages such as high shear , rapid mixing , short residence time , moderate barrel temperature , no furfural and hmf formation , no washing and conditioning , and adaptability to process modification .
a few extrusion pretreatments showed a significant improvement on sugar recovery from different biomasses such as corn stover [ 11 , 12 ] , miscanthus , switchgrass , prairie cord grass , big bluestem [ 14 , 15 ] , and douglas fir through enzymatic hydrolysis .
enzyme loading is an expensive input in biomass conversion [ 1719 ] ; hence , enzyme usage should be as low as possible .
typically enzymatic hydrolysis of cellulose is carried out using cellulase a complex system consists of three enzymes , namely , endoglucanase , exoglucanase , and cellobiase that act synergistically .
the cellobiase ( -glucosidase ) supplementation with cellulase enzyme was necessary to eliminate the inhibition effect of cellobiose [ 20 , 21 ] .
the removal of cellobiose by -glucosidase results in the absence of product inhibition [ 22 , 23 ] , thus , hydrolysis can be achieved at reduced enzyme levels .
reported a glucose conversion of 78.581.2% with complementation of -glucosidase whereas it was only 57.8% without complementation of -glucosidase when corn stover was pretreated using wet oxidation .
a wide range of enzyme dosage and hydrolysis conditions has been reported in literature for different biomasses depending on pretreatment . due
to variation in enzyme activity and its dose , making meaningful comparison between pretreatment methods is very difficult .
hence , there is a need to determine the suitable enzymes and their doses for economic reasons .
the objective of the current study is to address the two most expensive steps such as pretreatment and enzymatic hydrolysis in the biomass conversion process .
the first objective is to evaluate the effect of screw speed and barrel temperature on sugar recovery from warm season grasses such as switchgrass , big bluestem , and prairie cord grass .
the second objective is to select a suitable enzyme combination and their ratio for the enzymatic hydrolysis of pretreated switchgrass , big bluestem , and prairie cord grass .
switchgrass , big bluestem , and prairie cord grass are three major warm season grasses among the big four native grasses ( other : indiangrass ) in south dakota with potential as biomass feedstocks .
switchgrass , big bluestem , and prairie cord grass obtained from a local farm were ground in a hammer mill ( speedy jr , winona attrition mill co , mn ) using 4 mm sieve for further pretreatment .
the moisture content of ground biomass was adjusted to 21% ( w.b ) based on preliminary study .
extrusion was performed in a single screw extruder ( brabender plasti - corder extruder model pl2000 , hackensack , nj ) , which had a compression ratio of 3 : 1 , barrel length to screw diameter ratio ( l / d ) of 20 : 1 . in order to have a smooth biomass ( plug )
flow into the die section , the screw discharge end was fitted with conical metal piece .
the single screw extruder was fitted to a 7.5 hp motor , which had a provision to adjust the screw speed from 0 to 210 rpm .
the screw speed of the extruder was maintained at 100 , 150 , and 200 rpm during the extrusion of biomass samples .
the extruder barrel had provisions to control the temperature of the feed and transition zone in both barrel and die section . the transition zone and die section temperature of barrel was maintained between 50 , 75 , 100 , 150 , and 200c for different screw speeds depending upon the biomass .
the temperature inside the barrel and the speed of the screw were controlled by a computer ; and feeding to the extruder was done manually .
about 500 g of prairie cord grass was extruded under each pretreatment condition , divided into two batches accounting for variations due to extruder operation , and considered replicates .
the mean residence time varied between 30 and 90 sec depending upon the screw speed .
the power consumption for extrusion pretreatment of different feedstocks was measured clamp meter ( amprobe acd-4 , everett , wa ) .
the enzymatic hydrolyses were conducted in hungate glass tube ( bellco glass , inc , nj , usa ) with 0.3 g dry weight of pretreated biomass in a solution of citrate buffer ( 0.05 m , ph 4.8 ) and sodium azide ( 0.02 gl ) to maintain constant ph and to inhibit microbial contamination , respectively . in order to select an enzyme combination and ratio , multienzyme ( activity 100 fbgg ) , cellulase ( activity 70 fpug ) with -glucosidase ( activity 250 cbug ) in the ratio of 1 : 1 and 1 : 4 was added to the pretreated corn stover .
the amount of cellulase was maintained at 15 fpu / g of dry matter .
multienzyme complex consists of arabinose , -glucanase , cellulase , hemicellulase , pectinase , and xylanase , which has the ability to liberate bound materials and can degrade a variety of nonstarch polysaccharides .
hydrolysis was carried out for 72 hours at 50c and 150 rpm as described by selig et al . .
after hydrolysis , the samples were kept in boiling water for 10 min to inactivate the enzyme action .
the supernatant was centrifuged at 13000 rpm for 15 min and then frozen twice before injecting into the hplc to remove the impurities which contribute to the pressure increase in the hplc system .
soluble sugars and byproducts were quantified using hplc ( agilent technologies , santa clara , ca ; bio - rad aminex 87h column hercules , ca ) with a mobile phase of 0.005 m h2so4 at a flow rate of 0.6 ml / min at 65c and a sample volume of 20 l as mentioned by sluiter et al . .
ground biomass was also subjected to enzymatic hydrolysis and analyzed as the control . the sugar concentration obtained from chromatogram
was divided by dry weight of biomass taken for enzymatic hydrolysis in order to calculate the percentage of different sugars with respect to total biomass .
glucose and xylose are the major sugars present in the biomass as compared to arabinose . instead of reporting arabinose separately
acetic acid and glycerol were the byproducts found in the pretreated samples and their concentration was reported in gl :
( 1)yi = sipsir100,(2)yc=sipsir100,yi : individual sugar recovery , % , yc : combined sugar recovery , % , sip : individual sugar obtained from pretreated samples through hplc , sir : individual sugar from raw material . individual sugar recovery , % , combined sugar recovery , % , individual sugar obtained from pretreated samples through hplc , individual sugar from raw material .
the moisture balanced big bluestem and prairie cord grass were extruded using a screw with 3 : 1 compression ratio at varying screw speeds of 100 , 150 , and 200 rpm and barrel temperatures of 100 , 150 , and 200c , which resulted in nine treatment combinations ( i.e. , 3 3 = 9 ) per each biomass .
the switchgrass was pretreated at three different screw speeds ( 100 , 150 , and 200 rpm ) and the barrel temperatures of 50 , 75 , 100 and 150c .
each treatment run was divided into two batches and the samples collected were considered as replicates .
the collected data were analyzed with proc glm procedure to determine the main , interaction and treatment effect in sas 9.1 ( sas institute , cary , nc ) using a type i error ( ) of 0.05 .
the switchgrass , big bluestem , and prairie cord grass composition such as glucose , xylose , arabinose , lignin , ash content on dry matter basis ( % ) were determined and given in table 1 .
in general , glucose is referred as cellulose and xylose , arabinose , galactose , and mannose are combined together and termed as hemicellulose .
the switchgrass used in this study had lower glucose content than the values reported by many researchers [ 21 , 2931 , 33 , 36 , 37 ] and comparable with that of doe 's and jefferson et al .
however , the xylose content was lower ; arabinose and lignin content were higher than the values of studies listed in table 1 .
the big bluestem composition was in agreement with the doe 's value except for ash content .
the glucose , xylose , and arabinose content were less than the reported values [ 6 , 3840 ] , whereas the lignin and ash content were higher than those studies .
prairie cord grass used in this study had a lower cellulose and hemicellulose compared to the values reported by boe and lee . the lower glucose and higher lignin content offered more resistance for any pretreatment method .
lignin played a critical role not only in plant growth and development but also in biomass utilization ; lignin restricted the degradation of structural polysaccharides through enzymatic hydrolysis .
in general , the chemical composition of any biomass varies from place to place depending upon the agronomic practices , season , and varieties .
the main effect analysis of screw speed on sugar recovery from switchgrass , big bluestem , and prairie cord grass is depicted in figures 1(a)1(c)through 3(a)3(c ) when cellulase , multienzyme , and -glucosidase was used with different ratios during hydrolysis . in general , the screw speed had a positive influence on sugar recovery from switchgrass regardless of enzyme combinations and their ratios used during hydrolysis as seen from figure 1 .
muthukumarappan and julson reported sugar recovery increasing trend for switchgrass pretreated using a twin screw extruder while varying the screw speed from 200 to 400 rpm .
although the trend was same , they achieved lower sugar recovery than that of the present study . the difference might be due to type of extruder and pretreatment conditions , apart from chemical composition of switchgrass . in the present study ,
the change in sugar recovery was insignificant when the screw speed was increased from 100 to 150 rpm ; however , further increase in screw speed had a significant increase only for glucose recovery when cellulase and -glucosidase was used with 1 : 1 ratio ( figure 1(a ) ) .
when amount of -glucosidase was increased by four times with cellulase amount kept constant , similar sugar recovery pattern was observed as that of 1 : 1 cellulase with -glucosidase ratio ( figure 1(b ) ) .
karunanithy et al . reported a similar trend for corn stover pretreated in a single screw extruder .
the glucose recovery increasing trend found in this study is in agreement with previous study of alkali - microwave pretreated switchgrass and bermudagrass ; however , the glucose recovery was less in the present study .
the possible reason for higher amount of glucose recovered might be due to delignification of switchgrass by alkali .
statistical analysis showed that the increase in sugar recovery across the screw speed was not significant when cellulase and -glucosidase was used at 1 : 4 ratio .
the glucose recovery was comparable between cellulase with different -glucosidase ratios , whereas xylose recovery differed among them . when multienzyme with -glucosidase was used during hydrolysis
the sugar recovery was less than 50% as compared to that of 1 : 1 ratio of cellulase with -glucosidase ( figure 1(c ) ) . when the screw was increased from 100 to 200 rpm , the glucose , xylose , and combined sugar recovery increased between 1630 , 623 , and 1425% , respectively , depending upon the enzyme combinations and their ratios .
these results showed that the rate of shear development was more important than the mean residence time .
as observed from figure 2 the sugar recovery from big bluestem increased with an increase in screw speed across the enzyme combinations and ratios .
the screw speed showed a significant difference on sugar recovery when 1 : 1 cellulase and -glucosidase was used ( figure 2(a ) ) .
as the screw speed was increased from 100 to 200 rpm , the glucose , xylose , and combined sugar recovery also increased by 28.3 , 57.4 , and 32.2% , respectively .
a similar result was reported by muthukumarappan and julson for big bluestem pretreated using a twin screw extruder while varying the screw speed from 200 to 400 rpm
. however , the sugar recovery obtained by these authors is less than that of the present study . the difference might be due to type of extruder and pretreatment conditions , apart from chemical composition of big bluestem .
higher sugar recovery was recorded when -glucosidase amount was increased by four times while cellulase amount was maintained at same level .
however , the increase in sugar recovery was not statistically different across the screw speeds as noticed in figure 2(b ) .
when multienzyme with -glucosidase was used at a ratio of 1 : 1 , the sugar recovery from big bluestem increased irrespective of the screw speeds .
the increase in glucose and combined sugar recovery was negligible when 1 : 1 multienzyme with -glucosidase was employed during hydrolysis ( figure 2(c ) ) whereas , 1 : 1 cellulase and -glucosidase showed significant increase on sugar recovery .
a significant improvement on xylose recovery was observed as the screw speed was increased from 150 to 200 rpm ( figure 2(c ) ) .
multienzyme with -glucosidase ( 1 : 1 ) resulted in a lower sugar recovery among the enzyme combinations and ratios studied , and it was almost less than 50% of cellulase with -glucosidase ( 1 : 1 ) . these results showed that higher screw speed is required to disturb the cell wall structure of the big bluestem . the influence of screw speed on sugar recovery from prairie cord grass with different enzymes and ratios are depicted in figure 3 . in general , the sugar recovery decreased with an increase in screw speed when cellulase with -glucosidase was used during hydrolysis , whereas sugar recovery increased with screw speed when multienzyme with -glucosidase was used .
the sugar recovery slightly increased as the screw speed was increased from 100 to 150 rpm ; further increase in screw speed decreased the sugar recovery from prairie cord grass ( figure 3(a ) ) .
however , the change in sugar recovery was not statistically different across the screw speed .
as the amount of -glucosidase was increased by four times correspondingly the sugar recovery also increased .
the increase in glucose recovery might be attributed to the higher amount of -glucosidase , which effectively broke down the cellobiose to glucose .
a remarkable increase in xylose recovery was noted when -glucosidase amount was increased as evident from figure 3(b ) .
the statistical analysis showed that the decrease in sugar recovery across the screw speeds was not significant .
these results showed that any screw speed could be selected for the pretreatment of prairie cord grass among the screw speeds studied . when cellulase was replaced with multienzyme ,
the sugar recovery decrease might be due to lower amount of cellulase present in the multienzyme .
the screw speed showed a positive influence on glucose and combined sugar recovery from prairie cord grass when multienzyme with -glucosidase was used at a ratio of 1 : 1 , but screw speed had no influence on xylose recovery ( figure 3(c ) ) .
figures 1(d)1(f)through 3(d)3(f ) represent the effect of barrel temperature on sugar recovery from different biomasses studied when cellulase , multienzyme was added with -glucosidase during hydrolysis .
the temperature had a significant effect on sugar recovery regardless of enzyme combinations and their ratios , except 1 : 1 cellulase to -glucosidase ratio . among the temperatures studied ,
the maximum sugar recovery was obtained at a temperature of 75c . statistical analysis of sugar recovery showed that the difference was not significant across the barrel temperatures when 1 : 1 cellulase to -glucosidase ratio was employed during hydrolysis ( figure 1(d ) ) . when -glucosidase amount was increased by four times ,
a similar sugar recovery pattern was observed as compared to cellulase and -glucosidase at a ratio of 1 : 1 .
the sugar recovery initially increased by 25% when temperature was increased from 50 to 75c , and further increase in the barrel temperature reduced the sugar recovery ( figure 1(e ) ) .
the glucose recovery was higher with cellulase to -glucosidase ratio of 1 : 4 compared to that of 1 : 1 ratio .
when multienzyme was used instead of cellulase , the sugar recovery increased with an increase in temperature .
the maximum sugar recovery was noticed at a barrel temperature of 75c ( figure 1(f ) ) and it was similar to other enzyme combinations .
glucose , xylose , and combined sugar recovery increased by 19 , 103 , and 36% , respectively , when the barrel temperature was increased from 50 to 150c while multienzyme and -glucosidase was employed at ratio of 1 : 1 . the sugar recovery
was lowered by 45% when multienzyme and -glucosidase was used during hydrolysis compared to cellulase and -glucosidase with 1 : 1 ratio .
these result showed that the cell wall disturbance was maximum at 75c ; further increase in temperature might have contributed for thermal softening of switchgrass .
the present study results were contrary to the results reported for switchgrass pretreated in a twin screw extruder at a screw speed of 200 rpm while the temperature was increased from 25 to 100c .
this might be due to the difference in type of extruder , pretreatment conditions , and chemical composition of switchgrass .
the barrel temperature had a strong influence on the sugar recovery from big bluestem when cellulase and -glucosidase was used at a ratio of 1 : 1 as evident from figure 2(d ) .
as the barrel temperature was increased from 100 to 200c , the glucose , xylose , and combined sugar recovery correspondingly increased from 25.8 , 12.2 , 21.1 to 45.9 , 33.9 , and 40.5% , respectively . as the -glucosidase amount was increased by four times while cellulase amount was kept constant , the sugar recovery increased with an increase in barrel temperature .
higher sugar recovery with higher -glucosidase might be due to higher cellobiose broken into glucose .
a remarkable increase in xylose recovery was noticed between cellulase with different ratios of -glucosidase ( figures 2(d ) and 2(e ) ) .
statistical analysis showed a significant difference on sugar recovery between 100 and 150c , further increase of barrel temperature resulted in negligible sugar recovery increase as seen from figure 2(e ) .
when multienzyme and -glucosidase was used at a ratio of 1 : 1 , the sugar recovery pattern was different than the cellulase and -glucosidase combination .
the barrel temperature had a significant influence on xylose recovery , whereas temperature had no influence on glucose recovery as noted from figure 2(f ) .
these results showed that irrespective of enzyme combination and ratios , higher barrel temperature was required for higher sugar recovery from big bluestem . in contrary to an increasing trend with barrel temperature ,
a decreasing trend was reported by muthukumarappan and julson for big bluestem pretreated in a twin screw extruder when the barrel temperature increased from 25 to 100c at a screw speed of 200 rpm .
the glucose recovery from the present study was higher ( 37.1% ) compared to muthukumarappan and julson 's result ( 27.2% ) when big bluestem was pretreated at a barrel temperature of 100c and screw speed of 200 rpm ; the difference might be due to type of extruder and the chemical composition of big bluestem .
the barrel temperature negatively influenced the sugar recovery from prairie cord grass when cellulase and -glucosidase was used for hydrolysis .
the higher sugar recovery was obtained at lower barrel temperature as evident from figure 3(d ) .
when the temperature increased from 100 to 150c , the glucose , xylose , and combined sugar recovery was decreased by 31.7 , 27.9 , and 29.8% , respectively , and further increase of barrel temperature resulted in small increase of sugar recovery .
it was noted that xylose recovery was maximum at 200c whereas glucose and combined sugar recovery was maximum at 100c .
in general , the sugar recovery from prairie cord grass increased when the amount of -glucosidase was increased by four times ( figure 3(e ) ) which indicates the higher amount of -glucosidase enhanced / facilitated in the conversion of cellobiose to glucose .
xylose recovery showed a remarkable increase of four times when compared to cellulase with -glucosidase ( 1 : 1 ) . the barrel temperature had a negative impact on sugar recovery from cord grass .
. when multienzyme and -glucosidase was used at a ratio of 1 : 1 , the sugar recovery decreased considerably when compared to cellulase and -glucosidase at 1 : 1 ratio .
a different sugar recovery pattern was observed with 1 : 1 multienzyme and -glucosidase was employed during hydrolysis ( figure 3(f ) ) .
the barrel temperature of 100 and 150c had no influence on glucose recovery , whereas the glucose recovery decreased at 200c .
similar to glucose recovery , the lower temperature had no effect on xylose recovery , but the higher temperature showed a significant difference in xylose recovery . in contrary to cellulase enzyme combination ,
these results showed that the lower barrel temperature would result in higher sugar recovery with cellulase and -glucosidase combination . the screw speed and barrel temperature interaction effect on sugar recovery from switchgrass , big bluestem , and prairie cord grass when different enzyme combinations and ratios were used during hydrolysis
when cellulase or multienzyme with -glucosidase was used at a ratio of 1 : 1 , the temperature interaction with screw speed was different for switchgrass as evident from table 2 .
although temperature and screw speed as an independent variable had a significant effect on combined sugar from switchgrass , their interaction was not significant . a different interaction pattern was observed for big bluestem .
when 1 : 1 cellulase and -glucosidase combination was employed for hydrolysis , the temperature , screw speed , and their interaction had a significant impact on glucose , xylose , and combined sugar recovery . as the -glucosidase amount was increased , only temperature had a significant influence on sugar recovery .
the screw speed and temperature interaction was significant only for xylose recovery from big bluestem when multienzyme combination was used during hydrolysis .
although temperature had a significant influence on combined sugar recovery for big bluestem when it interacted with screw speed , the influence was negligible .
the barrel temperature and screw speed interaction was not significant on sugar recovery from prairie cord grass when cellulase enzyme combination was employed during hydrolysis . when multienzyme combination was used for hydrolysis , the interaction effect was significant for glucose and combined sugar recovery from prairie cord grass .
although temperature as an independent variable had no significant effect on combined sugar recovery from cord grass , when it interacted with screw speed the effect turned to be significant .
statistical analyses across the treatment combinations are presented in tables 3through 5 for different enzymes and ratios employed during hydrolysis of different biomasses .
the temperature , screw speed , and their interaction had influence on sugar recovery from switchgrass as confirmed from statistical analysis . in general , the barrel temperature of 75c showed higher sugar recovery from switchgrass ( table 3 ) across the screw speeds regardless of enzyme combinations and ratios .
the maximum glucose , xylose , and combined sugar recovery of 38.7 , 18.2 , and 28.2% , respectively , was obtained at a screw speed of 200 rpm and barrel temperature of 75c when cellulase and -glucosidase was employed at a ratio of 1 : 4 .
various pretreatments employed on switchgrass , enzyme dose , glucose , xylose , and total sugar yields are listed in table 6 .
the glucose recovery from switchgrass was less than that of the ammonia - water pretreatment results ( 55.2 and 43.7% ) for two switchgrass varieties ; 93% glucose from ammonia fiber expansion ; 91.4% glucose from dilute acid pretreatment and 7386% glucose from another dilute - acid pretreatment of switchgrass .
a glucose recovery of 68 and 87% for switchgrass pretreated in a combination of alkali and rf heating , alkali and microwave heating was reported by hu and wen and hu et al .
it is a well - known fact that alkali removes the lignin and acid solubilizes the hemicellulose thereby increasing the accessibility for the enzymes resulting in higher glucose recovery . as inferred from table 4 that the higher screw speed combined with higher temperature resulted in higher glucose , xylose , and combined sugar recovery from big bluestem .
however , the maximum glucose ( 57.5% ) , xylose ( 66.2% ) , and combined sugar recovery ( 57.6% ) from big bluestem were achieved at a barrel temperature of 150c and screw speed of 200 rpm when cellulase to -glucosidase ratio was maintained at 1 : 4 .
this result was higher than the glucose availability of 27.2 and 26.8% reported for 20% moisture content big bluestem extruded in a twin screw extruder at a barrel temperature of 100c with 200 and 400 rpm , respectively .
no regular trend was observed on sugar recovery from prairie cord grass ( table 5 ) .
a maximum glucose , xylose , and combined sugar recovery of 41.1 , 49.2 , and 44.6% , respectively , were recorded for prairie cord grass pretreated at a barrel temperature of 100c and screw speed of 150 rpm when cellulase and -glucosidase was employed at a ratio of 1 : 4 .
switchgrass and big bluestem showed a similar pattern ( increasing ) of sugar recovery with an increase in screw speed , whereas the sugar recovery pattern was different for prairie cord grass .
the sugar recovery from switchgrass showed an increasing trend with screw speed and it was similar to corn stover .
switchgrass and big bluestem exhibited an increasing sugar recovery trend with temperature increase from 100 to 150c .
the glucose , xylose , and combined sugar recovery differed among switchgrass , big bluestem , and prairie cord grass , which might be due the difference in their chemical composition and inherent nature of biomasses .
the lowest and highest sugar recovery was recorded for switchgrass ( 38.7 , 28.2% ) and big bluestem ( 57.5 , 57.6% ) , respectively . the lowest sugar recovery from switchgrass
might be attributed to the highest lignin content among the biomasses studied . in general ,
an increase in glucose recovery with -glucosidase from the biomasses studied was in agreement with microwave - alkali pretreated switchgrass and bermudagrass reported by keshwani .
glycerol and acetic acid were the byproducts found in a few pretreated switchgrass , big bluestem , and prairie cord grass samples .
the concentration of glycerol and acetic acid were in the range of 0.020.05 and 0.020.04 gl , respectively , recorded for switchgrass at a barrel temperature of 50c with screw speeds of 100 and 150 rpm .
the maximum concentration of glycerol and acetic acid was 0.03 and 0.04 gl , respectively , and was recorded for big bluestem at a screw speed of 150 rpm and barrel temperature of 100c .
the concentration of glycerol and acetic acid was similar to switchgrass but occurred at higher temperature and screw speed .
in contrary to other extrusion pretreatments of corn stover , miscanthus and douglas fir wood , acetic acid and glycerol were found in this study .
interestingly no furfural and hmf were found in any of the pretreatment conditions studied , which was in agreement with other extrusion pretreatments performed on different biomasses [ 11 , 13 , 16 ] .
the extruder has a power source of 7.5 hp ( 5595 w ) .
there are 4 heaters per extruder zone at 250 w each , so zone 1 = 1000 w and zone 2 = 1000 w. thus , the total 20 : 1 l / d extruder wattage is 2000 w. the total rated power to run this extruder for an hour is 7595 watt - hour . the ideal / no load drive and heater power consumption are 1.4 and 0.8 amps , respectively .
the actual drive power consumption is 3.5 , 4.4 , and 4.1 amps and heater power consumption is 1.6 , 2.4 , and 2 amps , respectively , for big bluestem , prairie cord grass , and switchgrass . for three phase alternate current , w = 3vi , where v is voltage ( 240 v ) and i is current in amps .
the total energy consumption for extrusion pretreatment of big bluestem , prairie cord grass , and switchgrass is 2120 , 2826 , and 2515 w , respectively , which is 27.9 , 37.2 , and 33.1% of the rated drive and heater power .
this extruder can pretreat 2 - 3 kgh depending on the types of feedstock .
amount of ethanol production can be calculated based on the availability of glucose in the raw feedstock and glucose recovered through extrusion pretreatment . assumping a thumb rule is 50% of the glucose will be converted into ethanol during fermentation with an efficiency of 90% .
ethanol density and its energy content are 0.79 g / ml and 21.1 mjl , respectively ( 1 mj = 278 watt - hour ) .
for example , glucose recovery of 57.5 , 38.7 , and 41.1% recorded for big bluestem , switchgrass , and prairie cord grass , respectively , would result in 1349 , 909 , and 809 watt - hour .
researchers across the world are working on utilizing hemicellulose fraction ; once the technology is developed , one can expect an additional energy of 60% that would narrow down the difference between input and output .
however , extruder used in this study is a lab - scale one ; the efficiency would improve as a result of scale - up process , and thereby one can expect net positive energy balance . in conclusion , technology development for hemicellulose utilization and scale - up process would be need of the hour .
this experiment was conducted to understand the influence of screw speed and barrel temperature on sugar recovery from switchgrass , big bluestem , and prairie cord grass .
when different enzyme combinations and ratios were employed during hydrolysis , it was confirmed that screw speed , barrel temperature and their combinations significantly influenced the sugar recovery .
based on the highest glucose , xylose , and combined sugar recovery , switchgrass could be pretreated at a screw speed of 200 rpm with barrel temperature of 75c with cellulase and -glucosidase at a ratio of 1 : 4 .
the big bluestem could be pretreated at a screw speed of 200 rpm with 150c , and prairie cord grass could be pretreated at a screw speed of 150 rpm and 100c while cellulase and -glucosidase was employed at a ratio of 1 : 4 . | biofuels from biomass have the potential to reduce the dependency on fossil fuels .
an efficient pretreatment method is required to accomplish the target of the energy act 2005 .
extrusion could be a viable continuous pretreatment method to be explored .
the objectives of the current study were to investigate the influence of screw speed and barrel temperature on sugar recovery from the selected warm season grasses and to select a suitable enzyme combination and dose for enzymatic hydrolysis .
the ground , moisture - balanced biomasses were pretreated using a single screw extruder at various screw speeds ( 100 , 150 , and 200 rpm ) and barrel temperatures ( 50 , 75 , 100 , 150 , and 200c ) .
cellulase or multienzyme with -glucosidase was varied from 1 : 1 to 1 : 4 during enzymatic hydrolysis to accomplish the second objective .
screw speed , barrel temperature , and their interaction had a significant influence on sugar recovery from the selected biomasses .
a maximum of 28.2 , 66.2 , and 49.2% of combined sugar recoverywasachieved for switchgrass , big bluestem , prairie cord grass when pretreated at a screw speed of 200 , 200 , and 150 rpm and at a barrel temperature of 75 , 150 , and 100c , respectively , using cellulase and -glucosidase at a ratio of 1 : 4 . extrusion pretreatment of these biomasses used only 2837% of the rated extruder power . |
actin filaments assemble into diverse three - dimensional arrays to execute a wide variety of processes in eukaryotic cells .
these actin filament arrays are characterized by distinct protein compositions and dynamic properties , but the underlying molecular mechanisms are incompletely understood [ 1 , 8 ] . to elucidate the principles by which functionally distinct actin filament populations can be generated in cells , we focused on analyzing stress - fiber - associated tropomyosin isoforms .
stress fibers are contractile actomyosin bundles that contribute to cell morphogenesis , adhesion , migration , and mechanosensing .
stress fiber assembly depends on several proteins promoting actin filament nucleation and polymerization , and different stress fiber types are decorated by partially non - overlapping sets of actin - binding proteins [ 7 , 10 , 11 , 12 , 13 , 14 ] .
six tropomyosin isoforms ( tpm1.6 , tpm1.7 , tpm2.1 , tpm3.1 , tpm3.2 , and tpm4.2 ) associate with stress fibers in osteosarcoma cells ( figures 1a and 1b ; figure s1a ) .
importantly , these isoforms display partially different localization patterns along stress fibers and , based on rnai experiments , are functionally non - redundant .
therefore , they may specify functionally distinct actin filament populations within the stress fiber network . however , whether distinct tropomyosin isoforms indeed segregate to different actin filaments has not been experimentally demonstrated .
moreover , the biochemical properties of actin filaments decorated by different tropomyosin isoforms have not been systematically studied . to examine the six stress - fiber - associated tropomyosin isoforms
, we expressed and purified them as non - tagged versions and as superfolder gfp ( sfgfp)- or mcherry - fusion proteins . in all cases ,
an alanine - serine extension was introduced to mimic amino - terminal acetylation . in the fluorescent fusion proteins ,
sfgfp / mcherry was linked to the n termini of the tropomyosins because such fusion proteins localize properly to the stress fiber network in cells and can rescue the knockout phenotype ( figure s1b ) .
moreover , a recent biochemical study demonstrated that an amino - terminal s. pombe tropomyosin fluorescent fusion binds actin filaments and forms similar end - to - end interactions compared to endogenous acetylated tropomyosin .
the actin filament co - sedimentation assay was first applied to examine the binding of non - tagged and sfgfp - tagged tropomyosins to non - muscle /-actin filaments .
consistent with earlier work , the stress - fiber - associated tropomyosins bound /-actin filaments with high affinity ( kd < 1 m ) and the n - terminal sfgfp fusion did not interfere with f - actin binding ( figure 1c ; figure s1c ) .
this was also supported by in vitro total internal reflection fluorescence ( tirf ) microscopy experiments demonstrating that the fluorescent fusions of tropomyosins decorated non - labeled /-actin filaments .
however , tropomyosins do not efficiently interact with rhodamine - labeled actin , because tropomyosins and rhodamine - actin segregated on filaments in experiments where only a small fraction ( 5% ) of actin monomers was labeled ( figure s1d ) . whereas sfgfp fusions of tpm1.6 , tpm1.7 , tpm2.1 , tpm3.1 , and tpm3.2 interacted strongly with individual actin filaments in the tirf microscopy assay , sfgfp - tpm4.2 bound only to actin filament bundles , induced either by methylcellulose or myosin ii , with high affinity ( figures s2a and s2b ; data not shown ) .
mechanistic principles by which tpm4.2 preferentially interacts with actin filament bundles ( at least in our tirf setup ) remain to be elucidated .
a simple interpretation is that , unlike the other tropomyosins , tpm4.2 promotes filament bundling and only interacts strongly with actin filaments that are bundled .
this property may be linked to its cellular function in myosin ii recruitment to stress fibers .
fluorescence recovery after photobleaching ( frap ) was applied to examine the dynamics of stress - fiber - associated tropomyosins on actin filaments .
the assay was first performed on individual actin filaments with tpm1.6 , tpm1.7 , tpm2.1 , tpm3.1 , and tpm3.2 .
interestingly , sfgfp fusions of tpm1.6 , tpm1.7 , and tpm2.1 displayed only very slow recovery on bleached actin filament segments , whereas the fluorescence recovery of sfgfp fusions of tpm3.1 and tpm3.2 on actin filaments was relatively rapid ( figures 1d and 1e ) . because tpm4.2 does not bind individual actin filaments with high affinity , the frap assay was also performed on actin filament bundles induced by methylcellulose . to confirm that we indeed measured the dynamics of actin - filament - associated tropomyosins ,
samples were spiked with 5% rhodamine - labeled actin to visualize the filament bundles .
importantly , sfgfp - tpm4.2 displayed similar rapid recovery on actin filament bundles compared to sfgfp - tpm3.1 and sfgfp - tpm3.2 , whereas the fluorescence recoveries of sfgfp - tpm1.6 , sfgfp - tpm1.7 , and sfgfp - tpm2.1 were very slow , further confirming the differences in the dynamics of actin filament association between these tropomyosins ( figures s2c and s2d ) .
together , these data reveal that the long stress - fiber - associated tropomyosin isoforms derived from the tpm1 and tpm2 genes display stable association with actin filaments , whereas the short isoforms derived from the tpm3 and tpm4 genes display dynamic association with actin filaments .
this correlates with the stronger cooperativity of actin binding observed with long tropomyosins compared with short isoforms .
we next asked whether the spatial segregation of tropomyosin isoforms is an intrinsic property of the tropomyosin proteins . to test this
, we examined whether different tropomyosin isoforms , when incubated in combination with each other and actin filaments , bind to same filaments or whether they segregate into different actin filaments or filament segments .
control experiments performed with sfgfp and mcherry fusions of the same tropomyosin isoform demonstrated that the two proteins with different fluorescent tags do not segregate along actin filaments .
this indicates that the n - terminal fluorescent fusions do not alter the ability of tropomyosins to form end - to - end oligomers .
please note that the number of tropomyosin - decorated actin filaments appeared to differ in the case of distinct tropomyosin isoforms .
this may arise from differences in the cooperativity of actin filament association between these tropomyosin isoforms .
importantly , when sfgfp and mcherry fusions of different tropomyosin isoforms were co - incubated with actin , the two proteins often segregated into different segments along the actin filaments ( figure 2a ; figure s3 ) .
quantification of co - localization indices of different tropomyosin pairs provided evidence that whereas the tropomyosin isoforms generated from the same gene ( tpm1.6 and tpm1.7 as well as tpm3.1 and tpm3.2 ) can co - polymerize on actin filaments , the isoforms generated from different tropomyosin genes are generally not capable of co - polymerizing on actin filaments and thus form segments along the filaments .
the only exception was tpm2.1 , which appeared to be able to co - polymerize with tpm3.1 and tpm3.2 but not with tpm1.6 and tpm1.7 ( figure 2b ) .
these data provide evidence that different tropomyosin isoforms can segregate into different actin filaments / filament segments and are capable of generating actin filament populations that are decorated by a specific tropomyosin isoform or a specific combination of tropomyosin isoforms .
we first approached this question by examining the effects of stress - fiber - associated tropomyosins on cofilin , because these two proteins have been shown to compete for f - actin binding both in vitro and in cells [ 5 , 18 , 19 , 20 ] .
the only exception reported so far is tpm1.2 , which , based on cell biological work , can associate with actin - depolymerizing factor ( adf)/cofilin on actin filaments .
co - sedimentation assays and in vitro tirf microscopy demonstrated that all stress - fiber - associated tropomyosin isoforms compete with cofilin for f - actin binding , and that the two proteins interact with filaments in a mutually exclusive manner ( figure 3a ; figure s4 ; data not shown ) .
dual - color tirf microscopy movies also demonstrated that in most cases , mcherry - cofilin clusters grew co - operatively along actin filaments toward the pointed end , simultaneously replacing sfgfp - tropomyosin from filaments ( figure 3b ; movie s1 ) .
these data are in agreement with recent high - speed atomic force microscopy work proposing that cofilin induces a unidirectional conformational change in actin filaments toward the pointed end of the cofilin - decorated segment , thus promoting the growth of a cofilin cluster toward the filament pointed end .
cofilin did not induce frequent severing of tropomyosin - decorated actin filaments in our tirf setup because non - labeled /-actin was used in our study , and severing events of bare filament segments could not be visualized .
thus , we examined the effects of tropomyosins on cofilin - mediated actin filament turnover by monitoring the decrease of pyrene - actin fluorescence resulting from filament disassembly in the presence of cofilin and vitamin d - binding protein ( dbp ) .
interestingly , the tropomyosin isoforms tested here had very different effects on actin filament disassembly by cofilin .
tpm2.1 , tpm3.1 , tpm3.2 , and tpm4.2 were relatively inefficient in protecting actin filaments from cofilin , and only two isoforms , tpm1.6 and tpm1.7 , efficiently protected actin filaments from cofilin - mediated disassembly ( figure 3c ) .
this is consistent with the tirf assays , suggesting that although all tropomyosins compete with cofilin for actin binding , tpm1.6 and tpm1.7 were the most efficient isoforms in competing with cofilin ( figures s4a and s4b ) .
similar to the non - tagged tropomyosins , also sfgfp - tpm1.6 and sfgfp - tpm1.7 protected actin filaments from cofilin more efficiently compared to other isoforms ( figure s4c ) .
tropomyosins and an actin filament cross - linking protein , -actinin , display typically mutually exclusive localizations in contractile actin filament structures such as myofibrils and stress fibers .
however , tpm1.6 co - localizes , at a light microscopy level , with -actinin in the non - contractile dorsal stress fibers of migrating cells .
thus , we performed actin filament co - sedimentation assays to test whether the stress - fiber - associated tropomyosins compete with non - muscle -actinin-1 for actin filament binding in vitro . in these assays , actin filaments were pre - incubated with tropomyosins , and the ability of -actinin to subsequently replace tropomyosins from filaments was examined .
the amount of all tropomyosin isoforms on actin filaments decreased in a concentration - dependent manner after addition of -actinin-1 , suggesting that all stress - fiber - associated tropomyosin isoforms compete with -actinin for actin filament binding ( figures 4a and 4b ; figure s4d ) .
the order in which the proteins were added on actin filaments did not affect their competition , because similar displacement was also observed when actin filaments were first incubated with -actinin-1 before addition of tropomyosins ( data not shown ) .
based on cryo - electron microscopy work , the binding sites of -actinin and tropomyosins on actin do not overlap [ 3 , 23 ] , and thus the molecular mechanism of competition between tropomyosins and -actinin for actin filament binding needs to be elucidated .
also , many non - muscle tropomyosin isoforms can affect the mgatpase activity and motility of myosins along actin filaments [ 24 , 25 , 26 , 27 , 28 ] .
however , the effects of different stress - fiber - associated tropomyosin isoforms on myosin ii have not been systematically compared . to elucidate how the decoration of actin filaments with different tropomyosin isoforms
influences their interaction with myosin ii , we measured the actin - activated atpase activity of non - muscle myosin iia heavy meromyosin ( nmiia - hmm ) during steady state .
these experiments revealed that whereas tpm4.2 , tpm3.1 , and tpm3.2 efficiently increased the actin - activated atpase activity of nmiia - hmm , the other tropomyosin isoforms had no detectable effect on myosin iia activity .
similar results were obtained in assays carried out with either skeletal muscle -actin or non - muscle -actin ( figures 4c and 4d ) .
together , these experiments demonstrate that actin filaments decorated by different tropomyosin isoforms display distinct functional properties .
filaments decorated with tpm1.6 and tpm1.7 do not activate myosin iia but efficiently protect filaments from cofilin - induced disassembly . on the other hand , tpm3.1 , tpm3.2 , and tpm4.2 stimulate the actin - induced atpase activity of myosin iia but do not efficiently protect actin filaments from cofilin .
the effects of tpm 3.1 and tpm4.2 on myosin iia activity are also consistent with cell biological data providing evidence that these tropomyosin isoforms can recruit myosin ii to stress fibers [ 6 , 7 ] .
interestingly , the functions of tropomyosins in filament stabilization versus myosin ii activation appear to correlate with the dynamics of their actin interactions .
this is because tpm1.6 and tpm1.7 display very stable association with actin filaments , whereas tpm3.1 , tpm3.2 , and tpm4.2 exhibit dynamic interactions with actin ( figure 4e ) . whereas other stress - fiber - associated tropomyosin isoforms either activate myosin ii or protect filaments from cofilin - mediated disassembly , tpm2.1 does not display strong effects on either of these activities .
we propose that this tropomyosin isoform regulates some other aspects of stress fiber assembly , dynamics , or contractility by controlling the activity or localization of another protein(s ) within the stress fiber network .
moreover , it is possible that tpm2.1 works only in concert with some other stress fiber proteins , such as caldesmon and calponins , in cells .
interestingly , tpm2.1 accumulates at focal adhesions at the distal ends of dorsal stress fibers , and recent studies demonstrated that it controls mechanosensitive sarcomere - like contractile units at these sites [ 7 , 29 ] . in the future
, it will be interesting to examine the interplay of different mammalian tropomyosin isoforms with a wider range of actin - binding proteins , including different filament cross - linkers , other myosins , the arp2/3 complex , and profilin , which in other organisms were shown to exhibit interplay with tropomyosins on actin filaments [ 30 , 31 , 32 , 33 ] .
please note that , although different tropomyosin isoforms display distinct localization patterns along non - contractile dorsal stress fibers , they all localize to myosin ii - containing foci in contractile ventral stress fibers .
thus , in addition to other actin - binding proteins , tropomyosins may also work in concert with each other to generate and maintain complex actin filament arrays in cells . in the context of the stress fiber network , it is possible that the non - contractile dorsal stress fibers need protection from adf / cofilin - induced filament disassembly and
actin filaments that are under myosin ii - generated tension are protected from adf / cofilins [ 14 , 34 ] . these myosin ii - associating actin filaments
may not , therefore , require tpm1.6 or tpm1.7 to inhibit their severing by adf / cofilins .
it is also important to note that filament severing by adf / cofilins is greatly enhanced by their co - factors , including coronin , and aip1 in cells , adding more complexity to the system .
our in vitro tirf microscopy experiments also provide the first evidence that most tropomyosins can not co - polymerize with each other on actin filaments but instead segregate into different filaments or filament segments .
thus , tropomyosins have the intrinsic ability to generate distinct actin filament populations that are decorated by a specific tropomyosin isoform(s ) , which subsequently can modulate the interactions of other proteins with these filaments
. these biochemical data also provide an explanation for distinct subcellular localizations of different tropomyosin isoforms within cellular actin filament networks of motile cells [ 7 , 36 ] .
this can arise from the chemical differences of tropomyosin n and c termini that either allow or prevent end - to - end association of different tropomyosin molecules . alternatively ( or in addition ) , this may involve different positioning of distinct tropomyosin isoforms on actin filaments or their effects on the conformation of actin filaments .
the latter alternative is supported by the fact that tropomyosins decorate the two structurally identical grooves along an actin filament , and thus their segregation can not be solely determined by head - to - tail associations ( i.e. , some coordination must exist between the two tropomyosin - binding grooves when filament segments decorated by only one tropomyosin isoform exist ) .
this may reciprocally also explain why tpm2.1 and tpm3.1/2 , which exhibit major differences in their n - terminal sequences , do not segregate to different actin filaments in our tirf assay .
furthermore , the mechanisms by which different tropomyosin isoforms are targeted to their specific cellular destinations remain to be elucidated .
good candidates for this task are formins , which at least in fission yeast can induce assembly of actin filament structures that are decorated by specific tropomyosin isoforms .
intriguingly , at least four different formins are linked to the assembly of the stress fiber network in motile cells [ 7 , 10 , 11 , 13 ] , and thus these formins may provide the link between actin filament nucleation and incorporation of specific tropomyosin isoforms into the different regions of the stress fiber network .
| summaryactin filaments assemble into a variety of networks to provide force for diverse cellular processes [ 1 ] .
tropomyosins are coiled - coil dimers that form head - to - tail polymers along actin filaments and regulate interactions of other proteins , including actin - depolymerizing factor ( adf)/cofilins and myosins , with actin [ 2 , 3 , 4 , 5 ] . in mammals ,
> 40 tropomyosin isoforms can be generated through alternative splicing from four tropomyosin genes .
different isoforms display non - redundant functions and partially non - overlapping localization patterns , for example within the stress fiber network [ 6 , 7 ] .
based on cell biological studies , it was thus proposed that tropomyosin isoforms may specify the functional properties of different actin filament populations [ 2 ] . to test this hypothesis
, we analyzed the properties of actin filaments decorated by stress - fiber - associated tropomyosins ( tpm1.6 , tpm1.7 , tpm2.1 , tpm3.1 , tpm3.2 , and tpm4.2 ) .
these proteins bound f - actin with high affinity and competed with -actinin for actin filament binding .
importantly , total internal reflection fluorescence ( tirf ) microscopy of fluorescently tagged proteins revealed that most tropomyosin isoforms can not co - polymerize with each other on actin filaments
. these isoforms also bind actin with different dynamics , which correlate with their effects on actin - binding proteins .
the long isoforms tpm1.6 and tpm1.7 displayed stable interactions with actin filaments and protected filaments from adf / cofilin - mediated disassembly , but did not activate non - muscle myosin iia ( nmiia ) .
in contrast , the short isoforms tpm3.1 , tpm3.2 , and tpm4.2 displayed rapid dynamics on actin filaments and stimulated the atpase activity of nmiia , but did not efficiently protect filaments from adf / cofilin .
together , these data provide experimental evidence that tropomyosin isoforms segregate to different actin filaments and specify functional properties of distinct actin filament populations . |
the
function of a parity generator / checker , which is an essential
operation for detecting errors in data transmission , has been realized
with multiphotochromic switches by taking advantage of a neuron - like
fluorescence response and reversible light - induced transformations
between the implicated isomers . | |
spontaneous dissection of the celiac artery with concurrent dissection of the splenic artery without abdominal aortic dissection is very rare .
spontaneous visceral artery dissection was first reported in 1947 , which occurs most frequently in the superior mesenteric artery and rarely in the celiac trunk . in the past ,
however , advances in diagnostic tools such as contrast - enhanced computed tomography ( ct ) , ct angiography , magnetic resonance angiography , and doppler ultrasonography have improved the rate of diagnosis .
we report two similar cases of isolated spontaneous splenic artery dissection in patients presenting to the emergency department ( ed ) and review the etiology , epidemiology , diagnosis , and treatment of this entity .
a 36-year - old healthy man with an unremarkable medical history was referred from a local hospital for an abdominopelvic ct finding that was compatible with splenic infarction due to splenic artery dissection .
the patient complained of abdominal pain in the left upper quadrant and the epigastric region as well as back pain . upon physical examination , he had mild tenderness to palpation in the upper left quadrant and the epigastric region without abdominal guarding .
vital signs were stable , with a blood pressure of 130/80 mmhg , heart rate of 80 beats / min , and body temperature of 36.8c .
his visceral arteries were evaluated in detail by mesenteric ct angiography , which showed dissections in the celiac trunk , common hepatic artery , and splenic artery with mural thrombi , and partial infarction of the spleen ( fig .
abdominal pain subsided after three days . a follow - up ct angiogram performed three days after admission showed no changes in the celiac trunk , common hepatic artery , splenic artery dissection , or partial infarction in the spleen .
after 10 days in the hospital , the patient was discharged with aspirin , with a follow - up after 12 days . a 42-year - old male patient presented to the ed with a complaint of abdominal pain persisting for one hour on january 21 , 2015 .
he had undergone a small bowel resection and open reduction internal fixation with a screw in the right femur due to a traffic accident in 2004 . since then
, he had been taking acetaminophen and tramadol to relieve lower back pain , with no other medications taken regularly .
his blood pressure was 130/80 mmhg , with a heart rate of 68 beats per minute and a body temperature of 36.8c . a contrast - enhanced ct scan of the abdomen and pelvis revealed celiac artery dissection with an intimal flap and dissection of the splenic artery with mural thrombus , accompanied by splenic infarction ( fig .
he was hospitalized in the vascular surgery department for conservative treatment with intravenous heparin for anticoagulation . the next day ,
his blood pressure increased to 170/110 mmhg and he received intravenous and oral calcium channel blockers to lower his blood pressure .
afterwards , his antihypertensive drug regimen was changed to an oral angiotensin ii receptor blocker based on consultation by a cardiologist .
a 36-year - old healthy man with an unremarkable medical history was referred from a local hospital for an abdominopelvic ct finding that was compatible with splenic infarction due to splenic artery dissection .
the patient complained of abdominal pain in the left upper quadrant and the epigastric region as well as back pain . upon physical examination , he had mild tenderness to palpation in the upper left quadrant and the epigastric region without abdominal guarding .
vital signs were stable , with a blood pressure of 130/80 mmhg , heart rate of 80 beats / min , and body temperature of 36.8c .
his visceral arteries were evaluated in detail by mesenteric ct angiography , which showed dissections in the celiac trunk , common hepatic artery , and splenic artery with mural thrombi , and partial infarction of the spleen ( fig .
abdominal pain subsided after three days . a follow - up ct angiogram performed three days after admission showed no changes in the celiac trunk , common hepatic artery , splenic artery dissection , or partial infarction in the spleen .
after 10 days in the hospital , the patient was discharged with aspirin , with a follow - up after 12 days .
a 42-year - old male patient presented to the ed with a complaint of abdominal pain persisting for one hour on january 21 , 2015 .
he had undergone a small bowel resection and open reduction internal fixation with a screw in the right femur due to a traffic accident in 2004 . since then
, he had been taking acetaminophen and tramadol to relieve lower back pain , with no other medications taken regularly .
his blood pressure was 130/80 mmhg , with a heart rate of 68 beats per minute and a body temperature of 36.8c . a contrast - enhanced ct scan of the abdomen and pelvis revealed celiac artery dissection with an intimal flap and dissection of the splenic artery with mural thrombus , accompanied by splenic infarction ( fig .
he was hospitalized in the vascular surgery department for conservative treatment with intravenous heparin for anticoagulation . the next day ,
his blood pressure increased to 170/110 mmhg and he received intravenous and oral calcium channel blockers to lower his blood pressure .
afterwards , his antihypertensive drug regimen was changed to an oral angiotensin ii receptor blocker based on consultation by a cardiologist .
other known risk factors are trauma , pregnancy , cystic medial degeneration ( marfan syndrome ) , connective tissue disease , periarterial inflammation , fibromuscular dysplasia , congenital disorders of the vascular wall ( such as ehlers - danlos syndrome ) , syphilis , polyarteritis nodosa , and fungal infections [ 3 - 5 ] .
. suggested that hypertension and smoking may also be associated with isolated dissection of abdominal aortic branches .
however , in more than half of all cases , none of the risk factors mentioned above were found , similar to the cases presented here .
about 83% to 88% of cases occur in men , with most cases occurring between 45 to 87 years ( average age , 55 years ) .
about half of splanchnic artery dissections present asymptomatically because there is little involvement of the small intestine .
for example , malabsorption is caused by dissection in the superior mesenteric artery , jaundice in the hepatic artery , and pancreatitis in pancreatic arterial arcades .
chronic dissection may present as intestinal angina , including postprandial abdominal pain and weight loss . in previous studies ,
recently , more advanced diagnostic techniques have been developed , such as contrast - enhanced ct , ct angiography , magnetic resonance angiography , doppler ultrasonography , and conventional angiography . among these techniques , enhanced ct and
ct angiography are considered to be the diagnostic imaging modalities of choice due to their convenience and reliability .
doppler ultrasonography is also widely used because it is the least invasive without exposure to radiation .
however , this intimal flap is not always easily found , and a mural thrombus may be the only indicator of splanchnic artery dissection .
the acute inflammatory process causes a high attenuation of fat around the dissection in symptomatic patients , though high attenuation is thought to be a nonspecific sign .
celiac trunk dissection can be managed by medical treatment , surgery , or endovascular intervention .
conservative management includes anticoagulation and antihypertensive drug regimens to prevent thrombotic complications such as visceral infarction and ischemia , and prevent progression of artery dissection .
surgical or endovascular treatment is indicated for complications such as arterial rupture and liver or bowel ischemia , and when artery dissection progresses despite medical treatment .
surgical treatments include small bowel resection , thrombectomy , resection of the dissected artery with anastomosis , and artery bypass grafting .
endovascular interventions consist of insertion of a bare stent or stent graft , fenestration , and transcatheter embolization .
splenic infarction is uncommon and incidence of splenic infarction due to celiac artery dissection is even rarer . as in our cases ,
medical treatment is indicated for cases of splenic infarction with concurrent celiac trunk dissection without any complications . however ,
if there are complications , such as abscesses or rupture , the patient should undergo surgical treatment .
isolated celiac artery dissection is uncommon , and can be misdiagnosed initially in the ed .
if a patient persistently suffers from unexplained acute abdominal pain , care providers should consider performing contrast - enhanced ct , ct angiography , or doppler ultrasound to exclude vascular events .
once a diagnosis has been made , ed providers should consult a vascular surgery specialist to tailor the treatment modality to the patient .
if the patient is hemodynamically stable without complications , conservative management is recommended as first - line treatment .
however , if the patient becomes hemodynamically unstable , complications arise , or medical treatment fails , caregivers should consider surgical or endovascular treatment . | isolated spontaneous dissection of the celiac trunk is rarely diagnosed in acute abdominal pain .
we present two cases of celiac trunk and splenic artery dissection with splenic infarction .
patients were successfully managed and stabilized by medical treatment .
isolated celiac trunk dissection can be fatal , therefore providers should be careful not to overlook this entity . |
silicon germanium ( sige ) has gained much attention in recent years thanks to its promising electrical and material properties .
the complete solubility of the two elements enables band gap engineering , and sige is relatively easy to integrate into silicon technology .
there are , however , still numerous issues regarding the electrical and material properties of sige that have to be clarified , e.g. the formation of electrically active defect complexes such as vacancy - type defects .
when a positron is implanted into condensed matter , it annihilates with an electron and emits two 511 kev -rays . the energy spectrum of the annihilation -rays is broadened due to the doppler effect associated with the momentum component of the annihilating electron
positrons tend to be localized in vacancy - type defects because of the coulomb repulsion from ion cores .
since the momentum distribution of electrons in such defects differs from that in bulk materials , one can detect the defects by measuring the doppler - broadening spectra of annihilation radiation .
a frequently adopted parameter used for characterizing the change in the doppler - broadening spectra is the so - called s parameter , which mainly reflects changes in the low - momentum region of the electron momentum distribution .
previous investigations comprise the study of semiconductor layers ( usually in the range of hundreds of nanometers ) and semiconductor / oxide systems .
the present contribution deals with positron implantation into utb - soi ( ultra - thin body - silicon - on - insulator ) and sige / soi multilayer structures ( partially presented in ref . ) .
the position and chemical environment of the sio2/si interface in the utb - soi was well characterized .
sige / soi have been proposed as an efficient way of producing strain - free substrates by strain equalization between the top crystalline layers or by strain transfer to the buried oxide .
chemical analysis of the annihilation site in the sige films suggests a prevalent decoration of the trapping sites ( vacancy - like defects ) with ge atoms associated with misfit dislocations and threading dislocations .
the capabilities of pas include the identification and analysis of different type of defects in epitaxial sige thin films and the utb - soi substrate in a nondestructive manner .
the utb - soi wafers , purchased from soitec , were prepared according to the smartcut process , based on implantation and wafer bonding .
all the soi wafers used have a ( 100 ) crystal orientation and are slightly p - doped ( boron 0.61.6 10 cm ) , as was the si reference sample .
the sige layers were grown on soi by means of lepecvd ( low - energy plasma - enhanced chemical vapor deposition ) .
the soi samples were heated to a temperature of 500c for 136 s during sige growth .
the sige layers were grown with a 36% germanium atomic concentration ( measured by x - ray diffraction ) . in order to promote relaxation ,
the samples have been submitted to a post - growth annealing treatment in vacuum ( ~10 mbar ) .
sige layer and silicon substrate thickness were optimized in order to give a substantial relative change ( from 18 to 56% ) of the degree of relaxation ( normalized difference between the measured lattice parameter and the strain - free lattice parameter dependent on the germanium concentration ) upon the annealing treatment
all samples were covered by a thin layer of natural oxide ( ~2 nm ) the measurement was performed by means of a slow positron beam , capable of implanting positrons with a kinetic energy variable from 0.05 to 18 kev . the mean positron implantation depth zm depends on e according to the formula with zm in nanometers when density and positron implantation energy e are expressed in grams per cubic centimeter and kev , respectively .
the gamma rays produced by positron annihilation were detected by means of a high purity germanium detector with resolution ( fwhm ) of about 1.32 kev at 511 kev .
the annihilation energy spectra have been analyzed both by extracting the full annihilation peak shape and by integrating the annihilation peak in the energy interval |e511 kev| 0.85 kev ( s parameter ) .
the area under the peak ( |e-511 kev| 4.25 kev ) was used for normalization . since the energy of the annihilation radiation is doppler shifted in the laboratory frame as a consequence of a finite momentum of the positron
electron annihilating pair along the line that connects the sample to the gamma ray detector , the annihilation peak changes its shape according to the momentum distribution of the electron cloud seen by the positron .
an increase of the s parameter thus reflects an increased annihilation rate with free ( i.e. valence ) electrons , while a broadening of the peak can be linked to an enhanced interaction rate with more bound ( i.e. core ) electrons .
figure 1 shows the results of positron implantation into a bare soi substrate with an extremely thin ( ~2 nm ) si layer on top . the s parameter evolution with the implantation energy is shown in panel a , while panel b shows the fraction of positrons annihilated into the oxide , the substrate , and into the buried interface as computed by the application of a positron implantation and diffusion algorithm by means of the vepfit program .
although positrons are not directly implanted at interfaces , a substantial fraction of positrons should annihilate into the buried interface at implantation energies higher than 2 kev .
the evident dip in the s parameter curve at about 3.5 kev is certainly related to strong positron trapping at interface .
this is confirmed by the excellent fit of the experimental data with the vepfit model ( solid line in fig .
this line is the outcome of several attempts with different models ( sets of vepfit input data ) , which in all cases imply the presence of a positron trapping region corresponding to the nominal position of the si / sio2 interface .
1a was obtained by assuming an oxide surface and four more layers : si , sio2 , sio2/si interface , and a semi - infinite si layer .
the corresponding best - fit values of positron diffusion lengths l+ , thicknesses , and s - parameters of the different layers are reported in table 2 .
it was necessary to fix some parameters ( labeled f in table 2 ) . in accordance with refs . and
, the interface was modeled as a very thin layer ( 1 nm ) with a short positron diffusion length ( l+~1 nm ) . the best - fit value of the depth of the interface coincides within the experimental error with the nominal value .
the introduction of an electric field near the interface region is also possible ( ~300 v / cm ) , but in this case , it is necessary to fix the interface position at the nominal value ( 147 nm ) .
attempts to introduce another absorbing layer at the first si / sio2 interface seem arbitrary . a s parameter as a function of the positron implantation energy in the soi sample .
bdashed lines represent the fractions of positrons implanted into the oxide ( blue ) and the silicon substrate ( red ) , calculated according to ref . .
the continue and dash - doted lines represent the fractions of positrons that annihilate after diffusion in the oxide ( blue ) , at the buried interface ( green ) and into the substrate ( red ) .
surface effects are not visible in the picture ( important only at low energies ) .
1 results obtained in the si / sio2/si soi heterostructure from the positron lineshape profile ( fig .
1 ) using vepfit positron diffusion length l+ , thickness t , and doppler s parameter .
fixed parameters are marked with the letter f figure 2 shows the ratio of the positron
electron annihilation peaks of the surface , the sio2 layer , and the sio2/si interface relative to the bulk silicon peak .
the surface and interface signals were obtained by means of a linear combination between the oxide , the interface , and a silicon bulk contributions , with the weights given by the fractions shown in fig .
1b . since both the positron diffusion coefficient and the lineshape parameters relative to the thermally grown oxide and to the silicon substrate
are known from the literature and from calibration experiments on bulk samples , the interface doppler peak shape can be extracted .
2 is characterized by a flat region at low momentum and by a peak at ~12 mrad ( 12 10m0c ) due to annihilation with tightly bound oxide electrons .
the absence of a clear signal of an increment of annihilation with nearly free electrons ( visible at momentum zero in fig .
2 ) and the need of a trapping layer at the interface convey the idea that positrons do not annihilate into voids , but rather that they are strongly localized at charged centers , most probably representing electron states created by the presence of silicon dangling bonds , as already demonstrated in positron implantation experiments with sio2/si samples and that they sense a relatively well - ordered oxide structure , typical of low quartz .
the same annihilation environment can be found more or less at any semiconductor surface covered with thermally grown or natural oxide , as seen in fig .
ratios of the positron electron momentum distribution at the surface of a silicon reference sample , in the silicon oxide and at the buried interface ( relative to bulk si ) figure 3 shows the experimental s parameter profiles of the sige / soi samples listed in table 1 . the approximated mean implantation depth was calculated according to eq .
1 ( the density of the sige layer has been estimated to be 3.52 g / cm ) .
the results indicate the existence of a wide plateau up to ~50 nm in both samples , which corresponds to the sige layer , and changes in height appear after the thermal treatment .
given the mixing between the signals coming from the oxide and from the sige / si layers , changes in the surface layers morphology can be appreciated by comparing the two s parameter evolutions relative to the sample before and after structural relaxation due to the annealing step .
the increase of the s parameter at low implantation energy can be thus directly related to an enhanced interaction with free electrons .
the exact positioning of the defected region is behind the resolution limits of the current measurements , given that the positron diffusion length in intrinsic semiconductors can be as high as 240 nm .
s parameter as a function of the mean implantation depth : sige a as grown
continuous and dashes lines are vepfit simulations , while vertical dashed lines mark the position of interfaces ( after ref . ) in order to gain more understanding on the environment of the positron annihilation site , we have averaged the doppler peak shape relative to a mean implantation depth between 2 and 30 nm ( the plateaus in fig .
3 ) . lower implantation energies have been neglected since they bring information also from the annihilation of positron from non - thermalised ensemble and from the natural oxide layer . according to both precise vepfit simulations and to simplified models of positron implantation , positrons implanted at energies lower than 2 kev
since the diffusion length of positrons into bulk semiconductors exceeds the actual thickness of these layers , the annihilation peak shape prevalently resembles the one characteristic of annihilation at the oxide / semiconductor interface .
the differences between the peak shape relative to the as - grown sample and to the annealed one are shown in fig .
4 and are related to an increased annihilation with low- and high - momentum electrons .
the former effect is reflected in the increase of the s parameter , as seen in fig . 3 and can be associated with an increase of positron annihilation at defects , and the latter can be interpreted as a modification of the chemical environment of the positron annihilation site .
ratios of the positron electron momentum distribution of the annihilating pair in sige a as grown
( full symbols ) and sige b annealed ( open symbols ) relative to bulk si .
the reference spectra of defected ge and surface / interface are shown with continue and dashed lines ( after ref . )
following the procedure outlined in , we have reproduced the measured annihilation peak shapes with a linear combination of doppler spectra of the semiconductor / oxide interface and the ge signal of a sample saturated of defects .
4 . as discussed in ref . , the results of this latter analysis to the data of fig .
4 show an increment in the germanium component from ( 22 10)% to ( 47 8)% after the annealing .
the indetermination of this component is associated with the spread of the experimental data , especially at high momentum . in particular , in order to better reproduce the measured doppler spectra on the whole momentum scale , we have employed , instead of the annihilation peak shape characteristics of annihilation into bulk germanium , the shape measured from a thick ( in the microns range ) layer of germanium grown on silicon .
the abrupt junction between germanium and silicon promotes the formation of misfit dislocations at the interface and threading dislocation that run across the whole germanium layer with an estimated density of about 10 cm .
positrons are trapped at defect sites causing a substantial reduction of the diffusion length and a slight increase of the s parameter .
this experimental finding can be explained by postulating positron annihilation at vacancies associated with dislocations , or at negatively charged centers associated with dislocations , given that the deposited ge layer was free of contaminants or dopant atoms , which are known to produce positron trapping defects .
the decomposition of the annihilation spectra into two terms ( interface and germanium defects ) gives an acceptable fit ( /[degrees of freedom ] values of 1.5 in the as - grown sample and 1.2 in the annealed sample ) and conveys the idea of a prevalent decoration of positron trapping centers with germanium atoms ( as already pointed out by rummukainen et al . in bulk sige layers ) , mainly associated with si vacancies .
( i ) the sio2/si interface in the utb - soi was identified with accuracy .
it was possible to estimate the depth where the interface is located with good precision .
the chemical analysis at the surface and the interface shows that positrons do not annihilate into large defects ( voids ) , but rather that they are strongly localized close to the silicon surface .
the observed momentum distribution is characteristic of annihilation in a relatively well - ordered oxide structure , typical of low quartz , and the strong localization can be explained with annihilation at negatively charged centers , like silicon dangling bonds .
( ii ) the process of strain relaxation in thin sige layers grown on soi substrates has been analyzed .
relaxation of the strained structure has been found to proceed via the introduction of new defects , presumably si vacancies , able to trap positrons .
chemical analysis of the annihilation site suggests a prevalent decoration of the trapping sites with germanium atoms .
the formation of lattice defects in the form of misfit dislocations between two adjacent layers and threading dislocations in the sige substrate are certainly associated with the identified defects .
it is demonstrated that the analysis of positron data coming from extremely thin surface layers is possible thanks to the reduced implantation range of positrons at low implantation energy and to the enhanced contrast due to the prevalent annihilation of not trapped positrons with strongly bound surface / interface oxide electrons .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited .
this article is distributed under the terms of the creative commons attribution noncommercial license which permits any noncommercial use , distribution , and reproduction in any medium , provided the original author(s ) and source are credited . | the potential of positron annihilation spectroscopy ( pas ) for defect characterization at the atomic scale in semiconductors has been demonstrated in thin multilayer structures of sige ( 50 nm ) grown on utb ( ultra - thin body ) soi ( silicon - on - insulator ) .
a slow positron beam was used to probe the defect profile .
the sio2/si interface in the utb - soi was well characterized , and a good estimation of its depth has been obtained .
the chemical analysis indicates that the interface does not contain defects , but only strongly localized charged centers . in order to promote the relaxation ,
the samples have been submitted to a post - growth annealing treatment in vacuum . after this treatment , it was possible to observe the modifications of the defect structure of the relaxed film .
chemical analysis of the sige layers suggests a prevalent trapping site surrounded by germanium atoms , presumably si vacancies associated with misfit dislocations and threading dislocations in the sige films . |
infection with human immunodeficiency virus ( hiv ) predisposes individuals to the development of neoplasms .
there are currently four acquired immune deficiency syndrome ( aids)-defining malignancies : kaposi 's sarcoma , non - hodgkin lymphoma ( nhl ) of high - grade pathologic type and b - cell or unknown immunologic phenotype , primary central nervous system lymphoma , and invasive cervical carcinoma .
mature t - cell and natural killer ( nk ) cell neoplasms make up only 12% of nhl cases worldwide . within this heterogeneous category of nhl , anaplastic large cell lymphoma ( alcl )
is listed revised european - american classification of lymphoid neoplasms ( real ) as an independent entity in the real .
alcl is a rare subtype of t - cell lymphoma that can involve mucocutaneous sites , primarily or secondarily , as part of the systemic disease .
alcl is usually comprised of large atypical cells with abundant cytoplasm , pleomorphism , and often having kidney shaped nuclei . in almost all
reported cases of alcl , tumor cells are cd30 and in most cases they express the cytotoxic granule - associated protein ( perforin ) .
based on the expression of resulting gene product : anaplastic lymphoma kinase ( alk ) alcl is divided into alk(+ ) and alk( ) categories .
this paper presents an unusual case of palatal alcl , which mimicked a benign neoplasm clinically but was found to be the first clinical manifestation of aids in this patient .
a 48-year - old male presented with a rapidly enlarging swelling in the right anterior palatal mucosa measuring 5.6 cm 4.5 cm with white coating , moderately painful , and bled upon touching [ figure 1 ] .
he had visited a local dentist with this swelling for which antibiotics and analgesics were prescribed for few days but did not get any relief .
intraoral anterior palatal swelling the patient was a nonsmoker , and his medical history did not reveal other relevant information .
results of laboratory testing showed hemoglobin , hematocrit , white blood cell , and platelet counts within normal range .
on intraoral examination , the swelling had not crossed the midline and appeared to be pedunculated , afebrile , nonfluctuant .
teeth associated with the swelling , i.e. , all premolars and molars on the right side of the upper jaw were noncarious , nontender , and nonmobile .
the first impression of the lesion suggested clinical characteristics of benignancy suggesting pyogenic granuloma , peripheral giant cell granuloma , peripheral ossifying fibroma , and even pleomorphic adenoma as differential diagnosis .
radiographic investigations were noncontributory as they showed mild bone loss which is usually seen in relation to reactive hyperplastic lesions .
histopathologic sections revealed an atypical lymphocytic infiltrate consisting of large , lymphoid cells with pleomorphic nuclei , numerous atypical mitoses , and infiltrative pattern of tumor cells .
these atypical round tumor cells showed vesicular nuclei having cleaved appearance with the presence of 2 or more nucleoli [ figure 2 ] .
immunohistochemical ( ihc ) studies were performed with appropriate positive and negative controls on the incisional processed tissue .
ihc analysis using streptavidin - biotin protocol was performed with antibodies : ckae1/ae3 , cd3 , cd5 , cd15 , cd20 , cd30 , cd56 , cd68 , cd99 , leukocyte common antigen ( lca ) , epithelial membrane antigen ( ema ) , synaptophysin , hmb-45 , s-100 , mpo , mum1 , and p80 .
tumor cells showed strong positivity with cd30 and lca , and ema positivity was seen only in 50% of tumor cells [ figure 3 ] .
negative stain was identified for ck , cd3 , cd5 , cd15 , cd20 , cd56 , cd68 , cd99 , synaptophysin , hmb 45 , s-100 , mum1 , mpo , and p80 .
( a ) sheet of atypical round tumor cells with vesicular nuclei ( h and e , 10 ) .
( b ) tumor cell with cleaved nuclei ( arrow head ) and mitotic figure ( arrow ) ( h and e , 40 ) tumor cells positive for ( a ) cd30 , membrane and cytoplasm ( ihc , 10 ) , ( b ) leukocyte common antigen , membrane ( ihc , 10 ) , ( c ) epithelial membrane antigen , membrane and cytoplasm ( ihc , 10 ) the patient was referred to oncology department where further laboratory and imaging tests were performed .
serology of the patient came out to be strongly positive for hiv based on elisa , negative for hepatitis b and c. further evaluation of his seropositivity by western blot confirmed elisa results of hiv . cd4
cell count had dropped down to 120 . full body scan to rule out the presence of any other malignancy associated with aids
r - epoch infusion chemotherapy was started for the patient in the oncology department with one cycle every 3 weeks for a total of six courses .
haart therapy ( efavirenz + tenofovir + emtricitabine ) was also initiated orally . despite consecutive follow - up , unfortunately , the patient did not cooperate and after three cycles of chemotherapy , the patient was lost .
however , it was observed during a follow - up period of chemotherapy that the swelling had decreased in size .
although , even after incisional biopsy the swelling had reduced but the remarkable reduction in size was seen after chemotherapy .
alcl is subdivided according to the ihc features in precursor t - cells or null cells , in 60% and 25% of cases , respectively .
cd3 , cd45 , perforin , and granzyme b. some alcls show positivity with alk whereas few do not show any expression .
the clinicopathologic features of alk carrying alcls were first investigated by shiota and mori and found that alk(+ ) is a distinct entity clinically and pathogenetically and should be differentiated from alk( ) tumors . who classification currently considers two alcl types
alk( ) and alk(+ ) . the tumors that show a positive reaction for alk have greater cell proliferation and show relatively better prognosis . on the other hand , cases of alk( )
the present case did not express alk ; hence , it was considered alk( ) alcl .
in addition , the tumor did not show expression of nk or t - cells therefore , it was also considered as null cell lymphoma .
alk( ) alcl normally occurs in adults ( 4065 years ) with male prevalence , as seen in our male patient of 48 years of age .
all persons infected with hiv , whether or not they undergo seroconversion illness and pass through a phase of symptomless infection ( clinical latency ) which may last up to several years . in the present case , he showed positivity for hiv antibody but had no other symptoms except the palatal swelling .
the period of clinical latency does not mean microbiological latency as virus multiplication goes on throughout .
cd4 t - cell count decreases steadily from over 1000/l to about 500 or less .
when the count falls to 200 or less , clinical aids sets in . in a healthy hiv negative person , normal cd4 t - cell count ranges from 460 to 1600 whereas in our patient it had dropped to 120 with no other acute infections and lymphadenopathy .
clinical aids with cd4 t - cell count dropped from 200 to 120 in the absence of acute illness made it an interesting case to be reported .
a search of the literature in the pubmed identified only 11 prior cases of cd30 alcl involving oral cavity .
of the 11 reported cases , all were of t - cell type except for one case with null cell type .
based on clinical cases found in the review of literature [ table 1 ] , there is a slight male prediction ( female : male , 1:1.5 ) , and the patients presented at a mean age of 53.4 years old .
most of the reported cases were located in the gingiva ( five cases ) , followed by the palate ( three cases ) , tongue ( two cases ) , retromolar trigone ( two cases ) , lip ( two cases ) , the floor of mouth ( one case ) , and buccal mucosa ( one case ) .
the main radiographic findings were bone resorption , and the main diagnostic hypothesis was a nonspecific infection . to the best of our knowledge ,
the present case is the first palatal alcl , which mimicked clinically as a benign tumor and was the first clinical manifestation of aids .
none of the alcl cases of palate reported in literature showed serological positivity to hiv .
out of all cases reported in literature only 14% were alk( ) in the oral cavity .
currently , there is only one case reported by notani et al . , which showed alk expression in alcl involving oral cavity .
synopsis of clinicopathological and anaplastic lymphoma kinase profile of oral anaplastic large cell lymphoma reported in pubmed literature the differential diagnosis of alcl includes hodgkin lymphoma ( hl ) and other forms of cd30(+ ) t- or b - cell lymphoma with anaplastic morphology and undifferentiated carcinoma or other malignancies .
other peripheral t - cell lymphomas show limited cd30 expression and lack hallmark cells which were seen in the present case .
undifferentiated carcinomas and melanoma can be identified using cytokeratin and melanoma markers which were negative for the present case .
interestingly , the present case of alk( ) alcl appears to be the first case reported in the palate of hiv - seropositive patient with alcl being the first clinical manifestation of aids . in our patient , it was the null cell type of lymphoma which did not show any positivity with cd3 and cd20 and happens to be the first such case .
alcl is a systemic malignant disease characterized by extranodal type which rarely occurs in the oral cavity as the first manifestation of acquired immunodeficiency syndrome .
hence , it is important to have accurate , clinical including hiv investigation , histological and ihc analysis of oral lesions to establish a correct diagnosis of such lesions which appear clinically benign .
| anaplastic large cell lymphoma ( alcl ) is an uncommon disease , accounting for < 5% of all cases of non - hodgkin 's lymphoma .
we report a case of 48-year - old male who presented a clinically benign swelling in the right anterior palatal region since last 2 months .
radiographic evaluation showed no bone loss in palatal area .
histological and radiological examination was in favor of a peripheral reactive lesion like pyogenic granuloma or a benign salivary gland tumor .
immunohistochemistry confirmed the diagnosis of anaplastic lymphoma kinase - negative ( alk( ) ) alcl .
further laboratory tests elisa for human immunodeficiency virus ( hiv ) and cd4 cell count was done which showed positivity for hiv . to the best of our knowledge ,
it is the first case of alk( ) alcl in the hard palate presenting as the first clinical manifestation of acquired immune deficiency syndrome . |
mechanical ventilation is one of the most frequently employed interventions in the intensive care unit ( icu ) .
although it is a life - saving measure , much time and effort is spent in trying to wean patients from the ventilator as quickly as possible , since mechanical ventilation is also a cause of numerous complications . in a recent issue of critical care , supinski and callahan report that infection is a significant risk factor for diaphragmatic weakness and failure to wean patients from mechanical ventilation .
the authors employed state of the art methods ( transdiaphragmatic pressure measurements during bilateral magnetic stimulation of the phrenic nerves ) , and found that patients with evidence of infection had less than half the diaphragmatic pressure - generating ability of uninfected patients . in addition , patients with the most severe diaphragmatic weakness had a markedly worse prognosis .
this consisted not only of a more prolonged need for ventilator support , but was also reflected in substantially higher mortality . indeed
, diaphragmatic function appeared to be a better prognostic indicator than other more conventional indices of critical illness severity , such as the sequential organ failure assessment score .
interestingly , the treating physicians in the icu dramatically underestimated ( in 90% of patients ) the degree of diaphragmatic weakness present in their mechanically ventilated patients .
they point to a need for greater awareness of the very high prevalence of diaphragmatic weakness in mechanically ventilated patients . the inability to successfully wean patients from mechanical ventilation has been closely linked to an unfavorably elevated level of the respiratory muscle work load / capacity ratio .
although great emphasis is appropriately placed upon reducing the numerator in this relationship through attempts at improving respiratory system mechanics , the denominator ( reflecting respiratory muscle function ) is more difficult to assess and often neglected .
nevertheless , several studies have now shown that diaphragmatic weakness is common and frequently profound in mechanically ventilated patients [ 4 - 6 ] , although the precise reasons for this are not well understood . based upon the fact that even uninfected patients in the study by supinski and callahan exhibited a large decrease ( to approximately 50% of normal values ) of diaphragmatic force - generating capacity , it seems clear that a major component of the diaphragmatic weakness observed in mechanically ventilated patients must be caused by additional factors other than infection . in this regard ,
another recent study reported that diaphragmatic weakness was present on the very first day of admission to the icu in patients requiring mechanical ventilation for a variety of conditions , including , but not limited to , sepsis .
the study by supinski and callahan did not specifically evaluate the time course for developing diaphragmatic dysfunction , either from the time of icu admission and initiation of mechanical ventilation , or from the onset of infection . when taken together
, however , the above studies strongly suggest that diaphragmatic dysfunction constitutes a distinct , common , and under - recognized form of organ failure that occurs with many types of critical illness , and especially during sepsis .
the results of these studies in icu patients are also consistent with a large body of data from different animal models , which have consistently demonstrated impaired diaphragmatic function during sepsis .
there is limited information about the impact of mechanical ventilation upon sepsis - induced diaphragmatic dysfunction , but the interaction between the two appears to be complex . although mechanical ventilation may mitigate the adverse effects of sepsis upon diaphragmatic function and oxygen demand to the muscle very early in its course , there are several reasons to believe that mechanical ventilation will either worsen or impede recovery from sepsis - induced diaphragmatic dysfunction over the longer term . in this
regard , mechanical ventilation itself leads to diaphragmatic atrophy and weakness in non - septic animals and humans , a phenomenon referred to as ventilator - induced diaphragmatic dysfunction ( vidd ) .
furthermore , sepsis - induced diaphragmatic dysfunction and vidd appear to share many of the same pathogenetic mechanisms , such as increased oxidative stress and mitochondrial dysfunction within diaphragm muscle fibers .
perfect storm , with mechanical ventilation either exacerbating the magnitude of diaphragmatic weakness caused by infection or slowing the subsequent recovery of diaphragmatic function once sepsis has resolved .
further studies will be required to specifically address these questions , and there is a clear need for novel therapeutic approaches that can either reverse or limit the development of diaphragmatic weakness in mechanically ventilated patients .
the presence of common cellular mechanisms implicated in sepsis - induced diaphragmatic dysfunction and vidd raises the possibility that pharmacologic agents directed at their shared molecular targets might be effective therapies for both conditions .
| accumulating evidence indicates that diaphragmatic weakness is common and frequently severe in mechanically ventilated patients .
supinski and callahan now report that infection is a major risk factor for diaphragmatic weakness in this patient population .
importantly , they show that patients with the greatest levels of diaphragmatic dysfunction have a much poorer prognosis in terms of more prolonged ventilation as well as higher mortality .
mechanical ventilation itself has also been found to induce diaphragmatic weakness along with cellular changes resembling those found in sepsis .
future studies should be directed at understanding the interaction between sepsis and mechanical ventilation , and to developing therapeutic approaches that target their common cellular pathways implicated in diaphragmatic weakness . |
in norway1 and most western societies,2 the major causes of long - term sick leave and work disability are chronic musculoskeletal and mental disorders . the numbers of disability pensioners are increasing and this represents a large economic burden for society .
in addition , not being able to participate in the labor force has negative consequences for the individual .
depression is the leading cause of disability3 and has a more negative prognosis for return to work ( rtw ) than other common mental health conditions.4,5 somatic symptoms and depression often coexist,6 and epidemiological findings support that there is a bidirectional association between depression and chronic musculoskeletal conditions.7 further , studies suggest that depression contributes to poor rtw in individuals referred to occupational rehabilitation , even controlling for pain severity.8 chronic pain is a common long - term condition that affects a person s physical and emotional functioning .
approximately 20%50% of individuals with musculoskeletal conditions show evidence of elevated depressive symptoms , further suggesting that comorbid depression is associated with poorer rehabilitation outcomes.8,9 multidisciplinary rehabilitation approaches have been shifting away from a restricted biomedical model toward a multifactorial model of interrelating physical , psychological , and social / occupational factors,10 with emphasis on function and work ability , disability management , and psychosocial interventions .
results support that participation in multidisciplinary rehabilitation programs increases quality of life and decreases anxiety and depression in employees with musculoskeletal disorders.11 several studies suggest that physical factors , such as improvements in strength , endurance , or flexibility , appear to have little relation to rtw rates after interdisciplinary treatment.12,13 other studies confirm that increased depressive symptoms are associated with decreased physical functioning.14,15 in a recent review , mcknight and kashdan16 stated that there is little research directly examining the relationship between depressive symptoms and physical ability .
the extent to which employees with chronic musculoskeletal disorders and depressive symptoms successfully adapt back into their job roles after participating in a multidisciplinary rehabilitation program remains unclear .
given the association between chronic pain , depression , and physical function,16,17 it is likely that rtw in persons with chronic musculoskeletal pain and comorbid depression would be associated with better physical functioning .
the aim of our investigation was to assess whether various measurements of physical functioning among persons with chronic musculoskeletal pain and comorbid depression were positively associated with rtw after participating in a 57-week multidisciplinary rehabilitation program .
participants with a long - term history of mixed chronic musculoskeletal pain were recruited by their general practitioners , who assigned them to a 57-week multidisciplinary rehabilitation program on the basis of interviews , observations , and clinical tests .
inclusion criteria for participation in the rehabilitation program were that the participants had chronic musculoskeletal pain ( longer than 3 months ) and were receiving national insurance in the form of sickness benefit or rehabilitation benefit . in norway one can start drawing a retirement pension at the age of 62 . to ensure that none of the participants were in any initial phase of retirement , we excluded those aged 60 years and older . further , to assess any difference in physical function and return to work among those with comorbid depressive symptoms at the end of the rehabilitation , we included only those with high depressive symptoms measured by the hospital anxiety and depression scale ( hads ) .
the participants provided written consent . to maintain the right to financial support in the form of sickness or rehabilitation benefits from the national health insurance office , an assessment of function and work capacity is required .
general practitioners recruited participants into the 57-week rehabilitation program in cooperation with the national health insurance office , in order to obtain a function and work capacity evaluation as required . in cooperation with the public health services , national health insurance office , employment office , and the employer , an individually tailored rehabilitation program was conducted .
the rehabilitation program included both individual counseling and group - based treatment , including physical components ( see table 1 ) .
the program consisted of 1 ) a 5-week intensive period that the participants attended approximately 6 hours a day , 4 days a week , and 2 ) a follow - up period of 52 weeks that the participants attended approximately 6 hours a day , 13 days a week .
symptoms of anxiety and depression were assessed using the hads , a self - rating scale commonly used for screening purposes in various clinical settings and in epidemiological studies.18 the symptom scale consists of seven items that cover anxiety and seven items that cover depression on a 4-point likert scale .
individual items are scored from 03 or 30 , depending on the direction of the wording of the items .
an optimal balance between sensitivity and specificity seems to be achieved when caseness is defined by a score of 8 on both the anxiety and depression subscales of the hads.18 four aspects of physical functioning ( muscle strength , endurance capacity , mobility , and balance ) and degree of physical pain were measured with single items on a visual analog scale ( vas ) .
the use of vas in employees with musculoskeletal disorders is well - established , and single - item measures frequently perform almost as well as multi - item scales.19,20 the vas is a line 10 cm in length where agreement or disagreement represents a continuum of the specific physical function measured . the vas variables were individually measured and scored in millimeters ( 0100 ) .
the participants were asked to rate the present condition / symptoms through the following questions : pain experience on a scale from 0100 where 0 is not troublesome at all and 100 is extremely troublesome , how troublesome do you experience the pain to be in everyday life ? muscle strength on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your muscle strength to be?21,22 endurance capacity on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your endurance capacity to be?21,22 mobility on a scale from 0100 where 0 is very limited flexibility and 100 is very flexible
, how flexible do you consider your muscles and joints to be?21,22 balance on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your balance to be?21,22 physical fitness was measured through the validated coop / wonca charts23 by the following question : during the past two weeks , what was the hardest physical activity you could do for at least 2 minutes ? the answering categories consist of a 5-point ordinal scale ranging from 1 ( very heavy ) to 5 ( very light ) .
age was used as a continuous variable , and marital status was measured as a dichotomous variable ( married / cohabiting versus unmarried / divorced ) .
disability status was registered in the following categories : rehabilitation benefit , vocational rehabilitation , part time work / rehabilitation benefit , sick leave , part - time disability pension , disability pension , social assistance , students , or unemployed .
active and passive strategies for work ability and work re - entry .
participants who reported working on a part - time or full - time basis were categorized as active and coded as on their way into / in work ( rtw ) , as were individuals in job retraining or an education program .
these latter two variables were categorized as active strategies by means of representing levels of functioning that in some ways mirror the work environment and bring forward work - related behavior in the participants eg , they have to go somewhere on a daily basis and keep functioning at a relatively consistent level .
the participants still on sick leave , receiving disability pension , or applying for disability pension after completing the 57-week multidisciplinary rehabilitation program were categorized as passive and coded as on their way out / out of work ( non - rtw ) .
the reason for this choice was that most participants in the rehabilitation program already had been on sick leave for several months before entering the 57-week rehabilitation program .
thus it was very likely that those still on sick leave after 57 weeks were in the initial phase of the process of being granted a disability pension .
the dependent variable rtw was self - reported and defined by being on their way into / in work .
a student s t - test for independent samples ( continuous variables ) and chi - square test ( categorical variables ) were used to compare those who were returning to work and those who were not .
bivariate associations between each domain of physical function and rtw were assessed through logistic regression models , estimating odds ratio ( or ) with 95% confidence intervals ( ci ) .
ibm spss statistics 20 ( ibm corporation , armonk , ny , usa ) was used for the statistical analyses .
participants with a long - term history of mixed chronic musculoskeletal pain were recruited by their general practitioners , who assigned them to a 57-week multidisciplinary rehabilitation program on the basis of interviews , observations , and clinical tests .
inclusion criteria for participation in the rehabilitation program were that the participants had chronic musculoskeletal pain ( longer than 3 months ) and were receiving national insurance in the form of sickness benefit or rehabilitation benefit . in norway one can start drawing a retirement pension at the age of 62 . to ensure that none of the participants were in any initial phase of retirement , we excluded those aged 60 years and older . further , to assess any difference in physical function and return to work among those with comorbid depressive symptoms at the end of the rehabilitation , we included only those with high depressive symptoms measured by the hospital anxiety and depression scale ( hads ) .
to maintain the right to financial support in the form of sickness or rehabilitation benefits from the national health insurance office , an assessment of function and work capacity is required .
general practitioners recruited participants into the 57-week rehabilitation program in cooperation with the national health insurance office , in order to obtain a function and work capacity evaluation as required . in cooperation with the public health services , national health insurance office , employment office , and the employer , an individually tailored rehabilitation program was conducted .
the rehabilitation program included both individual counseling and group - based treatment , including physical components ( see table 1 ) .
the program consisted of 1 ) a 5-week intensive period that the participants attended approximately 6 hours a day , 4 days a week , and 2 ) a follow - up period of 52 weeks that the participants attended approximately 6 hours a day , 13 days a week .
symptoms of anxiety and depression were assessed using the hads , a self - rating scale commonly used for screening purposes in various clinical settings and in epidemiological studies.18 the symptom scale consists of seven items that cover anxiety and seven items that cover depression on a 4-point likert scale .
individual items are scored from 03 or 30 , depending on the direction of the wording of the items .
an optimal balance between sensitivity and specificity seems to be achieved when caseness is defined by a score of 8 on both the anxiety and depression subscales of the hads.18
four aspects of physical functioning ( muscle strength , endurance capacity , mobility , and balance ) and degree of physical pain were measured with single items on a visual analog scale ( vas ) .
the use of vas in employees with musculoskeletal disorders is well - established , and single - item measures frequently perform almost as well as multi - item scales.19,20 the vas is a line 10 cm in length where agreement or disagreement represents a continuum of the specific physical function measured .
the participants were asked to rate the present condition / symptoms through the following questions : pain experience on a scale from 0100 where 0 is not troublesome at all and 100 is extremely troublesome , how troublesome do you experience the pain to be in everyday life ? muscle strength on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your muscle strength to be?21,22 endurance capacity on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your endurance capacity to be?21,22 mobility on a scale from 0100 where 0 is very limited flexibility and 100 is very flexible , how flexible do you consider your muscles and joints to be?21,22 balance on a scale from 0100 where 0 is extremely poor and 100 is extremely good , how do you consider your balance to be?21,22 physical fitness was measured through the validated coop / wonca charts23 by the following question : during the past two weeks , what was the hardest physical activity you could do for at least 2 minutes ?
the answering categories consist of a 5-point ordinal scale ranging from 1 ( very heavy ) to 5 ( very light ) .
age was used as a continuous variable , and marital status was measured as a dichotomous variable ( married / cohabiting versus unmarried / divorced ) .
disability status was registered in the following categories : rehabilitation benefit , vocational rehabilitation , part time work / rehabilitation benefit , sick leave , part - time disability pension , disability pension , social assistance , students , or unemployed .
active and passive strategies for work ability and work re - entry . participants who reported working on a part - time or full - time basis were categorized as active and coded as on their way into / in work ( rtw ) , as were individuals in job retraining or an education program .
these latter two variables were categorized as active strategies by means of representing levels of functioning that in some ways mirror the work environment and bring forward work - related behavior in the participants eg , they have to go somewhere on a daily basis and keep functioning at a relatively consistent level .
the participants still on sick leave , receiving disability pension , or applying for disability pension after completing the 57-week multidisciplinary rehabilitation program were categorized as passive and coded as on their way out / out of work ( non - rtw ) .
the reason for this choice was that most participants in the rehabilitation program already had been on sick leave for several months before entering the 57-week rehabilitation program .
thus it was very likely that those still on sick leave after 57 weeks were in the initial phase of the process of being granted a disability pension .
the dependent variable rtw was self - reported and defined by being on their way into / in work .
a student s t - test for independent samples ( continuous variables ) and chi - square test ( categorical variables ) were used to compare those who were returning to work and those who were not .
bivariate associations between each domain of physical function and rtw were assessed through logistic regression models , estimating odds ratio ( or ) with 95% confidence intervals ( ci ) .
ibm spss statistics 20 ( ibm corporation , armonk , ny , usa ) was used for the statistical analyses .
the mean duration of pain prior to entering the rehabilitation program was 15 years ( ranging from 3 months to 36 years ) .
most of the participants , 89.5% ( n=505 ) , reported pain in more than one location , with lower back , shoulders , and neck being the most common . to ensure that none of the participants were in any initial phase of retirement , we excluded those aged 60 years and older ( n=142 ) .
further , to assess any difference in physical function and return to work among those with comorbid depressive symptoms at the end of the rehabilitation , we included only those with high depressive symptoms measured by the hospital anxiety and depression scale ( hads ) .
the final sample consisted of 92 persons ( 21.7% of all participants < 60 years ) with chronic musculoskeletal disorders and comorbid depression under 60 years of age who had undergone the 57-week multidisciplinary rehabilitation program . of these
, 70 were returning to work ( rtw ) and 22 were not ( non - rtw ) .
all participants included in the present study had a hads depression score 8 at the end of the rehabilitation .
group ( rtw ) and the on their way out / out of work group ( non - rtw ) in terms of age , marital status , level of depression above the cut - off value of 8 , anxiety level , and pain experience ( table 2 ) . muscle strength , endurance capacity , mobility , balance , and physical fitness were significantly better among those who were not returning to work . in the bivariate analyses ,
four of the five domains of physical functioning measured by vas ( muscle strength , mobility , balance , and physical fitness ) were significantly negatively associated with rtw , indicating that better physical functioning was associated with not returning to work ( table 3 ) . among those with better function on muscle strength ( or 0.61 , 95% ci 0.450.83 ) , mobility ( or 0.73 , 95% ci 0.570.95 ) , and balance ( or 0.67 , 95% ci 0.500.91 ) , the odds of being on an active work re - entry strategy were 23%39% lower compared with those with poorer physical function .
the same pattern was seen for physical fitness measured by the coop / wonca charts ( or 1.75 , 95% 1.072.99 ) , indicating that the odds of returning to work were 75% lower among those who reported that they were able to perform hard physical activity for at least 2 minutes . in other words ,
each of these measures of physical functioning was worse among those reporting that they were on an active strategy for work re - entry .
endurance capacity ( or 0.77 , 95% ci 0.581.03 ) did not reach any statistical association with rtw ; even so , the or of 0.77 ( p=0.076 ) indicated that this result was in line with the other results and it would probably have become statistically significant in a larger sample .
in this study of persons with chronic musculoskeletal disorders and comorbid depression , we found higher levels of physical functioning to be significantly associated with non - rtw at the end of the rehabilitation period of 57 weeks . given that the multidisciplinary rehabilitation program in this study places a strong emphasis on function and work ability , our results are somewhat surprising .
additionally , we have not been able to find other studies confirming our results or the opposite ; that individuals with chronic musculoskeletal disorders and comorbid depression returning to work have better physical function compared with those who are depressed not returning to work . in the present study
, there were no differences in pain experience or severity of depression between those returning and those not returning to work .
hence , there are no straightforward explanations of our findings of reduced odds of rtw among those with best physical functioning . in the following discussion we present two important aspects that may be relevant to our findings .
physical limitations , physical ability and physical activity are all domains covering different aspects of human physical functioning .
most studies on mental health and physical function suggest an inverse association between decreasing levels of depressive symptoms and increasing physical functioning . however , an exhaustive literature review showed that the association between depressive symptoms and physical functioning is unexpectedly weak.16 this may be due to low consensus on what physical measurement to use , and whether such measures should be performance - based or patient - reported .
bean et al24 found that self - reported measures of physical function were associated with psychosocial factors such as depression , while performance - based measures were not .
nevertheless , the former study was among elderly persons with mobility limitations and results can not be generalized to the sample in our study .
an alternative explanation for the association found in the present study is that self - report of physical functioning may be influenced by psychological distress . despite participation in an occupational rehabilitation program
, some employees may lack motivation to return to a certain workplace . in a qualitative study of disability pensioners with chronic back pain ,
magnussen et al25 found that some of the barriers related to rtw were earlier negative work experiences , poor self - judgment of work ability , and low self - esteem .
further , many of the participants pointed to conditions at their former workplaces that they believed contributed to the disability process and that would make it difficult to return to work .
hence , the experience of being granted a disability pension , which means not returning to work , might be followed by the feelings of relief and satisfaction , while those , by definition , on their way into work might be subjected to remaining in an unsafe and uncertain situation , both economically and occupationally . in turn
, this emotional state may directly affect one s evaluation of physical functioning , despite being depressed .
several studies support that the rtw is a multi - factorial process8 and that physical functioning should not be mixed up with occupational functioning .
occupational functioning is more than being able to perform certain tasks at work,26 as it also involves non job - specific behaviors such as cooperation and dedication .
thus , occupational functioning can be related to factors at the individual level , at the group level , and at the organizational level . nevertheless , according to the literature most psychosocial interventions to achieve optimal rehabilitation outcomes in employees with musculoskeletal pain
have focused on risk factors within the individual ( eg , pain catastrophizing and expectancies ) and not on relevant factors at the workplace , such as interpersonal conflicts or job stress ( at the group or institutional level).27 in the vast majority of observational studies on factors related to the rtw process , as in this study as well , psychosocial interventions did not follow common evidence - based guidelines or treatment protocols.27,28 in turn , this makes any evaluation of treatment outcome after rehabilitation difficult , especially regarding assessment of interventions that focus on work - related factors outside of the individual.27 further , depression affects several areas related to occupational functioning , such as absenteeism , education , and employment opportunities .
depressed workers also report more conflicts and negative social outcomes.16 in a qualitative study of factors relevant in the assessment of the rtw process of employees on long - term sickness absence due to a depressive disorder , muijzer et al4 found that physical functioning , attitudes , competence , and environmental factors such as employer employee relationship have implications for the rtw process .
the study was , however , based on focus - group interviews with labor experts working at the social insurance institute in the netherlands . in a recent swedish study , sjstrm et al9 evaluated a 7-week multidisciplinary rehabilitation program in patients with musculoskeletal pain , with emphasis on mental symptoms measured by the hads .
they found that , even if stress levels improved , those with full - time sick leave showed no changes in anxiety and depression levels during the 2-year study period . according to the authors , the results indicate that persons with musculoskeletal pain and comorbid psychological distress may require rehabilitation with different content than that given to employees with low psychological symptoms . in a recent systematic review of factors associated with work participation and work functioning in depressed workers ,
lagerveld et al29 found that work - related and personal factors were less frequently studied .
further , the authors stated that gaps in knowledge exist on factors predictive of work participation and work functioning in depressed workers .
these results illustrate that the rtw process among depressed employees with musculoskeletal conditions is multifactorial and not merely a result of physical functioning or pain severity .
epidemiological studies from the nord - trndelag health study in norway suggest that approximately one - third of individuals reporting somatic health problems also have anxiety disorder and/or depression30 and that a statistical relationship exists between anxiety , depression , and functional somatic symptoms , independent of age and sex.6 at the same time , medical examination of patients with chronic musculoskeletal pain often fails to demonstrate any organic diseases.15 in a norwegian population - based cohort study , mykletun et al31 found anxiety and depression to be strongly and independently associated with disability pensions granted for physical conditions and diagnoses , suggesting that administrative data may have underestimated the contribution of mental disorders to the awarding of disability pensions .
the latter findings are partly supported by a recently published swedish study28 of biopsychosocial functioning and assessment of the ability to work in 174 patients on long - term sick leave due to chronic musculoskeletal pain .
all patients underwent orthopedic assessment of physical function and ability to work ; 83 were referred for psychiatric evaluation of diagnosis and function .
the final evaluation involved the orthopedist and the psychiatrist working together as a team to form a mutual opinion of the patients ` functional , physical , and psychological abilities in relation to occupational demands and the prognosis for returning to work .
interestingly , the main cause of sick leave changed from a somatic diagnosis to a psychiatric diagnosis in 69% of patients .
of these , 33% were considered not able to return to work . according to the authors,28 the study sheds light on the limitations related to the assessment of ability to work , because physicians , lacking an established medical protocol , evaluate the patient according to their own experience .
inappropriate assessment of work ability leads to the wrong choice of interventions , which may employ and maintain unethical practices regarding the rtw process in employees with chronic musculoskeletal pain and comorbid depression .
depression and pain share biological pathways and neurotransmitters , which has implications for the treatment . additionally , psychiatric disorders in patients with chronic musculoskeletal pain have commonly been undiagnosed.32 the combination of depression and pain is associated with worse clinical outcomes than either condition alone .
thus , a treatment model that incorporates treatment of both depression and pain seems necessary for more optimal outcomes.33 pain is also a subjective sensory experience that is closely associated with affect , cognition , and aspects of the employee s social environment .
thus , it is possible that depressive symptoms may act both as a cause and as a consequence of chronic musculoskeletal pain and absence from work .
further , it is possible , although not necessarily the case , that work - related factors are significantly involved in this complex process . according to the literature,16 few studies exist on the bidirectional relationship between depression and occupational functioning . in a recent evaluation of a swedish 7-week rehabilitation program ,
sjstrm et al9 followed 60 participants with musculoskeletal pain for a period of up to 2 years .
they found that the ten participants with full - time sick leave ( absence from work for medical reasons ) during the study period showed improved stress levels but no change in anxiety and depression levels ( assessed by the hads ) .
this group also had higher pain ratings and higher subjective disability ratings during the rehabilitation program than did those with no sick leave or those on part - time sick leave .
the study illustrates that mental health problems and musculoskeletal pain are interrelated and that traditional multidisciplinary rehabilitation may not meet any special needs of this selected group .
all data are self- reported ; thus , it is not possible to confirm whether participants actually did return to work or not . given the use of perceived measures , one might argue that it is uncertain whether the results are indicative of physical ability or rather perception of ability .
further , we had no information on medical diagnoses or daily use of prescribed drugs . still , as we found no difference in pain experience between those returning to work and those not returning to work , it is unlikely that the better physical functioning was due to the experience of less pain among those not returning to work .
the hads is useful in the assessment of symptoms of depression and anxiety ; however , it is not validated as a diagnostic tool for clinical diagnoses in accordance with the international classification of diseases and the diagnostic and statistical manual of mental disorders , fourth edition.34 the influence of occupational history , such as if the participants were unemployed or if they held a temporary or a permanent occupation position , may influence the results , but these factors were not taken into account in our study .
in addition , the cross - sectional design of this study makes it impossible to make any inference on causality .
however , the main focus of this investigation was to assess the association between physical function and rtw among participants with chronic musculoskeletal pain with elevated depressive symptoms at the end of a multidisciplinary rehabilitation program .
nevertheless , 22% of those younger than 60 years participating in this 57-week multidisciplinary rehabilitation program had elevated depressive symptoms at the end of the program .
it is possible that these participants constitute a marginalized group consisting of those who have had the longest duration of musculoskeletal pain and the longest sick leave prior to their participation in the multidisciplinary program .
this , however , gives reason to speculate whether the evaluation of the ability to work may not have taken mental health status into account prior to or during participation in the multidisciplinary rehabilitation program .
further , and in line with the suggestions from sjstrm et al,9 it is possible that this selected group of individuals with elevated depressive symptoms may require rehabilitation with different content other than what is usually offered through a traditional multidisciplinary rehabilitation program .
physical limitations , physical ability and physical activity are all domains covering different aspects of human physical functioning .
most studies on mental health and physical function suggest an inverse association between decreasing levels of depressive symptoms and increasing physical functioning .
however , an exhaustive literature review showed that the association between depressive symptoms and physical functioning is unexpectedly weak.16 this may be due to low consensus on what physical measurement to use , and whether such measures should be performance - based or patient - reported .
bean et al24 found that self - reported measures of physical function were associated with psychosocial factors such as depression , while performance - based measures were not .
nevertheless , the former study was among elderly persons with mobility limitations and results can not be generalized to the sample in our study .
an alternative explanation for the association found in the present study is that self - report of physical functioning may be influenced by psychological distress . despite participation in an occupational rehabilitation program ,
some employees may lack motivation to return to a certain workplace . in a qualitative study of disability pensioners with chronic back pain ,
magnussen et al25 found that some of the barriers related to rtw were earlier negative work experiences , poor self - judgment of work ability , and low self - esteem .
further , many of the participants pointed to conditions at their former workplaces that they believed contributed to the disability process and that would make it difficult to return to work . hence , the experience of being granted a disability pension , which means not returning to work , might be followed by the feelings of relief and satisfaction , while those , by definition , on their way into work might be subjected to remaining in an unsafe and uncertain situation , both economically and occupationally . in turn
, this emotional state may directly affect one s evaluation of physical functioning , despite being depressed .
several studies support that the rtw is a multi - factorial process8 and that physical functioning should not be mixed up with occupational functioning .
occupational functioning is more than being able to perform certain tasks at work,26 as it also involves non job - specific behaviors such as cooperation and dedication .
thus , occupational functioning can be related to factors at the individual level , at the group level , and at the organizational level . nevertheless , according to the literature most psychosocial interventions to achieve optimal rehabilitation outcomes in employees with musculoskeletal pain
have focused on risk factors within the individual ( eg , pain catastrophizing and expectancies ) and not on relevant factors at the workplace , such as interpersonal conflicts or job stress ( at the group or institutional level).27 in the vast majority of observational studies on factors related to the rtw process , as in this study as well , psychosocial interventions did not follow common evidence - based guidelines or treatment protocols.27,28 in turn , this makes any evaluation of treatment outcome after rehabilitation difficult , especially regarding assessment of interventions that focus on work - related factors outside of the individual.27 further , depression affects several areas related to occupational functioning , such as absenteeism , education , and employment opportunities .
depressed workers also report more conflicts and negative social outcomes.16 in a qualitative study of factors relevant in the assessment of the rtw process of employees on long - term sickness absence due to a depressive disorder , muijzer et al4 found that physical functioning , attitudes , competence , and environmental factors such as employer
the study was , however , based on focus - group interviews with labor experts working at the social insurance institute in the netherlands . in a recent swedish study , sjstrm et al9 evaluated a 7-week multidisciplinary rehabilitation program in patients with musculoskeletal pain , with emphasis on mental symptoms measured by the hads .
they found that , even if stress levels improved , those with full - time sick leave showed no changes in anxiety and depression levels during the 2-year study period . according to the authors ,
the results indicate that persons with musculoskeletal pain and comorbid psychological distress may require rehabilitation with different content than that given to employees with low psychological symptoms . in a recent systematic review of factors associated with work participation and work functioning in depressed workers , lagerveld
further , the authors stated that gaps in knowledge exist on factors predictive of work participation and work functioning in depressed workers .
these results illustrate that the rtw process among depressed employees with musculoskeletal conditions is multifactorial and not merely a result of physical functioning or pain severity .
epidemiological studies from the nord - trndelag health study in norway suggest that approximately one - third of individuals reporting somatic health problems also have anxiety disorder and/or depression30 and that a statistical relationship exists between anxiety , depression , and functional somatic symptoms , independent of age and sex.6 at the same time , medical examination of patients with chronic musculoskeletal pain often fails to demonstrate any organic diseases.15 in a norwegian population - based cohort study , mykletun et al31 found anxiety and depression to be strongly and independently associated with disability pensions granted for physical conditions and diagnoses , suggesting that administrative data may have underestimated the contribution of mental disorders to the awarding of disability pensions .
the latter findings are partly supported by a recently published swedish study28 of biopsychosocial functioning and assessment of the ability to work in 174 patients on long - term sick leave due to chronic musculoskeletal pain .
all patients underwent orthopedic assessment of physical function and ability to work ; 83 were referred for psychiatric evaluation of diagnosis and function . the final evaluation involved the orthopedist and the psychiatrist working together as a team to form a mutual opinion of the patients ` functional , physical , and psychological abilities in relation to occupational demands and the prognosis for returning to work .
interestingly , the main cause of sick leave changed from a somatic diagnosis to a psychiatric diagnosis in 69% of patients . of these
, 33% were considered not able to return to work . according to the authors,28 the study sheds light on the limitations related to the assessment of ability to work , because physicians , lacking an established medical protocol , evaluate the patient according to their own experience .
inappropriate assessment of work ability leads to the wrong choice of interventions , which may employ and maintain unethical practices regarding the rtw process in employees with chronic musculoskeletal pain and comorbid depression .
depression and pain share biological pathways and neurotransmitters , which has implications for the treatment .
additionally , psychiatric disorders in patients with chronic musculoskeletal pain have commonly been undiagnosed.32 the combination of depression and pain is associated with worse clinical outcomes than either condition alone .
thus , a treatment model that incorporates treatment of both depression and pain seems necessary for more optimal outcomes.33 pain is also a subjective sensory experience that is closely associated with affect , cognition , and aspects of the employee s social environment .
thus , it is possible that depressive symptoms may act both as a cause and as a consequence of chronic musculoskeletal pain and absence from work .
further , it is possible , although not necessarily the case , that work - related factors are significantly involved in this complex process . according to the literature,16 few studies exist on the bidirectional relationship between depression and occupational functioning . in a recent evaluation of a swedish 7-week rehabilitation program ,
sjstrm et al9 followed 60 participants with musculoskeletal pain for a period of up to 2 years .
they found that the ten participants with full - time sick leave ( absence from work for medical reasons ) during the study period showed improved stress levels but no change in anxiety and depression levels ( assessed by the hads ) .
this group also had higher pain ratings and higher subjective disability ratings during the rehabilitation program than did those with no sick leave or those on part - time sick leave .
the study illustrates that mental health problems and musculoskeletal pain are interrelated and that traditional multidisciplinary rehabilitation may not meet any special needs of this selected group .
all data are self- reported ; thus , it is not possible to confirm whether participants actually did return to work or not . given the use of perceived measures , one might argue that it is uncertain whether the results are indicative of physical ability or rather perception of ability .
further , we had no information on medical diagnoses or daily use of prescribed drugs .
still , as we found no difference in pain experience between those returning to work and those not returning to work , it is unlikely that the better physical functioning was due to the experience of less pain among those not returning to work .
the hads is useful in the assessment of symptoms of depression and anxiety ; however , it is not validated as a diagnostic tool for clinical diagnoses in accordance with the international classification of diseases and the diagnostic and statistical manual of mental disorders , fourth edition.34 the influence of occupational history , such as if the participants were unemployed or if they held a temporary or a permanent occupation position , may influence the results , but these factors were not taken into account in our study .
in addition , the cross - sectional design of this study makes it impossible to make any inference on causality .
however , the main focus of this investigation was to assess the association between physical function and rtw among participants with chronic musculoskeletal pain with elevated depressive symptoms at the end of a multidisciplinary rehabilitation program .
nevertheless , 22% of those younger than 60 years participating in this 57-week multidisciplinary rehabilitation program had elevated depressive symptoms at the end of the program .
it is possible that these participants constitute a marginalized group consisting of those who have had the longest duration of musculoskeletal pain and the longest sick leave prior to their participation in the multidisciplinary program .
this , however , gives reason to speculate whether the evaluation of the ability to work may not have taken mental health status into account prior to or during participation in the multidisciplinary rehabilitation program .
further , and in line with the suggestions from sjstrm et al,9 it is possible that this selected group of individuals with elevated depressive symptoms may require rehabilitation with different content other than what is usually offered through a traditional multidisciplinary rehabilitation program .
the findings of higher physical functioning among depressed employees not returning to work after participating in a multidisciplinary rehabilitation program in this study illustrate that self - reported physical functioning does not act as a proxy for occupational functioning in the rtw process . according to the literature
, there is a need for more research on the bidirectional association between depression and occupational functioning .
further , recent studies suggest that there is a need for standardized procedures in the evaluation of the ability to work and that this procedure needs to take mental health status into account
. longitudinal studies with the use of different registries ( employment history , sick leave , and use of prescribed drugs ) should preferably be used in the evaluation of factors promoting the rtw process among people with chronic musculoskeletal disorders and poor mental health . | backgroundthe aim of this investigation was to assess whether measures of physical functioning after multidisciplinary rehabilitation are associated with return to work among individuals with chronic musculoskeletal pain conditions and comorbid depressive symptoms.methodsincluded were 92 employees with chronic musculoskeletal disorders who had participated in a 57- week multidisciplinary rehabilitation program .
their ages ranged from 2559 years . the hospital anxiety and
depression scale was used to assess depressive symptoms .
different aspects of physical functioning ( muscle strength , mobility , endurance capacity , and balance ) were measured with single - item visual analog scales , and physical fitness was measured with the validated coop / wonca charts .
being on active work strategies , such as receiving rehabilitation benefit / vocational rehabilitation or being reported partly or completely fit , was defined as on their way into / in work .
cross - sectional associations were measured using logistic regression models , estimating odds ratio with 95% confidence intervals.resultsthere were no differences between the on their way into / in work group ( n=70 ) and the on their way out / out of work group ( n=22 ) regarding age , sex , or levels of anxiety or pain .
surprisingly , regression analyses showed that those with higher levels of physical functioning had significantly lower odds of returning to work.conclusionthe findings of an inverse relationship between self - reported physical function and returning to work in this sample illustrate that the return - to - work process among employees with chronic musculoskeletal pain and comorbid depressive symptoms is multifactorial and influenced by factors other than physical functioning at the individual level .
further research , especially longitudinal studies , is needed to assess the occupational trajectories among employees with chronic musculoskeletal pain and comorbid depressive symptoms after participation in a multidisciplinary rehabilitation program . |
periodontitis can be defined as the perpetuating inflammation and tissue damage into the tooth supporting structures .
there are several types of periodontitis such as aggressive and necrotizing but the adult periodontitis is the most common form .
this disease , mostly caused by gram negative bacteria , is known to be a risk factor for many systemic diseases [ 2 , 4 ] .
many studies have shown a clear relationship between periodontitis and low birth weight , preterm labor , pulmonary diseases and diabetes [ 4 - 7].there are also some studies showing the inter relationship between this disorder and cardiovascular diseases [ 4 , 8 , 9 ] .
the main goal of periodontitis treatment is to remove infection and prevention of the disease progression .
most forms of periodontitis are currently treated by nonsurgical methods such as scaling and root plaining which can lead to healthy periodontium for a long time . however , recurrence is not uncommon [ 3 , 10 ] .
over the past decade ,
antibiotic administration has been shown to have synergistic therapeutic effects in some types of periodontitis in combination with mechanical debriment [ 11 , 12 ] .
some other studies manifest effectiveness of alternative methods such as using lasers , interdisciplinary treatments and neuromuscular stimulation .
however , none of the aforementioned methods can thoroughly remove the infective agents [ 14 - 17 ] .
over the past years
, our laboratory has focused on studying the health effects of exposure to some common and/or occupational sources of electromagnetic fields such as mobile phones [ 18 - 25 ] and their base stations , mobile phone jammers , laptop computers , radars , dentistry cavitrons and mri [ 30 , 31 ] . in this work , we designed and produced helmholtz coils for enhancing the healing process in periodontitis .
magnetic fields can be demonstrated by lines of force and these fields are produced by electric current flow .
static magnetic fields ( smf ) are formed around a permanent magnet or by direct current ( dc ) flow , while time - varying magnetic fields are produced by alternating currents ( ac ) with a frequency above zero .
we know that all living cells preserve a membrane potential in the order of tens of mv across their plasma membrane . on the other hand , the energy transduction in mitochondria
furthermore , the action potentials generated in excitable cells along their membranes are in the order of -70 mv and the frequency modulation of nerve impulses plays a very basic role in conveying information along neural networks .
some studies indicated the adverse biological effects of electromagnetic fields [ 34 - 36 ] while numerous studies showed the beneficial effects of these fields on both hard and soft tissues [ 37 - 39 ] .
many studies show positive effects of pemf on healing both soft and hard tissues [ 40 - 43 ] .
enhanced healing of anastomoses and wounds by static electromagnetic fields has been reported by different investigators .
have recently shown that pulsed electromagnetic fields significantly enhanced wound closure and re - epithelialization .
the authors of this report hypothesized that pemf can increase the myofibroblast population , contributing to wound closure during diabetic wound healing .
there are also studies indicating positive effect of pemfs in treatment of osteoarthritis and ulcers [ 48 - 51 ] .
a study conducted by girgin et al . suggested that pemf will improve all physical features of the colonic anastomoses . in this study , we designed and produced helmholtz coils for enhancing the healing process in periodontitis in rodents .
this device is made up of two parallel coaxial circular coils that are separated from each other by a distance equal to the radius of one coil for producing an approximately uniform magnetic field in the space between the coils .
in a pair of helmholtz coils consisting of two current loops ( figure 1 ) , each with n turns and radius r , which are separated by a distance r , the magnetic field at any point along the axis of the coils ( z ) can be calculated by summing the individual magnetic fields of the coils using the superposition principle . in this configuration
, biot - savart law can be used to calculate the magnetic field at the center of this system when z=0 ( point o on figure 1 ) as follows :
b is the magnitude of the magnetic field
r is the radius of the loop
n is the number of turns in the current loop .
0 is the permeability of free space
schematic diagram of the helmholtz coil manufactured in this study .
the diameter of each loop was 25 cm and the distance between the two loops was 12.5 cm .
the peak to peak voltage was set at 2.1 v ( 50 hz , ac ) .
in this preliminary study , 10 male wistar rats ( 200 - 250 g ) were purchased from the sums animal laboratory .
all ethical codes of the sums regarding the use of animal models were applied . to induce periodontitis , 3 - 0
multi - filament braided silk suture ( supasil , supa medical devices , iran ) thread was placed at the cervical region of second maxillary molars and was knotted at the palatal side ( right side was considered as a positive control ) .
later , the knot was transferred to the subgingival palatal region of the second molars .
the thread was kept in the place for 21 days including 7 days of irradiation .
two pairs of identical helmholtz coils were designed and manufactured in the ionizing and non - ionizing radiation protection research center ( inirprc ) at shiraz university of medical sciences , shiraz , iran . as shown in figure 1 ,
the distance between the coils is 12.5 cm ( the coils are spaced apart at a distance equal to their radii ) .
these coils have an inner diameter of 12.5 cm and an outer diameter of 13.5 cm .
the coils are driven by a sinusoidal signal from a function generator ( gfg-8020h , gw instrument co. , ltd ) . the magnetic field ( b ) at the center , between the coils ,
the magnetic field was relatively uniform ( alterations less than 5% ) in the central area of the coils .
the windings of our helmholtz coil were made of standard 0.95 mm wire to provide the maximum possible current .
the peak to peak voltage was set at 2.1 v ( 50 hz , ac ) .
an oscilloscope ( gds 2202 , gw instrument co. , ltd ) was coupled to the system for monitoring the 50 hz sinusoidal mf waveform .
the strength of the magnetic field that is dependent on factors such as the number of turns in the helmholtz coil and the current applied to the coils was measured by a recently calibrated emf meter ( lutron 828 , taiwan ) .
rats were exposed to pemf 4h / day for 7 days . during the exposure rats
the temperature inside the coil was monitored by a thermometer continuously during the experiment . in all animals ,
the left side was considered as experimental periodontitis side and the right side as the control ( no intervention ) .
in this preliminary study , 10 male wistar rats ( 200 - 250 g ) were purchased from the sums animal laboratory .
all ethical codes of the sums regarding the use of animal models were applied . to induce periodontitis , 3 - 0
multi - filament braided silk suture ( supasil , supa medical devices , iran ) thread was placed at the cervical region of second maxillary molars and was knotted at the palatal side ( right side was considered as a positive control ) .
later , the knot was transferred to the subgingival palatal region of the second molars .
the thread was kept in the place for 21 days including 7 days of irradiation .
figure 2 shows the irradiation protocol used in this study .
the ligature and irradiation protocol used in this study
two pairs of identical helmholtz coils were designed and manufactured in the ionizing and non - ionizing radiation protection research center ( inirprc ) at shiraz university of medical sciences , shiraz , iran . as shown in figure 1 ,
the distance between the coils is 12.5 cm ( the coils are spaced apart at a distance equal to their radii ) .
these coils have an inner diameter of 12.5 cm and an outer diameter of 13.5 cm .
the coils are driven by a sinusoidal signal from a function generator ( gfg-8020h , gw instrument co. , ltd ) . the magnetic field ( b ) at the center , between the coils ,
the magnetic field was relatively uniform ( alterations less than 5% ) in the central area of the coils .
the windings of our helmholtz coil were made of standard 0.95 mm wire to provide the maximum possible current .
the peak to peak voltage was set at 2.1 v ( 50 hz , ac ) .
an oscilloscope ( gds 2202 , gw instrument co. , ltd ) was coupled to the system for monitoring the 50 hz sinusoidal mf waveform .
the strength of the magnetic field that is dependent on factors such as the number of turns in the helmholtz coil and the current applied to the coils was measured by a recently calibrated emf meter ( lutron 828 , taiwan ) .
rats were exposed to pemf 4h / day for 7 days . during the exposure rats
the temperature inside the coil was monitored by a thermometer continuously during the experiment . in all animals ,
the left side was considered as experimental periodontitis side and the right side as the control ( no intervention ) .
helmholtz coils generated a uniform magnetic field between its coils which provided a highly homogenous field ( 5% ) .
the magnetic field strength at the center of the space between two coils was 97.6 t ( figure 3 ) .
a. the helmholtz coils manufactured in this study generated a uniform magnetic field between its coils which provided a highly homogenous field ( 5% ) .
b. the magnetic field strength at the center of the space between two coils was 97.6 t .
the purpose of this preliminary study was to compare tooth mobility at different time periods before and after the exposure phase . according to the american academy of periodontology ( aap )
tooth mobility can be defined as the movement of a tooth in its socket resulting from an applied force .
a modified version of miller s mobility index ( modified for rats ) was used for assessing and scoring the mobility of the teeth considering miller s mobility index , tooth mobility is divided into three classes ; score i- mobility up to 1 mm , score ii- mobility of 12 mm and finally score iii- mobility over 2 mm and/or rotation or depression .
our preliminary results showed that exposure to emf could significantly decrease the inflammation and mobility of the animals teeth .
these findings are generally in line with those of other investigators who reported the healing effects of exposure to magnetic fields in cases such as fractures [ 53 , 54 ] .
he exposed rabbits after osteotomy of the femur to low - frequency magnetic field produced by a pair of helmholtz - coils .
he came to this conclusion that magnetic fields have significant positive effects on fracture healing .
however , he stated that the mechanisms of interaction of magnetic field and bone fracture healing should be clarified by further studies .
it is worth mentioning that there are reports indicating that electromagnetic fields used clinically for accelerating bone healing also affect the proliferation of lymphocytes in vitro .
johnson have previously shown that t - cell proliferation can be modulated by in vitro exposure to electromagnetic fields .
based on the preliminary findings of this study , exposure of laboratory animals to pemf enhances the healing of periodontitis . although we can not directly extrapolate our findings on laboratory animals to humans , our results prompt us to perform further experiments on this issue .
| background : effects of electromagnetic fields on healing have been investigated for centuries .
substantial data indicate that exposure to electromagnetic field can lead to enhanced healing in both soft and hard tissues .
helmholtz coils are devices that generate pulsed electromagnetic fields ( pemf ) .
objective : in this work , a pair of helmholtz coils for enhancing the healing process in periodontitis was designed and fabricated .
method : an identical pair of square helmholtz coils generated the 50 hz magnetic field .
this device was made up of two parallel coaxial circular coils ( 100 turns in each loop , wound in series ) which were separated from each other by a distance equal to the radius of one coil ( 12.5 cm ) .
the windings of our helmholtz coil was made of standard 0.95 mm wire to provide the maximum possible current .
the coil was powered by a function generator .
results : the helmholtz coils generated a uniform magnetic field between its coils .
the magnetic field strength at the center of the space between two coils was 97.6 t .
preliminary biological studies performed on rats show that exposure of laboratory animals to pulsed electromagnetic fields enhanced the healing of periodontitis .
conclusion : exposure to pemfs can lead to stimulatory physiological effects on cells and tissues such as enhanced healing of periodontitis . |
in humans , intracerebral hemorrhage ( ich ) causes secondary damage in the brain through the induction of cerebral edema and perihematomal injury [ 1 , 2 ] .
perihematomal edema with mass effect is an almost universal complication of ich , elaborated over several days after the initial insult .
glutamate release , bioenergetic failure , inflammation , and apoptosis play a key role in the pathogenesis of secondary injury [ 3 , 4 ] . in animal models of ich
, molecular and cellular studies also indicate that hemorrhage induces inflammation , apoptotic cell death , and progressive tissue destruction in perihematomal tissue [ 57 ] .
various knockout mice have been bred to study the gene expression of the pro - oxidants heme and ferrous iron , proteolytic enzymes such as metalloproteinases , thrombin , and inducible nitric oxide synthase , as well as leucocyte adhesion molecules ( cd18 ) in the perihematomal area .
their genetic variability has turned the mouse into a key study animal for intracerebral hemorrhage despite the small size of its brain .
the hematoma is either induced by stereotactic injection of collagenase or autologous blood into the basal ganglia .
longitudinal studies of hematoma kinetics in mice are sparse and generally use one modality only .
the purpose of our pilot study was ( i ) to develop t1-and t2-weighted mri protocols for high - resolution studies of mouse brain in vivo which allow for the identification of hematoma and edema , and ( ii ) to perform a direct comparison of hematoma and edema volumes on mri with morphometric studies on cryosections .
serial mr imaging would allow longitudinal studies of the temporal and spatial evolution of intracerebral hemorrhages and perihematomal injury .
that would not only substantially reduce the amount of animals needed , but also better depict the time - dependent action mechanism of various pathogenetic factors .
c57bl/6 mice ( jackson laboratories , bar harbor , me , usa ) were maintained in the department of laboratory animal research at harvard medical school with access to food and water ad libitum .
all experiments were performed in accordance to the national institutes of health guide for the care and use of laboratory animals as well as the institutional guidelines established by the institutional animal care and use committee ( iacuc ) at harvard medical school .
a total of 11 male adult mice , 1012 weeks of age , with a body weight of 2030 g , were used .
mice were anesthetized by intraperitoneal injection of 0.5 mg / g body weight of avertin ( tribromoethanol , sigma - aldrich , st .
, mice were injected with 0.075u collagenase type vii - s ( sigma - aldrich , st .
louis , mo , usa ) in 500 nl saline into the left caudate putamen using the following stereotactic coordinates : 0.5 mm posterior , 3.0 mm lateral to the bregma , and 4.0 mm in depth .
collagenase was infused over 2 minutes using a stereotaxic injector ( stoelting , wood dale , il , usa ) and the 1 l - syringe ( hamilton , reno , nv ) stayed in place for 10 minutes to prevent reflux .
, two mice underwent mr imaging 2 , 8 , and 24 hours after collagenase injection to define the best early time points for mr imaging . in part 2
, nine mice were subjected to an imaging schedule from days 1 ( n = 9 ) , 3 ( n = 7 ) , 10 ( n = 5 ) , and 21 ( n = 2 ) based on the different phases of edema ( hyperacute , acute , subacute , chronic ) that bear their own pathophysiology .
animals were evaluated using a modified 28-point neurological scoring system before isoflurane anesthesia for the mr . the test included body symmetry , gait , climbing , circling behaviours , front limb asymmetry , and compulsory circling .
each point was graded from 0 to 4 , establishing a maximum deficit score of 24 .
mr imaging was performed with a high - field 4.7 tesla mr scanner ( biospin , bruker , germany ) at the center for basic mr research at beth israel deaconess medical center .
anesthesia was induced with 2% isoflurane and maintained with 1.6% isoflurane in 100% o2 via an mr - compatible nose cone apparatus to minimize motion artifacts .
mice were positioned headfirst and prone inside a plexiglass cradle with respiratory monitoring and a constant flow of isoflurane delivered directly to the nose of the mouse .
the magnet compatible apparatus allows a precise and reproducible fixation of the head at the center of the bruker linear birdcage radiofrequency coil with an inner diameter of 22 mm .
t1- and t2-weighted images were acquired at a 256 256 matrix and a 2.56 2.56 cm field of view . a relaxation enhancement ( rare )
sequence was used for both t1-weighted ( echo time / relaxation time [ te / tr ] = 12/500 , rare factor = 1 ) and t2-weighted ( te / tr = 75/2000 , rare factor = 12 ) imaging . t2
* -weighted imaging parameters were te = 14 ms , tr = 1500 ms , fov = 2.56 2.56 cm , and fa = 30 , matrix 128 128 . in all acquisitions ,
hematoma size was determined from t1 and t2 * images , and t2 images were used for edema quantification .
the measurements were performed by an examiner blinded to the exam date using a computer - assisted image analysis program ( imagej , nih , available at website http://rsb.info.nih.gov/ij/ ) .
the values for hematoma and edema volumes were compared with those determined from cryosections . following final mr imaging , brains were removed , placed in 4% paraformaldehyde and cryoprotected in 30% sucrose for 3 days at 4c before they were frozen .
coronal sections of 20 m - thickness were taken at 300 m intervals over the rostral - caudal extent of the lesion .
sections were stained with luxol fast blue and cresyl violet according to previously published protocols [ 15 , 17 , 18 ] .
lesion volumes ( mm ) were computed as running sums of lesion area multiplied by the thickness of each section ( 300 m ) over the extent of the lesion expressed as an orthogonal projection . given the small sample size at different examination dates , the correlation coefficient ( spearman coefficient ) for non - parametric correlations and two - tailed p values at a confidence interval ci = 95% were analyzed using one - way anova .
in two animals , mr imaging was performed as early as 2 hours after hematoma induction , and the hematoma was observed as a focal hypointensity on t1- , t2- , and t2 * -weighted images .
the hematoma remained stable without any evidence of expansion when scanned again 8 hours and 24 hours following collagenase injection ( figure 1 ) . in mice with serial mr imaging up to 72 hours , 10 and 21 days ,
the predominantly low - signal hematoma within the left caudate / putamen in t1- , t2- , and t2 * -weighted images were consistent with deoxyhemoglobin and intracellular methemoglobin after 24 hours .
the signal converted to predominantly high intensity at 72 hours to 10 days on t2-weighted rare spin - echo images , consistent with extracellular methemoglobin ( figure 2 ) .
the initially hypointense hematoma on t1-weighted images became hyperintense on days 3 and 10 . even on t2
* -weighted images , the signal of the hematoma core became hyperintense at 10 days . on day 21 ,
a low - signal streak was left at the site of the former space - occupying hematoma on t1- , t2- , and t2 * -weighted images ( figures 3 and 4 ) .
table 1 illustrates the mean hematoma sizes on t1- and t2 * -weighted images and the mean edema sizes on t2 on day 1 for nine mice , on day 3 for seven mice , on day 10 for four mice , and for the remainder ( n = 2 ) on day 21 .
after final imaging , the equivalent hematoma and edema volumes were compared with the hematoma and edema areas on histology as also shown in table 1 .
the spearman correlation coefficient between hematoma volume on t2 * -weighted images and histology was r = 0.61 ( p < 0.09 ) over all times points , thus not reaching statistical significance .
the spearman correlation coefficient between hematoma volume on t1 and histology , however , was statistically significant r = 0.7 ( p < 0.04 ) .
so was the coefficient for edema volume on t2 and histology on all examination dates with r = 0.73 ( p < 0.03 ) .
ongoing research in intracerebral hemorrhage and the prospects of therapy have raised the need for a noninvasive method to investigate ich in rodent models .
the quantification of hematoma volume is currently based on spectrophotometry assays [ 8 , 20 ] , histomorphometry on stained brain slices , or computer - assisted outlining of brain slices , thus excluding follow - up measurements during the disease progression and for the evaluation of novel therapeutic interventions .
the same accounts for edema volumetry that uses brain water content measurements [ 11 , 16 , 17 ] , blood - brain barrier permeability for evans blue dye on histology , or hemispheric enlargement for quantitation . among the biological imaging techniques , magnetic resonance imaging ( mri )
constitutes an excellent tool for neuroimaging in rats and pigs [ 22 , 23 ] .
owing to the variety of knockout genotypes in mice , repeated studies of the same animal are important research tools to study hematoma and edema evolution in a longitudinal fashion .
previous mri studies of the mouse brain have either been postmortem imaging of excised specimen with a near - microscopic spatial resolution [ 24 , 25 ] or in vivo studies with a high in - plane resolution at the expense of a much poorer section thickness . at high magnetic fields ,
the ratio between surface and volume in the head is important because of the presence of high susceptibility effects and poor magnetic field homogeneity in the whole head .
even with optimization of the shimming procedure and a reduction in tr , it was not possible to suppress this artifact completely . for t1-weighted sequence , the echo time was minimized to remove any t2 weighting and minimize susceptibility artifacts at air / tissue interfaces . in t2-weighted sequence ,
a rare factor of 12 increased the effective echo time and reduced the scan time .
the echo time in t2 * sequence was optimized to provide adequate signal - to - noise ratio while maximizing the contrast for measurement of hematoma .
a review of the literature revealed only two reports [ 13 , 27 ] with murine experimental ich and quantitation of hematoma size on t2 * alone or t2-weighted images .
given these extremely limited data on the changes in signal intensities in the course of hematoma resorption and the lack of data for edema formation , we undertook this study to describe the signal characteristics of hematoma resorption and edema evolution in mice over time . in our pilot study
, we show for the first time that intracerebral hemorrhages appear hypointense on t1 at high field strength as early as 2 hours after the ictus .
although less well demarcated compared to t2 * images , the hematoma remained discernable on t1 in the course of hematoma resorption .
hematoma volumes on t1 correlated well with those on histology ( p < 0.04 ) .
t2 images approximated edema size well in the acute to chronic phase of edema formation in mice when compared with histology ( p < 0.03 ) .
van der weerd et al . had found t2-weighted images useful for quantitation of lesion volume 24 hours after stroke in a mcao model .
as our results indicate , t2 * changes do not correlate with hematoma volumes on histology .
that might be due to blooming artifacts that render the suitability of t2 * images for quantification of hematoma at this field strength questionable .
knight et al . assessed the evolution of intracerebral hemorrhage in rats by mri estimates of t2 relaxation time and hematoma - induced changes in cerebral blood flow and blood - brain barrier permeability over 14 days using a 7-tesla , 20-cm bore magnet .
the lesion core and adjacent rim were identified by windowing of t2 values and changed in a consistent manner over time .
the mri and histological estimates of tissue loss were well correlated . despite the novel approach for following the temporal and spatial resolutions of intracerebral hemorrhages in mice by high field mr imaging ,
the high costs for experimental mr imaging at high field are a major drawback to large study groups .
the reproducibility of the murine ich model used in our study that was pioneered by clark et al . made it one of the principal models of murine ich [ 11 , 12 , 1517 , 30 ] .
theoretical concerns that bacterial collagenase might induce an inflammatory response , independent of that elicited by parenchymal blood , have not been confirmed for the activation of microglia in cell cultures after the addition of collagenase at various concentrations . whether the morphological substrate of what we depict as hyperintensity on t2-weighted images exclusively represents edema , remains questionable .
alternate techniques imply the injection of autologous blood into the striatum [ 8 , 10 ] , but lack the pathophysiologic event of vessel rupture .
repetitive multimodal mri scanning at 4.7 t in mice with collagenase - induced ich allows studying the individual kinetics of signal intensities underlying hematoma resolution and edema formation in a noninvasive manner .
that would allow studying a wide range of therapeutic treatments in a longitudinal fashion in mice . | background and purpose . pilot study to examine the use of t1- , t2- , and t2 * -weighted images for evaluating hematoma size and extent of edema in mouse brain at high field . methods .
following collagenase - induced intracerebral hemorrhage , nine mice were imaged at 4.7 t using t1- , t2- , and t2 * -weighted images for hematoma and edema quantitation on days 1 , 3 , 10 , and 21 after surgery .
values were compared with morphometric analysis of cryosections at the time of final mr imaging .
results . for hematoma quantitation , the spearman correlation coefficient ( r ) between t1 signal change and histology was 0.70 ( p < 0.04 ) compared with r = 0.61 ( p < 0.09 ) for t2*. the extent of perihematomal edema formation on cryosections was well reflected on t2 with r = 0.73 ( p < 0.03 ) .
conclusions . within the limits of our pilot study ,
mr imaging on 4.7 t appears to approximate the temporal changes in hematoma and edema sizes in murine ich well , thus laying the groundwork for longitudinal studies on hematoma resorption and edema formation . |
there is no doubt that our aging population presents society with a number of economic and public health challenges .
recent figures released by the uk automobile association predict that , in 20 years , 90% of men and 80% of women aged over 70 years will hold a driving licence .
contrast this with the situation today , where three - quarters of men and only 31% of women in that age group drive . in absolute terms , the number of drivers over 70 is set to double in 20 years and hit 10 million by 2050 .
it is also widely agreed that , per vehicle mile travelled , older drivers are at a higher risk of a fatal crash ( e.g. , ) .
these figures raise concerns for road safety with more older drivers driving more miles , and , for more years , there could be a profound impact on absolute numbers of road casualties in the future .
but in an enlightened society , road safety is only one half of the older driver
problem , as evans puts it ; we have a responsibility to meet the mobility needs of a growing population of older adults .
well - being in older people depends to a large extent on their ability to successfully engage with various practical and recreational activities in daily life . in turn , many of these activities are dependent on being able to drive .
driving thus enables older adults to keep on living independently and maintain their quality of life [ 7 , 8 ] .
is actually a problem for society not just a case of getting them off the roads .
indeed , many older people actually restrict their driving boundaries to conditions where they feel safe and comfortable [ 911 ] , further compounding the problem .
a solution is required which not only supports older drivers but also balances their requirements with road safety targets to continue reducing the number of killed and seriously injured on the roads . adopting a more user - centred approach
, such an alternative would be to compensate for the cognitive limitations of older drivers by making changes to the driving environment to make driving safer for the older person , both inside the car in terms of design factors , and perhaps advanced driving information systems , but also outside in terms of traffic system design [ 12 , page 5 ] .
both academic ( e.g. , [ 10 , 13 ] ) and policy reports ( e.g. , [ 1 , 3 , 7 , 12 ] have suggested that such a solution should exploit vehicle design and safety technology innovations inside the car , underpinned by a sound understanding of the older driver 's cognitive abilities and information requirements .
there is a very clear need for such research addressing appropriate technology to aid safe car driving behaviour amongst the older driver population .
a major objective of this paper is to consider the literature from several perspectives to understand and propose solutions for the older driver problem .
research from cognitive neuropsychology and driving ergonomics is examined , and important synergies and parallels between the two fields are identified as a basis for taking this work forward . specifically , we suggest that a promising basis for future research stems from work into cognitive inconsistency in neuropsychology and interest in driving performance consistency in ergonomics .
firstly , though , we look at road safety statistics to determine the extent of the problem .
although there is some debate over the prevalence of older drivers in road accident statistics , it is widely agreed that , when controlling for exposure , drivers over 70 are at increased risk of an at - fault accident with the data being comparable to those for the under-25 age group ( e.g. , [ 2 , 4 , 9 , 11 , 1315 ] ) .
casualty rates per mile driven increase with age after 7075 years , and the risk increases exponentially for drivers in their 80s [ 4 , 11 , 14 ] . whilst physical frailty is undoubtedly a factor in these statistics ,
the evidence for an association between age and crash risk is growing ; any remaining doubt is probably more due to the smaller population of older drivers and self - regulation of their driving behaviours ( cf . ) .
indeed , we currently rely on self - regulation to control the risk expecting older drivers to declare for themselves when they are unfit to drive .
but the evidence suggests this does not always work many drivers are not aware of ( or do not recognise ) their own limitations [ 9 , 11 ] , and they either do not cease driving early enough or conversely cease driving too soon . because of ( a ) lack of information , feedback , and insight , elderly drivers are not , i believe , in a good position to determine for themselves when they should reduce or cease driving [ 9 , page 171 ] .
moreover , as the older driving population grows and becomes more mobile , it is anticipated that mileages will increase , as will the need to face more challenging traffic conditions .
we have already said that , per mile driven , older drivers are almost at a similar risk of a crash as young drivers .
when the types of accidents are analysed , though , it is clear that older drivers differ from younger groups in that their accidents are less about taking risks , but more about errors of perception or judgement [ 2 , 4 , 11 , 14 ] . rather than single - vehicle accidents involving speed , alcohol , or fatigue ,
older drivers have multiple - vehicle accidents at junctions involving giving way , or when turning or changing lanes .
this tends to be due to deficits in bottom - up visual and cognitive processing , as opposed to top - down failures of experience or expertise .
verhaegen argues that this decrement in performance is consistent with the notion that cognitive abilities decline with age .
there is a wide body of scientific evidence to suggest that age - related declines in cognitive functions such as attention and decision making can be a source of increased crash risk on the roads ( e.g. , [ 1820 ] ) .
aging leads to declines in many perceptual and cognitive functions related to driving , with visual , spatial , and attentional abilities all having been shown as potential sources of increased risk . whilst the reduction in driver capabilities with age can be offset with experience [ 4 , 21 ] , as age increases beyond 65 years , fitness to drive ( in terms of sensory , perceptual , and cognitive abilities )
it has been shown that older drivers ( over 60 ) are slower , less accurate , and less coordinated in their responses .
tsimhoni and green used visual occlusion to demonstrate that drivers over the age of 55 experience more demand during driving on curved road sections , while brouwer et al . found that drivers in their mid-60s were less able to divide attention and integrate their responses in a dual - task scenario . in terms of driving tasks , negotiating junctions and merging traffic
undoubtedly , many of the problems that older drivers face are in part due to declining visual capabilities with age , affecting visual search at junctions ( e.g. , [ 14 , 25 ] )
. typically , it is the ambient or peripheral visual field which degrades [ 25 , 26 ] , leading some researchers to argue for a useful field - of - view
one such test has demonstrated sensitivity to crash risk in older drivers and shows promise as a screening instrument .
however , there is a significant body of evidence to show that visual acuity and the ufov test do not predict all aspects of driving performance ( e.g. , [ 28 , 29 ] ) and that central cognitive processing plays a key role alongside visual perception and decision making [ 5 , 11 , 13 , 18 ] . attention and executive function have both been implicated as predictors of driving performance [ 19 , 28 , 30 ] , with older drivers being more susceptible to errors under conditions of high mental workload [ 9 , 11 ] .
such cognitive declines are an inevitable part of the aging process : age - related decline in cognitive functions such as attention , anticipation , executive functioning and information processing means that older drivers tend to have difficulty in dealing with complex traffic situations and reduced capacity to respond quickly and flexibly to changing traffic situations [ 3 , page 45 ] .
adrian drew upon an established model of executive functioning and a standardised battery of tests to show that this is probably due to older drivers having difficulties with distraction and focusing their attention .
the idea of investigating independent cognitive measures in relation to driving performance in older adults is not new , and although meta - analyses and reviews ( e.g. , [ 31 , 32 ] ) suggest that tests of visuospatial skills may have some promise , the majority of studies have produced mixed findings .
efforts to develop metrics of individual cognitive abilities as correlates of driving performance have met with varying degrees of success [ 11 , 3133 ] . in particular ,
low to moderate correlations between cognitive measures and driving performance make it difficult to distinguish between those who are fit and unfit to drive , and those studies reporting high correlations have not been replicated .
reasons for these discrepant findings include the often small sample sizes , the wide variety of cognitive measures that have been assessed , and the different approaches to assessment of driving performance , their varying realism and their rigour .
the most apparent consequence of cognitive decline with aging is reaction time , which is slower for older drivers [ 4 , 9 ] .
recently though , there has been considerable interest in the cognitive aging field for investigating intraindividual variability , or inconsistency , of reaction time ( rt ) performance .
such measures index moment - to - moment fluctuations in performance over successive trials of a given cognitive task and are thought to reflect neurobiological integrity ( e.g. , [ 34 , 35 ] ) .
consistent with this view , increased variability is associated with older age ( e.g. , [ 36 , 37 ] ) and a range of neurological conditions including dementia and is a sensitive metric of cognitive function in older adults ( e.g. , ) .
importantly , measures of rt inconsistency are sensitive to relatively subtle effects when standard measures of accuracy and mean rt from the same tasks are not .
for example , recent work [ 39 , 40 ] found that the cognitive effects of increasing age and mild mental health disorders ( anxiety and depression ) were detected by measures of inconsistency , but not by measures of accuracy and mean rt .
data supporting the view that inconsistency is associated with neurobiological integrity were produced in a recent neuroimaging study of apparently healthy 60- to 64-year olds ; rt inconsistency was related to the degree of white matter lesioning in the frontal cortex , whereas mean rt was not .
this work has now been extended to demonstrate the same association in 44- to 48-year olds .
these studies clearly suggest that measures of rt inconsistency are sensitive not only to subtle effects in aging contexts but also to neurobiological integrity .
meanwhile , ergonomics research into driving performance has developed along parallel lines , with an important and distinguishing aspect of performance being consistency in driving both in terms of perceptual judgements and vehicle control .
bloomfield and carroll pioneered measures of lateral and longitudinal inconsistency in a driving simulator , arguing that these variables were more appropriate metrics of driving performance than traditional measures of mean or standard deviation .
their measures have since been successfully applied in several of our studies in the brunel university driving simulator ( e.g. , [ 44 , 45 ] ) and clearly distinguish good from poor drivers , in line with best practice for safe driving which suggests that smoothness and consistency is key . whilst the ecological validity of simulator studies
may be called into question , many modern simulators offer realistic and immersive representations of driving and provide a safe , replicable , and controllable environment for studies of this nature . moreover , with relevance to the present discussion , the higher - level cognitive and performance markers associated with driving are directly testable in a simulator . given the conceptual overlap with measures of cognitive inconsistency above , these studies together suggest that measures of rt inconsistency have huge potential in the present context .
it is striking , though , that to date no work has drawn these two lines of research together .
we believe the parallels between consistency - based metrics of driving and pc - administered measures of cognitive performance provide a promising and innovative basis from which to develop metrics and models that can help us better understand the cognitive limitations of older drivers .
pilot studies in our laboratory indicate that older adults ( in comparison to a younger group ) exhibit higher inconsistency on a neuropsychological test battery as well as higher inconsistency on driving performance metrics in a simulator . future research is planned to investigate and validate these associations in more detail . in terms of their application
the first is in response to calls for compulsory screening tests for older drivers : instead of asking whether and why older people have more accidents perhaps we should be asking which older drivers are more likely to have accidents [ 12 , page 45 ] .
previous assumptions about identifying a threshold age beyond which deterioration in cognitive functioning presents an unacceptable risk to driving have met with limited success , as individual differences make crude age - related cut - offs inappropriate [ 1 , 4 , 7 , 11 , 48 ] .
a more detailed instrument , tailored to individual differences , could be of use for drivers themselves in self - diagnosis , as well as by general practitioners ( gps ) as part of a wider battery assessing medical - psychological fitness to drive .
such cognitive testing could be in addition to existing practices for gp assessments , which include visual acuity , general health , and medications .
anecdotal evidence suggests that gps are currently uncomfortable with screening patients for driving ability , since they are not experts in driving standards . a cognitive testing instrument ,
as suggested here and elsewhere ( e.g. , ) , used in conjunction with standard medical assessment may help reassurance and reliability in these processes .
however , such screening remains contentious and does not accord with the spirit of solving the older driver problem maintaining safety and mobility .
the aim is to prolong independence , rather than try to remove older drivers from their cars . in that respect
, we can turn to user - centred design and a raft of technological support systems that are becoming available in cars .
numerous in - car technologies are coming on stream now that could support the cognitive functioning of older drivers ( cf . [ 3 , 10 ] ) , such as blind spot warning systems , lane keeping assistance , adaptive cruise control , speed limit displays , and collision mitigation braking systems .
nevertheless , such systems are very much a result of technology push rather than user pull ; what is needed is a balanced , user - centred assessment of these technologies .
the user - centred approach would argue that the cognitive limitations of older drivers may be compensated for by technological interventions which support the older driver in maintaining their independence and mobility .
for instance , vision enhancement systems could assist visual impairment associated with night - time driving ; similarly a head - up display ( hud ) could relieve the visual accommodation problems of eyesight in old age .
collision avoidance systems could help with speed and gap judgements at junctions , while adaptive cruise control or active steering could help reduce the demands of challenging driving situations .
earlier research in this area with younger drivers ( i.e. , under 55 years ) has shown that these systems can reduce driver workload and bring some improvements to driving performance [ 44 , 4951 ] .
extrapolating such results to older drivers may not be straightforward , though ; waller notes that the extent to which new technology could assist ( older drivers ) is not known . nevertheless , if new technology is designed , taking into account the abilities and limitations of older users , it holds promise of extending the self - sufficiency of many elderly drivers
( page 24 ) . lees and lee suggest that emerging vehicle technologies can be exploited to enhance the safety of older and younger drivers , by tailoring such systems to support bottom - up or top - down processing , respectively .
previous research on younger drivers supports this , indicating that advanced driver assistance systems ( adass ) can bring some improvements to driving performance [ 45 , 49 ] , while european projects such as prevent and eddit have explored the potential for extending these findings for the specific needs of older drivers . moreover , a recent uk project explored this very issue and reported that most new in - car technologies have so far ignored older drivers ' needs . using participatory methods , older drivers identified systems that enhanced feedback as having potential to assist their driving .
however , this approach could exacerbate problems of high mental workload with older drivers [ 9 , 11 ] .
in particular , the diminished capacities of older drivers could render them more susceptible to overload with poorly designed assistance ( cf .
earlier work in the drivage project ( e.g. , ) set out to evaluate the driving abilities of older people and to examine the potential benefits and distractions of providing additional information to the driver .
more recently , a government report noted that in - vehicle systems could specifically help older drivers but also cautioned that interface design and divided attention limitations might cancel out such benefits .
that said , horberry et al . found that older drivers were no more susceptible to distraction from in - car systems than younger drivers .
moreover , the technological limitations of earlier systems are rapidly being overcome , and new advances in multisensory displays offer enhanced feedback whilst avoiding distraction or overload for the older driver ( cf . ) .
results from the drivage project and elsewhere suggest that technological assistance inside the car is only of benefit if designed from a user - centred perspective .
more recent research has indicated the potential of developing high technology vehicular interfaces using participatory methods specifically to meet the needs of older drivers .
this research also highlighted that the theoretical opportunity for a technology to assist with specific limitations of older drivers could not always be accessed by the older driver group for a variety of reasons , including poor user interface design and technology immaturity .
more research is clearly necessary to develop technologies and interfaces which not only support older drivers but are also acceptable and accessible to the population of older drivers . thus we see that , on the one hand we have technological options , and on the other we have the needs and wants of older drivers ( e.g. , [ 10 , 57 ] ) .
but so far we do not have a complete user - centred solution based not only on the desires of older drivers but also their objective information processing requirements .
to finish , we bring together the themes covered in this paper to argue for a new stream of research addressing the older driver problem .
we have seen that , mile for mile , older drivers are at greater risk of a collision than those between the ages of 25 and 55 . one reason for this increase in risk is the decline in cognitive abilities with aging , particularly executive function .
but individual differences in the aging process , as well as societal acceptability , make it unreasonable to set an age - related threshold for dictating when older drivers should give up their licence . indeed , the user - centred solution searches for technologies to support older drivers in maintaining safety and mobility .
there are many technologies offered by vehicle manufacturers now , with more arriving in the near future , that could fulfil this purpose . however , few if any of these have been designed specifically for older drivers , and so their benefits may be limited .
participatory research has identified the needs and wants of older drivers when it comes to technology ; it is our contention that this is only half of the story , as systems should be specifically designed to compensate for the cognitive decrements associated with aging .
many researchers have tried to identify those decrements with respect to driving performance , with varying degrees of success .
we believe that we have discovered a promising avenue of investigation in the conceptual overlap between measures of intraindividual variability from the cognitive neuropsychology of aging and metrics of driving inconsistency from ergonomics . future research is planned to explore these overlaps in rigorous empirical studies and to use the output as a basis for specifying technological support systems for older drivers .
if we can identify systems to improve consistency in driving , we can go some way to solving the older driver problem . | the rise in the aging driver population presents society with a significant challenge how to maintain safety and mobility on the roads .
on the one hand , older drivers pose a higher risk of an at - fault accident on a mile - for - mile basis ; on the other hand , independent mobility is a significant marker of quality of life in aging . in this paper , we review the respective literatures on cognitive neuropsychology and ergonomics to suggest a previously unexplored synergy between these two fields .
we argue that this conceptual overlap can form the basis for future solutions to what has been called the older driver problem .
such solutions could be found in a range of emerging driver assistance technologies offered by vehicle manufacturers , which have the potential to compensate for the specific cognitive decrements associated with aging that are related to driving . |
oral squamous cell carcinoma ( oscc ) is the most common malignancy of the oral cavity , accounting for approximately 90% of oral neoplasms . despite aggressive treatment modalities
the overall 5-year survival rate of oscc , which is less than 50% , has not changed much in the past decades . as oscc is generally asymptomatic at an early stage ,
most cases are diagnosed at the advanced stage , when the treatment options are limited and the clinical outcome is rather poor .
unfortunately , highly sensitive and specific biomarkers for oscc are still not available for clinical use and exploring novel molecular markers is urgently needed .
micrornas are a family of small , non - coding rnas ( approximately 2125 nt in length ) with the ability to regulate protein output via silencing the expression of targeted genes at the posttranscriptional level .
mirnas play important roles in regulation of most cellular processes , such as cell proliferation , differentiation , migration , and survival .
deregulation of mirnas is closely linked to the pathological mechanisms of many human diseases , including cancer .
mirnas can not only regulate the expression levels of oncogenes and tumor suppressor genes to influence the progression of cancer , but also might directly function as oncogenes or tumor suppressor genes .
tu et al . reported that mir-372 and mir-373 were up - regulated in oscc tissues .
in addition , overexpression of these 2 mirnas was closely correlated with nodal metastasis , lymphovascular invasion , and poor survival , indicating that mir-372 and mir-373 might act as oncogenes in oscc and promote its progression .
mir-181a was down - regulated in oscc and enforced expression of mir-181a can inhibit tumor cell behavior , suggesting that mir-181a might play a tumor suppressive role in oscc .
dysregulation of mir-9 has been shown to participate in the carcinogenesis of many types of cancers , including oscc [ 1315 ] .
although previous studies have demonstrated that mir-9 is significantly down - regulated in oscc tissues and cell lines and might function as a tumor suppressor in oscc , whether the expression level of serum mir-9 is changed in patients with oscc has until now remained unknown .
therefore , the aim of this study was to evaluate the serum mir-9 expression level in oscc patients and its potential clinical values .
the study was approved by the ethics committee of the affiliated hospital of binzhou medical college and written informed consent was obtained from all the participants : 104 oscc patients , 30 olk patients , and 40 healthy volunteers .
study subjects were recruited from patients who received treatment at the department of oral surgery , the affiliated hospital of binzhou medical college and all cases were pathologically confirmed by at least 2 pathologists . in addition , subjects had not received any kind of therapy before serum sample collection .
up to 4-ml blood samples were drawn from subjects and then the specimens were centrifuge at 3500 g at room temperature for 5 min .
serum was transferred into rna - free ep tubes and stored at 80c until use . the qiagen mirneasy kit (
qiagen , hilden , germany ) was used to isolate total rna from serum samples according to the manufacturer s protocol .
then the miscript sybr green pcr kit ( qiagen ) was used to generate cdna and the subsequent pcr amplification .
the pcr reaction was performed using a 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) .
rnu6b was used as internal control for normalization of data , and the expression level of serum mir-9 was calculated and determined using the 2ct method .
the primers for mir-9 were : sense 5-cggagatcttttctctcttcaccctc-3 , antisense 5-caagaattcgcccgaaccagtgag-3. because the data were not normally distributed , the expression levels of serum mir-9 in patients with olk , patients with oscc , and healthy volunteers were analyzed using the kruskal - wallis test .
the chi - square test was used to evaluate the correlation between serum mir-9 expression level and clinical parameters of oscc .
the association between serum mir-9 and overall survival / disease - free survival was examined using the kaplan - meier method and log - rank test .
all data were analyzed using spss v21.0 software ( spss inc . , chicago , il , usa ) .
the study was approved by the ethics committee of the affiliated hospital of binzhou medical college and written informed consent was obtained from all the participants : 104 oscc patients , 30 olk patients , and 40 healthy volunteers .
study subjects were recruited from patients who received treatment at the department of oral surgery , the affiliated hospital of binzhou medical college and all cases were pathologically confirmed by at least 2 pathologists . in addition , subjects had not received any kind of therapy before serum sample collection .
up to 4-ml blood samples were drawn from subjects and then the specimens were centrifuge at 3500 g at room temperature for 5 min .
serum was transferred into rna - free ep tubes and stored at 80c until use . the qiagen mirneasy kit (
qiagen , hilden , germany ) was used to isolate total rna from serum samples according to the manufacturer s protocol .
then the miscript sybr green pcr kit ( qiagen ) was used to generate cdna and the subsequent pcr amplification .
the pcr reaction was performed using a 7500 real - time pcr system ( applied biosystems , foster city , ca , usa ) .
rnu6b was used as internal control for normalization of data , and the expression level of serum mir-9 was calculated and determined using the 2ct method .
because the data were not normally distributed , the expression levels of serum mir-9 in patients with olk , patients with oscc , and healthy volunteers were analyzed using the kruskal - wallis test .
the chi - square test was used to evaluate the correlation between serum mir-9 expression level and clinical parameters of oscc .
the association between serum mir-9 and overall survival / disease - free survival was examined using the kaplan - meier method and log - rank test .
all data were analyzed using spss v21.0 software ( spss inc . , chicago , il , usa ) .
the expression levels of serum mir-9 in patients with oscc and those with olk were significantly decreased compared with healthy controls ( p<0.01 ) . in addition , olk patients had a higher serum mir-9 expression level than oscc patients ( p<0.05 ) ( figure 1 ) .
the average expression level of serum mir-9 was first evaluated in healthy volunteers , and then the relative expression level of serum mir-9 in all oscc patients was determined by comparing it with the average expression level of serum mir-9 in healthy controls . the median expression level of serum mir-9 ( 0.58 fold ) was used to divide the oscc patients into a high serum mir-9 expression group and a low serum mir-9 expression group .
the chi - square test showed that the expression level serum mir-9 was associated with t stage ( p=0.013 ) , lymph node metastasis ( p=0.002 ) , and tnm stage ( p=0.007 ) .
however , it was not correlated with sex ( p=0.918 ) , age ( p=0.095 ) , smoking status ( p=0.225 ) , tumor site ( p=0.398 ) , or tumor grade ( p=0.060 ) ( table 1 ) . the survival analysis revealed that oscc patients in the low serum mir-9 expression group had poorer overall survival ( p=0.022 ) ( figure 2 ) and disease - free survival rates ( p=0.004 ) than the patients in the high serum mir-9 expression group ( figure 3 ) . for overall survival ,
univariate analysis showed that t stage ( p=0.038 ) , lymph node metastasis ( p=0.006 ) , tnm stage ( p=0.026 ) and serum mir-9 expression level ( p=0.015 ) were significant prognostic factors for oscc .
all the above clinical parameters were also independent risk factors for oscc ( table 2 ) in disease - free survival , t stage ( p=0.043 ) , lymph node metastasis ( p=0.005 ) , tnm stage ( p=0.031 ) , and serum mir-9 expression level ( p=0.011 ) were significant prognostic factors in univariate analysis .
lymph node metastasis ( p=0.006 ) , tnm stage ( p=0.040 ) , and serum mir-9 expression level ( p=0.018 ) were independent risk factors ( table 3 ) .
the expression levels of serum mir-9 in patients with oscc and those with olk were significantly decreased compared with healthy controls ( p<0.01 ) . in addition , olk patients had a higher serum mir-9 expression level than oscc patients ( p<0.05 ) ( figure 1 ) .
the average expression level of serum mir-9 was first evaluated in healthy volunteers , and then the relative expression level of serum mir-9 in all oscc patients was determined by comparing it with the average expression level of serum mir-9 in healthy controls . the median expression level of serum mir-9 ( 0.58 fold ) was used to divide the oscc patients into a high serum mir-9 expression group and a low serum mir-9 expression group .
the chi - square test showed that the expression level serum mir-9 was associated with t stage ( p=0.013 ) , lymph node metastasis ( p=0.002 ) , and tnm stage ( p=0.007 ) .
however , it was not correlated with sex ( p=0.918 ) , age ( p=0.095 ) , smoking status ( p=0.225 ) , tumor site ( p=0.398 ) , or tumor grade ( p=0.060 ) ( table 1 ) .
the survival analysis revealed that oscc patients in the low serum mir-9 expression group had poorer overall survival ( p=0.022 ) ( figure 2 ) and disease - free survival rates ( p=0.004 ) than the patients in the high serum mir-9 expression group ( figure 3 ) .
for overall survival , univariate analysis showed that t stage ( p=0.038 ) , lymph node metastasis ( p=0.006 ) , tnm stage ( p=0.026 ) and serum mir-9 expression level ( p=0.015 ) were significant prognostic factors for oscc .
all the above clinical parameters were also independent risk factors for oscc ( table 2 ) in disease - free survival , t stage ( p=0.043 ) , lymph node metastasis ( p=0.005 ) , tnm stage ( p=0.031 ) , and serum mir-9 expression level ( p=0.011 ) were significant prognostic factors in univariate analysis .
lymph node metastasis ( p=0.006 ) , tnm stage ( p=0.040 ) , and serum mir-9 expression level ( p=0.018 ) were independent risk factors ( table 3 ) .
in the present study , our results showed that the expression level of serum mir-9 was significantly down - regulated in patients with oscc or olk , indicating that mir-9 might be involved in regulating the initiation and progression of oscc .
detecting serum mir-9 expression level in patients with oral pre - cancer might help screen and identify the high risk population that has the potential to develop into oscc .
in addition , low serum mir-9 expression level was associated with advanced stage and poor prognosis of oscc , and serum mir-9 was an independent risk factor for oscc . based on our study results
, mir-9 probably acts as a tumor suppressor in oscc and this finding is consistent with previous reports .
yu et al . showed that mir-9 was under - expressed in oscc tissues and oral cancer cell lines .
in addition , overexpression of mir-9 could suppress the proliferative capacity of oral cancer cells both in vitro and in vivo .
mir-9 might have a tumor - suppressive role by downregulating the expression of cxc chemokine receptor 4 via the wnt/-catenin signaling pathway .
curcumin , a phytochemical derived from the rhizome of curcuma longa , has demonstrated antitumor activity in many types of tumors .
recently reported that curcumin inhibited proliferation of oral cancer cells by upregulating mir-9 expression and inhibiting wnt/-catenin signaling , indicating that the regulatory role of mir-9 in oscc closely interacts with wnt/-catenin signaling .
in addition to oscc , mir-9 has also been shown to suppress tumorigenesis in various types of cancers .
demonstrated that the expression of mir-9 was reduced in nasopharyngeal carcinoma ( npc ) tissues and cell lines , and the reduction of mir-9 in npc specimens was correlated with clinical stage and metastasis .
moreover , ectopic expression of mir-9 could significantly suppress proliferation , migration , and invasive capacity of npc cell lines , indicating that mir-9 might be a negative regulator of npc progression .
however , overexpression of mir-9 inhibited growth and induced monocytic differentiation in specific aml cell lines but not in all types of aml cells investigated , indicating the tumor suppressive role of mir-9 was strictly cell context - dependent .
similarly , wan et al . showed that mir-9 was downregulated in human gastric adenocarcinoma and that enforced expression of mir-9 could inhibit proliferation of cancer cells both in vitro and in vivo .
moreover , the tumor - related gene nf - kappab1was identified as a downstream targeted gene of mir-9 in gastric cancer . although mir-9 can suppress tumorigenesis in some types of cancers , it might also act as an oncogene .
wu et al . reported that the expression level of mir-9 was significantly upregulated in gliomas tissues compared with normal adjacent tissues .
in addition , mir-9 expression level was closely associated with who grade , karnofsky performance score , and overall survival , indicating that mir-9 may have an important role in tumor progression in human gliomas .
zhu et al . showed that the expression level of mir-9 was higher in colorectal cancer ( crc ) with distant metastasis than that in non - metastasis crc .
overexpression of mir-9 changed the morphological appearance of colorectal and enhanced the motility , suggesting mir-9 might be crucial for crc metastasis . several reasons might explain the contradictory role of mir-9 in different types of cancers .
firstly , the concrete function of mir-9 may be cancer - dependent . because a single mirna can target a number of downstream genes ,
it is common to observe the phenomenon that a specific mirna function as an oncogene in a certain type of cancer while it acts as a tumor suppressor in another type of cancer .
however , it was upregulated in patients with mixed - lineage leukemia - rearranged aml compared with the controls and could promote the progression of this specific type of aml .
the genetic changes in the leukemia cells might be responsible for the opposite role of mir-9 in different subtypes of aml .
the expression level of serum mir-9 was downregulated in patients with olk and those with oscc .
collectively , our data demonstrate that mir-9 is a tumor suppressor in oscc and can serve as a potential therapeutic target to treat this malignant disease . | backgrounddysregulation of mir-9 is a common feature of many types of cancers , including oral squamous cell carcinoma ( oscc ) .
however , whether the expression level of serum mir-9 is changed in patients with oscc remains unknown.material/methodsquantitative real - time polymerase chain reaction ( qrt - pcr ) was performed to examine the expression level of serum mir-9 in oscc patients , oral leukoplakia ( olk ) patients , and healthy volunteers , then we evaluated the association between serum mir-9 expression level and clinical outcome of oscc patients.resultsthe expression level of serum mir-9 was significantly downregulated in patients with oscc or olk in comparison with healthy controls ( p<0.01 ) .
serum mir-9 expression level was associated with various clinicopathological parameters , including t stage ( p=0.013 ) , lymph node metastasis ( p=0.002 ) , and tnm stage ( p=0.007 ) .
in addition , the oscc patients in the low serum mir-9 expression group had poorer overall survival rate ( p=0.022 ) and disease - free survival rate ( p=0.004 ) compared with those in the high serum mir-9 expression group .
multivariate analysis showed that serum mir-9 was an independent prognostic factor for oscc.conclusionsserum mir-9 was downregulated in patients with oscc and patients with olk .
in addition , low serum mir-9 was correlated with poor prognosis of oscc , indicating mir-9 might play a tumor suppressive role in oscc and can serve as a promising biomarker for this deadly disease . |
osteogenesis imperfecta is a heritable disorder characterized by bone fragility , deformity of the spine , and long bones .
additional abnormalities such as short stature , blue sclera , and dentinogenesis imperfecta have been described .
skeletal manifestations are due to a generalized deficiency of development of both membranous and endochondral bone and include markedly thinned calvarium with delayed closure of fontanelle and sutures and excessive wormian bone formation .
developed a classification of oi subtypes : oi type i with blue sclerae ; perinatal lethal oi type ii , also known as congenital oi ; oi type iii , a progressively deforming form with normal sclera ; and oi type iv , with normal sclerae .
levin et al . suggested that oi subtypes could be further divided into types a and b based on the absence or presence of dentinogenesis imperfecta . in the large majority of patients with oi types i iv , the disease is caused by mutation in the two genes that encode collagen type i alpha chains .
oi type v is characterized radiologically by interosseous membrane calcification of the forearms and a radiodense band visualised at the growth plate .
the aim of this paper is to further delineate the phenotypic characterization of osteogenesis imperfecta type v.
a - one - year - old boy was referred to our department for clinical evaluation .
progressive upper and lower limbs swellings have been noted since birth and were an unpleasant dilemma for the parents and the paediatrician .
the mother was a 28-year - gravida one - abortus 0 married to a 32-year - old unrelated man .
his subsequent course of development has been within normal limits . at the age of one year ,
craniofacially , there were sparse hair , frontal bossing , faint eye brows , depressed nasal bridge , full cheeks , and thin and transparent skin .
long philtrum was associated with thin upper lip and neither dentinogenesis imperfecta nor blue sclerae were present .
his neurological examination was normal , and there were no skin stigmata suggestive of a neurogenetic disorder .
the following biochemical parameters were within the normal range : serum and urinary oligosaccharides , mucopolysaccharides , serum lactate , pyruvate , creatine phosphokinase , alkaline phosphatase , calcium , phosphorus , and vitamin d metabolism and chromosomal study .
hormonal investigations included thyroid hormones ; adrenocorticotropic hormone and growth hormone were negative as well .
genetic testing revealed absence of mutation in col1a1 or col1a2 genes , respectively . on the bases of skeletal survey showed low mineralization of the skull bones associated with persistent opening of the anterior fontanelle .
the thorax showed generalized osteoporosis associated with thin and somehow gracile and short ribs ( figure 2 ) .
bilateral calcified interosseous membranes of the forearms associated with bilateral anterior subluxation of the radial heads , and the ends of the long bones were thick .
osteoporosis and broad and short bones of the hands and the carpal bones were small and dysplastic associated with apparent swelling of the forearms ( figure 3 ) .
the pelvis showed dysplastic ischium ; the femora were broad and short particularly over the distal parts .
calcification of the interosseous membrane was apparent bilaterally associated with overwhelming osteoporosis and the presence of a radiodense band visualized at the growth plate ( figure 4 ) .
supracondylar femoral fracture in the same patient at the age of 3 years note the profound development of callus around the lower third of the shaft of the femur .
this feature looks like a characteristic finding in patients with oi type v ( figure 5 ) .
nonaccidental swelling over the limbs is a constant feature in children with infantile cortical hyperostosis .
it is characterized by an infantile episode of massive subperiosteal new bone formation that typically involves the diaphyses of the long bones , mandible and clavicle .
there is onset of pain and swelling before the age of 6 months , often in the lower limbs or jaws , and sometimes accompanied by fever .
osteogenesis imperfecta is a rare inherited disorder of connective tissue with varying degrees of severity .
orthopaedic complications include osteopenia , recurrent fractures which are prone to nonunion and malunion resulting in deformities , protrusio acetabuli , and malalignment of the limbs .
there has been an increase in the lifespan of patients with osteogenesis imperfecta [ 1 , 6 , 7 ] .
osteogenesis imperfecta type i is the commonest form of osteogenesis imperfecta and is inherited as an autosomal dominant condition .
affected individuals may have blue sclerae with a tendency to fractures of the long bones , although healing occurs without deformity .
the majority of patients ( about 90% ) with a clinical diagnosis of oi have a mutation in col1a1 or col1a2 , the genes encoding collagen type i [ 8 , 9 ] .
osteoporosis pseudoglioma , cole - carpenter , and bruck syndromes have severe bone fragility with low bone mineral content .
idiopathic juvenile osteoporosis ( ijo ) is a transient nonhereditary form of bone fragility in children which is self - limiting and may be difficult to distinguish from oi type i. however collagen studies are negative in these individuals and iliac bone biopsy specimens show low remodelling rate in cancellous bone in ijo , whereas in oi there is high turnover in both cancellous and cortical bone .
reported 7 people who might have been classified as having type v , but there was calcification of the interosseous membrane in the forearm , hyperdense metaphyses , a coarsened pattern of matrix lamellae , and a tendency to develop hyperplastic callus .
cheung et al . reported a long - term followup of a child with hyperplastic callus formation and noted the considerable mobility .
anderson jr and hauge described the natural history of the hyperplastic callus formation in 23 patients with osteogenesis imperfecta type v. they concluded that hyperplastic callus formation is one of the conspicuous features of oi type v. the diagnosis of oi type v was based on interosseous membrane calcification of the forearms and a radiodense band visualized at the growth plate .
histopathologic analyses of rapidly growing hpc have shown distinctive zones with the outer regions of callus containing oedematous region showing hypercallus trabeculae of woven bone and small cartilaginous islands .
cho et al . encountered a single recurrent mutation in the 5-untranslated region of iftm5 encoding interferon - induced transmembrane protein 5 .
multiple swellings over the limbs associated with radiographic features of callus - like were the reason for suspecting nonaccidental abuse .
but , nevertheless , the constellation of long bone deformities and osteoporosis , with ossification of the interosseous membrane of the forearms , and anterior bilateral radial subluxation were distinctive features in favor of osteogenesis imperfecta type v. features of rapid periosteal apposition were noted almost all over the long bones albeit with varying degrees .
the short bones of the hands were short and broad and the carpal bones were small and dysplastic .
the normalcy of the parents could just as easily be a fresh dominant mutation or even an x - linked condition .
interestingly , our patient was tested for mutations affecting collagen type i , but no sequence abnormalities were detected . | swellings over the upper and lower limbs were encountered in a one - year - old child .
skeletal survey showed a constellation of distinctive radiographic abnormalities of osteoporosis , hyperplastic callus and ossification of the interosseous membrane of the forearm , femora , and to lesser extent the tibiae .
neither wormian bones of the skull nor dentinogenesis imperfecta was present .
genetic tests revealed absence of mutation in col1a1 or col1a2 genes , respectively .
the overall phenotypic features were consistent with the diagnosis of osteogenesis imperfecta type v ( oi - v ) .
the aim of this paper is to distinguish between swellings because of intrinsic bone disorders and these due to child physical abuse . |
goniometry , the measurement of joint angles , has been used as a staple in human physical therapy since the 1970s [ 15 ] .
it is commonly used as an objective measure of joint and muscle disease in addition to being used for patient assessment following joint or muscle trauma [ 68 ] .
goniometry is also regularly used in the human sector as an objective assessment of healing / improvement in cases of neurologic rehabilitation .
specifically , goniometry has successfully been used in patients receiving rehabilitation for diseases ranging from cerebral palsy [ 9 , 10 ] , to duchenne muscular dystrophy , to spinal cord injury . in veterinary medicine
, goniometry is used to assess outcome objectively in canine and feline patients undergoing physical therapy while recovering from orthopedic and neurologic disease [ 1316 ] . in the veterinary literature , there remain only a few published reports related to physical rehabilitation and the neurologic patient [ 1719 ] .
one of the most common neurologic diseases for which rehabilitation is a component of therapy is type 1 intervertebral disc disease .
type 1 intervertebral disc disease was originally described by hansen in the 1950s [ 20 , 21 ] .
disc desiccation leads to weakening of the annulus fibrosus and eventual herniation of the nucleus through the annulus and subsequent spinal cord compression .
dachshunds are the most common breed of dog affected by disc disease and herniation [ 22 , 23 ] . between 19 and 24% of dachshunds , within their lifespan , will suffer from thoracolumbar disc disease [ 22 , 23 ] .
the majority of disc herniation occurs in the thoracolumbar spine and results in pelvic limb weakness , leading to possible surgery and postoperative rehabilitation therapy .
for this reason , there is a significant need for reported range of motion measurements in miniature dachshunds , as a breed .
having published joint angle measurements for the pelvic limbs in miniature dachshunds would allow veterinary physical rehabilitation practitioners an objective means to guide and assess physical rehabilitation in their patients .
having objective data on limb girth measurements for miniature dachshunds presenting with various degrees of pelvic limb paresis would also be prudent as muscle atrophy is a realized sequela to neurologic injury including thoracolumbar disc extrusion .
one difficulty with joint angle measurements in animals versus humans is the variety of limb shape and girth differences among breeds and between animal species .
joint angles differ not only between dogs and cats but also between different dog breeds [ 13 , 14 , 25 , 26 ] . to date
, the use of goniometry has been validated in both labrador retrievers and cats .
joint angles have also been measured and reported for mixed breed dogs and greyhounds . a study by benson et al . compared joint angles between two breeds of dog ,
this study determined that there was variability between values for both dog breeds ; the final conclusion being that one universal table for normal joint angle values may not be applicable between dog breeds .
thus , there is a need for published range of motion measurements in a variety of dog breeds .
the aims of this paper are ( 1 ) to report the mean and median pelvic limb joint angles and limb girth measurements in miniature dachshunds presenting with ambulatory paraparesis , nonambulatory paraparesis , and paraplegia secondary to thoracolumbar intervertebral disc extrusion and ( 2 ) to compare joint angle and limb girth measurements between miniature dachshunds who present with ambulatory paraparesis , nonambulatory paraparesis , and paraplegia secondary to thoracolumbar intervertebral disc extrusion .
it is our hypothesis that we will be able to successfully report the mean and median goniometry and limb girth measurements in miniature dachshunds with varying degrees of neurologic dysfunction and that there will be no significant difference in these values between groups .
dachshunds that presented to the washington state university veterinary teaching hospital between april 2011 and february 2012 for thoracolumbar disc extrusion were included in the study .
dogs were being recruited for a physical rehabilitation study involving surgically addressed thoracolumbar disc herniation and hydrotherapy .
the project was approved by the animal care and use committee at the washington state university .
dogs were excluded from the study if they did not have a thoracolumbar disc extrusion on mr imaging and if they were not taken to surgery .
group 1 consisted of 3 dogs ; these dogs were ambulatory with pelvic limb paresis at the time of study inclusion .
group 2 consisted of 6 dogs ; these dogs were nonambulatory with pelvic limb paresis at the time of study inclusion .
group 3 consisted of 6 dogs ; these dogs were paraplegic at the time of study inclusion .
all measurements were taken within 24 hours of presentation , diagnostic imaging , and surgery .
awake dogs were put in lateral recumbency ; the angles of extension and flexion on the up pelvic limb were measured at the hock , stifle , and hip ( see figures 1 and 2 ) .
the dog was rotated to the contralateral side and the measurements were repeated on the opposite leg .
joint angle measurements were performed utilizing a previously published and validated technique . while in lateral recumbency , the girth of both the right and left pelvic limb was also measured in centimeters using a spring tape measure ( gulick ii tape measure , fitness mart , gays mills , wi ) .
the technique utilized to perform girth measurements was based on a previously published and validated technique .
thigh length from the greater trochanter to the distal femur at the level of the lateral fabella was measured using the spring tape measure .
the thigh circumference was measured 70% distal to the greater trochanter ( see figure 3 ) .
measurements were conducted by one of two certified physical rehabilitation individuals ( lal , sat ) , or a resident in neurosurgery , or a board certified neurologist ( avc , sat ) .
the statistical analyses were performed using a statistical software package ( sas , version 9.3 , sas institute , cary , nc ) .
mean and standard deviation as well as median values for joint flexion and extension in addition to thigh girth were calculated .
mean and median flexion and extension angles were compared between groups 13 for each joint .
each value was viewed independently : dog 1 left limb hock extension was one variable and right limb hock extension was a second variable .
the mean and median values for joint flexion and extension in addition to thigh girth were calculated regardless of group . a standard t - test
joint angle values were compared for hip , stifle and hock flexion , and extension ; girth was also compared . both goniometry values and girth were compared with a paired t - test .
the median and mean angles of flexion at the hip , stifle , and hock for all three groups of dogs are recorded in table 1 .
flexion angles differed between groups to a greater degree when measurements at the hip ( median values of 49 to 55 degrees and mean values of 49.1 to 54.3 degrees ) and stifle ( median values of 43.5 to 54 degrees and mean values of 46 to 56 degrees ) were made as compared to measurements at the hock ( median values of 39 to 40 degrees and mean values of 39.2 to 39.5 degrees ) .
the median and mean angles of extension at the hip , stifle , and hock for all three groups of dogs are recorded in table 2 .
similar to flexion angles , extension angles differed between groups to a greater degree when measurements at the hip and stifle were made as compared to the hock .
median extension values ranged from 151.5 to 160 degrees for the hip and mean values ranged from 152.5 to 156.5 degrees .
median values ranged from 160 to 163.5 degrees for stifle extension , while mean values were 157.3 to 164.2 degrees .
median values for hock extension were 167.5 to 172.5 degrees , while mean values were 167.5 to 171.5 degrees .
the median and mean pelvic limb girth measurements for all three groups of dogs are recorded in table 3 .
median limb girth ranged from 22.5 to 24 cm , while mean girth ranged from 22.7 to 23.7 cm .
there were no significant differences in joint angles or girth among the three groups ( ambulatory paraparetic , nonambulatory paraparetic , or paraplegic ) ; however , statistical significance was not reached ; p values ranged from 0.27 to 0.99 . when group was disregarded and values for extension , flexion , and girth combined , no differences existed .
we were able to successfully measure joint angles and girth in miniature dachshunds in this study .
it was found that when angles of flexion and extension between the three groups of dogs were compared , there was a trend toward measurements being more similar when made at the hock versus the stifle or hip . in our study , a variety of individuals made joint angle measurements , the trend in the data would support the idea of less variability when measurements of flexion and extension at the hock are made as compared to measurements at the stifle or hip in miniature dachshunds .
this finding is different than previous canine goniometry studies wherein measurements at all joints were found to be repeatable between individuals .
. found there to be high intraobserver agreement between staff members when goniometry was performed in labrador retrievers .
one explanation for this difference in miniature dachshunds could be the shape of the pelvic limb in this breed and its associated muscle mass . in this chondrodystrophoid breed , the bony landmarks utilized to make reliable goniometry measurements are more challenging to palpate and reliably locate as compared to the same structures in a long - legged breed , such as the labrador retriever .
similarly , the location of the thick pelvic limb thigh musculature in relationship to the inguinal fold , inherent to the miniature dachshund pelvic limb , made acquisition of limb girth measurements challenging .
it is unlikely that the trend toward increased range of joint angles measured in the hip and stifle as compared to the hock between groups was secondary to the neurologic status of the patients .
there was no trend toward one group of dogs having greater or lesser joint angles at one joint as compared to the next .
additionally , there was no significant degree of variability in limb girth between groups of miniature dachshunds enrolled in our study .
patients in this study suffered from thoracolumbar disc herniation and presented acutely after injury for work up and surgery .
thus , it is unlikely either disuse or neurogenic atrophy , both of which would affect limb girth measurements , was contributing to limb girth values in these dogs .
limb girth measurements proved to be challenging in this population of dogs . in this study ,
a previous study looking at circumference as measured at two separate locations along the femur ( 50% versus 70% of femur length ) showed it to be technically easier to make reliable measurements at a distance 70% of femur length .
the researchers postulated that because this more distal location is farther from the skin of the flank it is easier to get reliable measurements .
the unique shape of the dachshund leg and the close proximity of flank skin to the stifle made measurement of girth challenging .
some major goals of rehabilitation in neurologic patients are maintenance of muscle strength and joint mobility in addition to the reduction of muscle atrophy .
objective data such as joint angle and limb girth measurements is vital to gaging rehabilitation success in miniature dachshunds suffering from both orthopedic and neurologic injuries .
we conclude that joint angle and limb girth measurements can successfully be made in the miniature dachshund but that the unique shape and muscle distribution of the breed 's pelvis make obtaining these values challenging .
we also conclude that miniature dachshunds , with varying neurologic dysfunction at the time of range of motion and limb girth assessments , show no significant difference in values when measurements are made within 24 hours of acute onset of neurologic signs . | purpose . to report the mean and median pelvic limb joint angles and girth measurements in miniature dachshunds presenting with varying degrees of pelvic limb weakness secondary to thoracolumbar intervertebral disc extrusion
. methods . 15 miniature dachshunds who presented to wsu - vth for thoracolumbar disc extrusion .
dachshunds varied in neurologic status from ambulatory paraparetic to paraplegic at the time of measurements . results .
there were no significant differences in joint angles or girth among the three groups ( ambulatory paraparetic , nonambulatory paraparetic , or paraplegic ) ( p > 0.05 ) . when group was disregarded and values for extension , flexion , and girth combined , no differences existed . conclusions .
goniometry and limb girth measurements can successfully be made in the miniature dachshund ; however , the shape of the dachshund leg makes obtaining these values challenging .
there were no differences in joint angle or girth measurements between dogs with varying neurologic dysfunction at the time of measurement . |
malassezia species are lipophilic yeasts which are as part of the normal flora of human skin , and are found in mostly of healthy adults .
these yeasts are the cause of pityriasis versicolor and malassezia folliculitis , and also implicated in the pathogenesis of common skin disorders such as seborrheic dermatitis , psoriasis , and atopic dermatitis ( 1 ) .
pityriasis versicolor is a superficial fungal infection that usual clinical feature is slightly scaly patches of variable color ( either pink , brown , or white ) with irregular margin , most commonly found on the trunk and shoulders .
the relationship between malassezia species and their pathogenetic relationship was determined . in 1996 , guho et al .
classified malassezia genus into seven species , m. furfur , m. obtusa , m. globosa , m. slooffiae , m. sympodialis , m. pachydermatis , and m. restricta , based on morphological and biochemical characteristics ( 2 ) .
however , with the development of molecular methods such as polymerase chain reaction ( pcr ) , additional five species have been described which are m. yamatoensis , m. nana , m. japonica , m. equi , and m. dermatis(38 ) .
however , these phenotypic methods are usually time consuming , lack sufficient discriminatory power , and are unable to unambiguously differentiate newly identified species .
various dna - based molecular methods have been described to overcome this problem ( 9 ) .
the aim of this study was to identify malassezia species on their colonies , which were grown in culture media using 26s rdna polymerase chain reaction restriction fragment length polymorphism ( pcr - rflp ) .
patients with clinically suspected with pityriasis versicolor , referred to the mycology laboratory of the arak university of medical sciences were recruited .
seventy patients ( 85.2% ) were confirmed with pityriasis versicolor , based on the presence of both hyphae and yeasts in direct microscopy .
the sampled skin was inoculated in modified leeming and notman culture medium ( 1% w / v peptone , 1% w / v glucose , 0.2% w / v yeast extract , 0.8% desiccated ox bile , 0.1% v / v glycerol , 0.05% w / v glycerol monostearate , 0.5% v / v tween 60 , and 2% v / v oleic acid , 1% w / v agar in distilled water ) supplemented with cyclohexamide ( 0.5% ) and chloramphenicol ( 0.05% ) , and was incubated at 32c for two weeks .
isolated yeasts in 30% glycerol solution at -70 c were stored in the freezer .
their sequences were : forward , 5-taacaaggat - tcccctagta-3 and reverse , 5-attac - gccagcatcctaag-3 ( 11 ) . pcr amplification was performed in a final volume of 50 ul .
each reaction contained 1 ul of template dna , 0.5 of each primer , 0.20 mm of each deoxynucleoside triphosphate ( dntps ) , 5 ul of 10x pcr buffer , and 1.25 u of taq polymerase .
aninitial denaturation step at 94 c for 5 min was followed by 30 cycles of denaturation at 94 c for45 s , annealing at 55 c for 45 s , and extension at 72 c for 1 min , with a final extension step of 72 c for 7 min .
amplified products were visualized by1.5% ( w / v ) agarose gel electrophoresis in tbe buffer , stained with ethidium bromide ( 0.5 gml ) , and photographed under uv trans illumination .
the cfo i enzyme ( roche diagnostics , mannheim , germany ) was used in this study ( 7 ) .
the restriction enzyme reaction was performed by incubating a 21.5 ul aliquot of pcr product with 10 u of the enzyme in a final reaction volume of 25 ! at 37 c for 3 h. after the reaction , rflp pattern was analyzed with dna fragments in 2% agarose gel electrophoresis and staining with ethidium bromide .
patients with clinically suspected with pityriasis versicolor , referred to the mycology laboratory of the arak university of medical sciences were recruited .
seventy patients ( 85.2% ) were confirmed with pityriasis versicolor , based on the presence of both hyphae and yeasts in direct microscopy .
the samples were collected by scraping . the sampled skin was inoculated in modified leeming and notman culture medium ( 1% w / v peptone , 1% w / v glucose , 0.2% w / v yeast extract , 0.8% desiccated ox bile , 0.1% v / v glycerol , 0.05% w / v glycerol monostearate , 0.5% v / v tween 60 , and 2% v / v oleic acid , 1% w / v agar in distilled water ) supplemented with cyclohexamide ( 0.5% ) and chloramphenicol ( 0.05% ) , and was incubated at 32c for two weeks .
isolated yeasts in 30% glycerol solution at -70 c were stored in the freezer .
their sequences were : forward , 5-taacaaggat - tcccctagta-3 and reverse , 5-attac - gccagcatcctaag-3 ( 11 ) .
each reaction contained 1 ul of template dna , 0.5 of each primer , 0.20 mm of each deoxynucleoside triphosphate ( dntps ) , 5 ul of 10x pcr buffer , and 1.25 u of taq polymerase .
aninitial denaturation step at 94 c for 5 min was followed by 30 cycles of denaturation at 94 c for45 s , annealing at 55 c for 45 s , and extension at 72 c for 1 min , with a final extension step of 72 c for 7 min .
amplified products were visualized by1.5% ( w / v ) agarose gel electrophoresis in tbe buffer , stained with ethidium bromide ( 0.5 gml ) , and photographed under uv trans illumination .
the cfo i enzyme ( roche diagnostics , mannheim , germany ) was used in this study ( 7 ) .
the restriction enzyme reaction was performed by incubating a 21.5 ul aliquot of pcr product with 10 u of the enzyme in a final reaction volume of 25 ! at 37 c for 3 h. after the reaction , rflp pattern was analyzed with dna fragments in 2% agarose gel electrophoresis and staining with ethidium bromide .
from 70 skin samples , which were microscopically positive for malassezia elements , 60 samples were grown on culture medium ( 85.7% ) . using pcr reaction to amplify the 26s rdna region of all malassezia species ,
rflp analysis of pcr products of the its1 region with restriction enzyme cfo i was undertaken on 60 isolates .
restricta , lane 4 : m. furfur , lane 5 : m. sympodialis , lane 6 : m. restricta , m : 50 bp ladder using enzyme cfoi ( roche diagnostics , mannheim , germany ) , we could distinguish four different species , including 37 ( 61.6% ) m. globosa , 14 ( 23.3% ) m. furfur , 5 ( 8.4% ) m. sympodialisand 4 ( 6.7% ) m. restricta
. in one case was isolated m. globosa along with m. restricta . in this study m. pachydermatis ,
restricta , lane 4 : m. furfur , lane 5 : m. sympodialis , lane 6 : m. restricta , m : 50 bp ladder
the malassezia genus has undergone several taxo - nomic revisions since 1996 ( 2 ) .
recently , on the basis of dna relatedness , five new species have been included : m. dermatis , m. nana , m. japonica , m. yamatoensis , and m. aequi ( 38 ) .
various molecular methods of characterizing species of malassezia including pulsed - field gel electrophoresis ( pfge ) of malassezia species , internal transcribed spacer 1 ( its1 ) ribosomal dna sequences , aflp genotyping and random amplified polymorphic dna ( rapd ) have been used ( 1214 ) .
but this methods are time consuming and too expensive for use as a routine diagnostic method .
recently , pcr - rflp method used for identification of malassezia species from colony and patient skin scales ( 11 , 13 , 1517 ) .
thus in this study , we used pcr - rflp for identification of malassezia species in arak city , iran . in our study
, 70 skin samples were microscopically positive for malassezia elements and 60 samples were grown on culture medium ( 85.7% ) .
the commonest of species was m. globosa , that was similar to other studies ( 11 , 1724 ) .
however , makimura et al . , isolated m. furfur as the commonest of species ( 9 ) . in this study , in one case m. globosa a long with m. restrictawas isolated . among malassezia species of human skin flora ( 2527 ) ,
predominance of m. globosa from patients with pityriasis versicolor can be due to pathogenic characteristics of this species .
m. globosa was the commonest of malassezia species isolated from patients with pityriasis versi - color in arak city .
this study provides important data for epidemiological status of malassezia species in center of iran .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors . | abstractbackgroundthe lipophilic yeasts of malassezia species are members of the normal skin microbial that are cause of pityriasis versicolor .
pityriasis versicolor is a common superficial fungal infection with world - wide distribution .
the phenotypic methods for identification of malassezia species usually are time consuming and unreliable to differentiate newly identified species .
but dna - based techniques rapidly and accurately identified malassezia species .
the purpose of this study was isolation and identification of malassezia species from patients with pityriasis versicolor by molecular methods in markazi province , central iran in 2012.methodsmycologic examinations including direct microscopy and culture were performed on clinical samples .
dna extraction was performed from colonies .
the its1 region of rdna from isolates of malassezia species were amplified by pcr reaction .
the pcr were digested by cfo i enzyme.resultsfrom 70 skin samples , were microscopically positive for malassezia elements , 60 samples were grown on culture medium ( 85.7% ) . using pcr - rflp method , that was performed on 60 isolates , 37(61.6% ) m. globosa , 14(23.3% ) m. furfur , 5(8.4% ) m. sympodialis and 4(6.7% ) m. restrictawere identified . in one case was isolated m. globosa along with m. restricta.conclusionthe pcr - rflp method is a useful and reliable technique for identification of differentiation of malas - sezia species . |
4% of adults in the united states have attention - deficit hyperactivity disorder ( adhd ) , a neurobiological disorder associated with learning disorders , psychiatric comorbidity , and substantial education , occupation , and interpersonal impairments .
central nervous system stimulants , which include formulations of methylphenidate and amphetamines , are recommended as first - line medications in the pharmacologic treatment of adhd and have been demonstrated to be effective in the treatment of adhd .
, however , as with other drugs affecting the central nervous system , they have the potential to be misused , abused , and diverted for nonmedical purposes .
nonmedical use of prescription adhd stimulants has been documented among young adults in general and college students in particular , with rates of past - year nonmedical use ranging from 5% to 35% in various studies of college - age individuals .
kroutil et al . found the past - year prevalence of nonmedical use of adhd stimulants to be higher among young adults aged 18 to 25 years ( 1.3% ) than among individuals aged 12 to 17 years ( 0.9% ) or those aged 26 years or older ( 0.1% ) .
several studies have examined the correlates of nonmedical use of prescription stimulants , finding that those who engaged in non - medical use of prescription stimulants may also have engaged in nonmedical use and abuse of other drugs. for example , in a study of college students , mccabe et al . reported that students who engaged in nonmedical use ( defined as use not under a doctor 's orders ) of at least one of three prescription stimulant brands ( ritalin , dexedrine , and adderall ) were more likely to report illicit drug use , including use of marijuana , ecstasy , cocaine , and nonmedical use of prescription opioids .
for example , herman - stahl et al . analyzed young adult 's responses ( age range , 1825 years ) to the 2002 national survey on drug use and health ( nsduh ) and found that using marijuana and other illegal drugs increased the odds of both methamphetamine and nonmedical prescription stimulant use .
similarly , wu et al . found that 16 to 25 year olds reporting prescription stimulant misuse ( defined as nonmedical use , use that was not prescribed , or use only for the experience or feeling it caused ) were likely to report using three or more illicit drugs at least once in their lifetime .
although multi - drug use is well documented among nonmedical users of prescription stimulants , research to date has typically been limited to young adults or college students .
furthermore , limited analyses have been performed of drug use initiation patterns ( i.e. , whether the other drug use occurred before , during , or after the same time period as the non - medical prescription stimulant use ) . using data from the 20022004 nsduh , colliver et al .
examined the order of first nonmedical use of prescription stimulants and other prescription drugs and illicit drugs among lifetime users and found that the age at initial nonmedical use of stimulants was more often earlier than initial use of several other drugs , including crack cocaine , heroin , ecstasy , cocaine , prescription tranquilizers , pcp , and prescription pain relievers .
initial use of alcohol , cigarettes , marijuana , and inhalants preceded initial misuse of stimulants for most users of stimulants and other drugs . however , the broad category of stimulant medications , as measured in the nsduh and reported by colliver et al . ,
is composed of not only adhd medications , but also prescription diet pills and methamphetamine .
we sought to provide insight into the drug use histories of those specifically reporting nonmedical use of prescription adhd stimulants in an attempt to discern whether nonmedical use of prescription adhd stimulants was the first step in a pattern of abusing behavior or , conversely , whether it is a later development following a preexisting history of drug abuse .
we also sought to examine and compare the demographic characteristics of those reporting nonmedical use of prescription adhd stimulants , those reporting other drug use , and those reporting no drug use .
this study examined the drug use patterns and correlates of those reporting nonmedical use of prescription adhd stimulants in a general population sample , based on data from the 20022009 nsduh .
unlike prior datasets , which were largely restricted to young adults , the nsduh affords an opportunity to examine nonmedical use in a broad u.s .
we conducted an analysis of the public use data from the nsduh , an annual household - based survey sponsored by the u.s .
the nsduh collects information on the prevalence , patterns , and consequences of drug and alcohol use and abuse via in - person , computer - assisted interviews from a representative sample of the civilian , noninstitutionalized u.s .
population aged 12 years and older . specifically , nsduh collects information from residents of households and noninstitutional group quarters ( e.g. , shelters , rooming houses , dormitories ) and from civilians living on military bases .
the survey excludes homeless individuals who do not use shelters , military personnel on active duty , and residents of institutional group quarters , such as jails and hospitals .
the data used in this analysis are based on information obtained from a total of 443,041 respondents to the 2002 through 2009 surveys .
the nsduh solicits information about the nonmedical use of four broad categories of prescription medications : stimulants , pain relievers , tranquilizers , and sedatives .
nonmedical use is defined in the nsduh as use of these medications without a prescription of the respondent 's own or use simply for the experience or feeling the drug caused .
the survey includes questions that allow for estimation of nonmedical use of these broad categories of prescription drugs in the participant 's lifetime , the past year , and the past month .
the nsduh also contains detailed questions about the nonmedical use of a large set of specific medications .
however , this information is generally limited to use at any time in the individual 's life ( i.e. , lifetime use ) . additional information ( e.g. , age of first use ) is generally only obtained for the broad drug categories , not for specific medications . for questions regarding nonmedical use of prescription stimulants , respondents are presented with a single placard containing pictures of the common dosage forms of several different prescription stimulants and a listing of only the names of some others .
the stimulant category includes prescription - type metham - phetamine ( e.g. , desoxyn ) , prescription diet pills , and adhd medications .
our focus was on assessing the nonmedical use of stimulants with specific indications for the treatment of adhd ( table 1 ) .
prescription stimulants for adhd reported in the nsduh and used in the calculation of lifetime nonmedical use of prescription adhd stimulants dexedrine ( drug - specific question , open - ended responses ) dextroamphetamine ( drug - specific question , open - ended responses ) ritalin / methylphenidate ( drug - specific question , open - ended responses ) adderall ( drug - specific question from 2006 through 2009 , open - ended responses prior to 2006 ) dexmethylphenidate , focalin adhd / add medication ; otherwise unspecified abbreviations : adhd = attention - deficit hyperactivity disorder ; nsduh = national survey on drug use and health .
two recently approved stimulants for the treatment of adhd ( daytrana , approved in 2006 , and vyvanse , approved in 2007 ) were not included in this analysis because they were not available throughout the entire time period of study . however , an examination of nsduh data revealed no reports of nonmedical use of daytrana in response to open - ended questions in 20062009 ; < 0.01% of respondents reported nonmedical use of vyvanse in both 2009 and 2008 in response to open - ended questions , although there were no reports of nonmedical use in 2007 .
several adhd medications are presented in one of three groups ( ritalin and methylphenidate constitute one such group ) .
the remaining medications ( including dexedrine and dextroamphetamine ) are presented individually on the placard .
respondents are instructed to review the stimulant placard and are asked the following for each group :
have you ever , even once , used [ ritalin or methylphenidate ] that was not prescribed for you or that you took only for the experience or feeling it caused ? have you ever , even once , used [ ritalin or methylphenidate ] that was not prescribed for you or that you took only for the experience or feeling it caused ? for the medications presented individually on the card ( including dexedrine and dextroam - phetamine ) , respondents are told to look at all of the pictured or listed medications and are asked :
which of the stimulants shown below the red line on card c have you used when they were not prescribed for you or that you took only for the experience or feeling they caused ? which of the stimulants shown below the red line on card c have you used when they were not prescribed for you or that you took only for the experience or feeling they caused ?
nonmedical use for the remaining adhd stimulants ( e.g. , biphetamine , dextrostat , concerta , dexamyl , dexmethylphenidate / focalin , metadate , methylin ) was captured via the following open - ended question :
have you ever , even once , used any other prescription stimulant , besides the ones shown on card c when it was not prescribed for you or that you took only for the experience or feeling it caused ? have you ever , even once , used any other prescription stimulant , besides the ones shown on card c when it was not prescribed for you or that you took only for the experience or feeling it caused ? if the respondent answers yes to this question , then he or she is asked to state which other prescription stimulant they used . the medications in the previously described questions could also be captured in this open - ended question .
thus , information on the nonmedical use of prescription adhd stimulants is captured via responses to both close - ended questions that directly ask about nonmedical use of specific adhd medications and open - ended questions that ask broadly about any other prescription stimulant use .
prior to 2006 , information on nonmedical use of adderall ( any formulation ) was captured via the open - ended question about some other stimulant . however , in 2006 a series of questions specifically about adderall were added to a section of the survey entitled special drugs in which respondents are asked the following :
adderall is a prescription stimulant that comes in tablet or capsule form .
have you ever , even once , used adderall that was not prescribed for you or that you took only for the experience or feeling it caused ?
have you ever , even once , used adderall that was not prescribed for you or that you took only for the experience or feeling it caused ?
those who reported any nonmedical use of adderall were further asked when they had last used adderall nonmedically ( i.e. , within the past 30 days , more than 30 days ago but within the past 12 months , or more than 12 months ago ) . for our purposes , a person was considered to have engaged in nonmedical use of prescription adhd stimulants if they reported any non - medical use of a prescription stimulant specifically indicated for the treatment of adhd ( i.e. , any of the medications listed in table 1 ) during their lifetime , whether based on a drug - specific , closed - ended question ( including the adderall - specific questions added in 2006 ) or an open - ended question .
questions regarding nonmedical use of other prescription medications ( i.e. , prescription pain relievers , tranquilizers , and sedatives ) are asked in a similar manner .
specifically , respondents are shown visual examples of brand name pills and asked :
have you ever , even once , used [ any of the following medications when they were ] not prescribed for you or that you took only for the experience or feeling they caused ? have you ever , even once , used [ any of the following medications when they were ] not prescribed for you or that you took only for the experience or feeling they caused ?
respondents are also asked a follow - up question to assess nonmedical use of other prescription medications :
have you ever , even once , used any other [ prescription medication ] , besides the ones shown when it was not prescribed for you or that you took only for the experience or feeling it caused ?
have you ever , even once , used any other [ prescription medication ] , besides the ones shown when it was not prescribed for you or that you took only for the experience or feeling it caused ?
respondents who answer this question affirmatively are then asked to provide the names of other prescription medications used non - medically .
the names of up to five other prescription drugs may be provided for each category ( pain relievers , tranquilizers , stimulants , and sedatives ) .
the nsduh also collects information on age at first nonmedical use for the four broad categories of prescription drug type ( how old were you the first time you used any prescription [ pain reliever / tranquilizer / stimulant / sedative ] that was not prescribed for you or that you took only for the experience or feeling it caused ? ) .
this information is only collected at the category level , and not for specific medications within the broader class .
age at first use of illicit street drugs ( e.g. , cocaine / crack , inhalants , marijuana / hashish , heroin , and hallucinogens ) is also collected .
we calculated the percentage of those who reported ever having engaged in nonmedical use of a prescription adhd stimulant in the nsduh surveys of 20022009 .
sample sizes reported are unweighted ; all percentages and mean ages that are reported are weighted .
table 1 includes the list of specific adhd stimulants that were incorporated in this measure . among those who reported ever having engaged in nonmedical use of prescription adhd stimulants
, we calculated the age at first non - medical use for the broad category of prescription stimulants ( because information on age at first use for specific stimulant medications , including adhd medications , was not available ) .
thus , for those who engaged in nonmedical use of prescription adhd stimulants only ( and did not report nonmedical use of any other prescription stimulant medication ) the reported age at first use specifically represents the age at first nonmedical use for an adhd medication . for those who reported engaging in nonmedical use of other stimulants , as well as nonmedical use of prescription adhd stimulants ,
it is not possible to determine whether the reported age at first use refers to nonmedical use of an adhd medication or another type of stimulant medication . in this case
, the reported age at first use is only a proxy for age at first nonmedical use of prescription adhd stimulants . among those who reported ever having engaged in nonmedical use of prescription adhd stimulants , the reported age at first nonmedical use of any other prescription drug class ( i.e. , tranquilizers , pain relievers , and sedatives ) and age at first use of any illicit drug use
the reported average age for first nonmedical stimulant use was compared with the average age for first use of other drugs to determine the proportion of those engaged in nonmedical use of prescription adhd stimulants for whom initial use of other drugs ( marijuana , cocaine / crack , heroin , hallucinogens , and nonmedical use of tranquilizers , pain relievers , and sedatives ) occurred prior to their initial nonmedical use of prescription stimulants .
we also examined the following potential correlates of nonmedical use of prescription adhd stimulants : age , sex , race / ethnicity , marital status , education , employment status , and population density .
chi - square statistics and corresponding p values were calculated to compare the demographic characteristics of those reporting nonmedical use of prescription adhd stimulants with the following two groups : those reporting other nonmedical use or illicit drug use ( but no nonmedical use of prescription adhd stimulants ) and those reporting no drug use ( meaning no nonmedical use of prescription adhd stimulants and no nonmedical use of other pharmaceuticals or use of illicit drugs ) .
these statistics were calculated using suddaan version 10.0.1 , which accounts for the complex weighting scheme used by the nsduh .
over the study , 3.4% of americans aged 12 years and older reported ever having engaged in nonmedical use of prescription adhd stimulants . among those , 16.8% reported nonmedical use of adhd stimulants exclusively , whereas the remaining 83.2% reported nonmedical use of other stimulants in addition to adhd stimulants ( e.g. , prescription diet pills and methamphetamine ) . table 2 compares the demographic characteristics of ( 1 ) those reporting any lifetime nonmedical use of prescription adhd stimulants ; ( 2 ) those reporting other nonmedical use or illicit drug use ( including nonmedical use of non - adhd prescription stimulants for those not reporting any use of cocaine ( 61.4% ) and nonmedical use of prescription tranquilizers ( 52.2% ) .
prescription adhd stimulants used to calculate lifetime nonmedical use ( n = 443,041 ) abbreviations : adhd = attention - deficit hyperactivity disorder .
table heading percentages are weighted percentages based on the total sample and summed to 100% .
p values are determined by x tests vs. nonmedical use of prescription adhd stimulants ( i.e. , nonmedical use of prescription adhd stimulants ) .
nonmedical use of prescription adhd stimulants ) ; and ( 3 ) those reporting no drug use ( i.e. , no nonmedical use of prescription adhd stimulants , no nonmedical use of other pharmaceuticals and no use of illicit drugs ) .
overall , compared with those reporting other nonmedical use or illicit drug use and those reporting no drug use , those reporting lifetime nonmedical use of prescription adhd stimulants tended to be younger , male , caucasian , and unmarried ; 57.7% of those reporting lifetime nonmedical use of prescription adhd stimulants were 26 years and older compared with 78.3% for those reporting other drug use and 76.2% for those reporting no drug use . compared with the other two groups , a significantly larger percentage of those reporting nonmedical use of prescription adhd stimulants were men ( 57.4% vs. 53.1% for other drug users and 44.2% for those reporting no drug use ) . of those reporting nonmedical
use of prescription adhd stimulants , 87.0% were caucasian , non - hispanic , versus 72.8% for those reporting other drug use and 64.5% for those reporting no drug use . among those reporting nonmedical
use of prescription adhd stimulants , 69.0% were unmarried versus 49.3% for those reporting other drug use and 44.1% for those reporting no drug use . the majority ( 95.3% ) of those reporting nonmedical use of prescription adhd stimulants also reported use of at least one illicit drug ( i.e. , marijuana , cocaine / crack , heroin , hallucinogens , inhalants ) or nonmedical use of at least one other prescription drug ( i.e. , tranquilizers , pain relievers , sedatives , or non - adhd stimulants ) .
such use preceded nonmedical use of prescription adhd stimulants in 77.6% of cases ; on average , 2.40 ( 95% ci = 2.352.45 ) drugs were used prior to nonmedical use of prescription adhd stimulants . among those reporting nonmedical use of prescription adhd stimulants who also reported use of an illicit drug or nonmedical use of another prescription drug , the most commonly reported other drugs were marijuana ( 90.6% ) , hallucinogens ( 68.2% ) , and prescription pain relievers ( 66.1% ) ( table 3 ) .
these users also frequently reported age of first nonmedical or illicit drug use among those reporting nonmedical use of prescription adhd stimulants ( n = 21,465 ) abbreviations : adhd = attention deficit hyperactivity disorder ; se = standard error ; ci = confidence interval .
individual drugs are sorted by increasing age of onset statistics for any other drug use take into account all other drugs used by each individual : ( 1 ) age of first nonmedical use is the minimum age of first nonmedical use among all of the other drugs a person used ; ( 2 ) any individual who used at least one of the other drugs listed here is included as a yes in the percent other drug use calculation ; and ( 3 ) any individual who used at least one of the other drugs listed here prior to initial nonmedical use of prescription adhd stimulants is included as a yes in the percentage of other drug use preceding nonmedical use of prescription adhd stimulants calculation . among those reporting any nonmedical use of prescription adhd stimulants , the average age at first nonmedical use of a prescription stimulant was 19.45 years ( table 3 ) compared with an average age of 15.69 years for the initiation of other drug use . considering those who only reported nonmedical use of prescription adhd stimulants ( i.e. , those who did not report nonmedical use of any other stimulant type aside from adhd stimulants ) ,
the reported mean age at first nonmedical use of a prescription stimulant was 21.06 years . considering those who reported nonmedical use of other prescription stimulants , in addition to nonmedical use of adhd stimulants , the reported mean age at first nonmedical use of a prescription stimulant was 19.38 years . reported first use of marijuana ( average age = 15.92 years ) , inhalants ( average age = 17.48 years ) , hallucinogens ( average age = 17.95 years ) , and nonmedical use of prescription pain relievers ( average age = 19.37 years ) occurred at a younger age compared with the first reported nonmedical use of a prescription stimulant .
our findings suggest that nonmedical use of prescription adhd stimulants is not commonly the initiating factor leading to the abuse of other drugs .
rather , nonmedical use of prescription adhd stimulants appears to be adopted by individuals already engaged in broader patterns of drug abuse and nonmedical use . extending previous research on the correlates of nonmedical use of prescription stimulants among young adults and college students , we found in a general population sample that those who reported having engaged in nonmedical use of prescription adhd stimulants also reported having engaged in other drug use . among those reporting lifetime non - medical use of prescription adhd stimulants in our study , 95.3% also reported use of an illicit drug or nonmedical use of another prescription drug class , with such use typically ( in 77.6% of cases ) preceding nonmedical use of prescription adhd stimulants .
this suggests a broad trend of individuals already engaged in drug abuse and misuse who seek out new drugs as they become available .
it does not suggest that prescription stimulants indicated for the treatment of adhd have any particular appeal for first - time or nave nonmedical users of drugs .
in fact , available research suggests that individuals who are treated for adhd are at a lower risk for developing a subsequent substance abuse disorder than those with untreated adhd .
our study also extends prior research by providing some insight into the patterns of drug use over a user 's career . for those reporting both nonmedical use of prescription adhd stimulants and other drug use , the other drug use preceded nonmedical use of prescription adhd stimulants in 77.6% of cases , with an average of 2.40 drugs reportedly used prior to the first nonmedical use of prescription adhd stimulants .
because the nsduh question about age of first use pertains to all prescription stimulants ( not adhd stimulants specifically ) , this indicates that nonmedical use of prescription adhd stimulants first occurred after at least 2.40 other drugs were used non - medically or illicitly .
it was not possible to tell how often nonmedical use of prescription adhd stimulants occurred only after nonmedical use of other prescription stimulants had already been initiated because the nsduh only characterizes initiation of nonmedical use of the prescription stimulant class , without regard to brand .
this likely results in under - counting of drug use preceding nonmedical use of prescription adhd stimulants .
for example , someone who engaged in nonmedical use of prescription diet pills prior to nonmedical use of prescription adhd stimulants would not be counted as having engaged in nonmedical use prior to non - medical use of prescription adhd stimulants .
consequently , the data underestimate the extent that drug use preceded nonmedical use of prescription adhd stimulants .
it is unknown how many of those reporting nonmedical use of prescription adhd stimulants in our study had also been prescribed the drug legitimately ; that is , the data are not able to distinguish between the misuse of adhd medications by those for whom it is prescribed and nonmedical use by those without a prescription
. however , prior research has found misuse among both groups to be associated with multidrug use .
for example , in a college sample , similar proportions of those with and without a prescription for stimulants ( 16%18% ) reported nonmedical use of stimulants ( defined as use of any form of prescription stimulants that were not prescribed for you or that you took only for the experience or feeling they caused or overuse of a medically prescribed stimulant ) , and lifetime nonmedical use was independently and significantly associated with past year use of marijuana , inhalants , cocaine , hallucinogens , heroin , amphetamines or methamphetamine , ecstasy , prescription analgesics , and prescription tranquilizers . this study has several limitations .
first , results can not be generalized to those population groups excluded from the nsduh sampling frame , including homeless individuals who do not use shelters , active military personnel , and residents of institutional group quarters , such as jails and hospitals .
the nsduh only obtains information on lifetime nonmedical use of specific brands of prescription stimulants ; it is not designed to estimate current use at the brand - specific level . because the nsduh question about age of first use pertains to all prescription stimulants ( not adhd stimulants specifically ) , it was not possible to tell how often nonmedical use of prescription adhd stimulants occurred only after nonmedical use of other prescription stimulants had already been initiated .
furthermore , the information on age at first use is based on retrospective self - report , and therefore is subject to recall bias .
in addition , the calculated percentage of other drug use preceding nonmedical use of prescription adhd stimulants could be affected by older adults who may have used other substances in adolescence or early adulthood before the prescribing of stimulants for the treatment of adhd became more prevalent .
the specific survey question on which estimates of lifetime nonmedical use are based does not allow for differentiation between what might typically be considered abuse of prescription stimulants ( e.g. , for psychotropic effect ) and situations involving use by an individual for whom the medication was not prescribed , even if it was taken only once and for a purpose consistent with the therapeutic effect ( e.g. , an adult with suspected adhd who took the medication prescribed for their child , experienced the medication benefits , and then went on to receive a legitimate prescription after consulting a physician ) .
finally , the nsduh does not collect information on motives for nonmedical use , which would provide better understanding of the behavior .
nonmedical use of prescription adhd stimulants does not appear to be an initiating factor leading to the nonmedical use of other prescription medications or abuse of illicit drugs .
rather , nonmedical use of prescription adhd stimulants appears to be adopted by individuals already engaged in broader patterns of drug abuse and misuse .
the definition of nonmedical use can vary by study but is defined by the samhsa and in this article as use that was not prescribed or that you took only for the experience or feeling it caused . in cases where the term is used in other published article , we specify the exact definition of the term as used by the study authors . | multidrug use is well documented among nonmedical users of prescription stimulants .
we sought to provide insight into the drug use patterns of those reporting nonmedical use of prescription attention - deficit hyperactivity disorder ( adhd ) stimulants in an attempt to discern whether such use is a first step in a pattern of drug - abusing behavior or , conversely , is a later development accompanied or preceded by a history of drug abuse . a cross - sectional , population - based survey of the u.s .
civilian , non - institutionalized population aged 12 years and older was analyzed for lifetime nonmedical use of prescription adhd stimulants , lifetime nonmedical use of another prescription drug , illicit drug use , and drug use initiation patterns .
this included 443,041 respondents from the 20022009 national survey on drug use and health .
lifetime nonmedical use of prescription adhd stimulants was reported by 3.4% of those aged 12 years and older . of these ,
95.3% also reported use of an illicit drug ( i.e. , marijuana , cocaine / crack , heroin , hallucinogens , inhalants ) or nonmedical use of another prescription drug ( i.e. , tranquilizers , pain relievers , or sedatives ) , and such use preceded nonmedical use of prescription adhd stimulants in 77.6% of cases . on average , 2.40 drugs were used prior to the first nonmedical use of prescription adhd stimulants .
these data suggest that nonmedical use of prescription adhd stimulants is not commonly an initiating factor leading to the nonmedical use of other prescription medications or abuse of illicit drugs .
rather , nonmedical use of prescription adhd stimulants appears to be adopted by individuals already engaged in broader patterns of drug abuse and misuse . |
to examine the putative relationship between ptsd and osa , we determined the apnea hypopnea indices ( ahi ) , body mass index ( bmi ) , ptsd severity , and nightmare complaints in veterans with ptsd , veterans without ptsd , and healthy controls .
twenty - three male veterans with ptsd were recruited through the outpatient clinic of the military mental healthcare , utrecht , the netherlands .
ptsd patients with habitual benzodiazepine usage ( n=1 ) and substance abuse were excluded ( n=2 ) .
patients who used benzodiazepines , alcohol , or drugs weekly or monthly were instructed to refrain from these substances on the day of the sleep recordings .
twenty - four trauma controls ( tcs ; veterans without ptsd ) and 17 healthy controls ( hcs ; civilians or service members who were nave for deployment ) were recruited through advertisements in veteran - related magazines and newspapers .
tcs were matched for age , year , and region of deployment with the ptsd group and were excluded when they had a clinician - administered ptsd scale ( caps ) score of 18 or higher .
hcs were matched for age with the ptsd group and were excluded when reporting significant psychotrauma in the past according to the caps .
all control subjects were medically healthy males without a history of psychiatric disorders and without sleep complaints .
written consent was obtained from all participants , after a complete written and verbal description of the study .
the study was approved by the institutional review board of the university medical centre of utrecht , the netherlands .
all veterans were screened for psychiatric illness using the structured clinical interview for dsm iv axis i disorders [ severe combined immunodeficiency ( scid ) ] ( spitzer , williams , gibbon , & first , 1992 ) .
the diagnosis of ptsd ( assessed with the scid ) was confirmed by the caps ( blake et al . , 1995 ) and
, e.v . ) . only patients with a caps score above 50 were included .
trauma controls were included if they met the a1 criterion for ptsd ( the person experienced , witnessed , or was confronted with an event or events that involved actual or threatened death or serious injury , or a threat to physical integrity of self or others ) but had a caps score below 18 and did not meet dsm iv criteria for ptsd or any other life time axis i disorder .
subjects were screened for medical conditions by history taking and physical examination . as a high bmi
psg recordings were obtained during one night at kempenhaeghe , the netherlands , with brainlab real time , including electrooculography for vertical and horizontal eye movements , electromyography ( chin , left and right m. tibialis anterior ) , electrocardiography , and electroencephalography ( f0-c0 , f3-c3 , p3-o1 , c4-a1 , o2-a2 ) , airflow at the nose ( nasal pressure ) , movement of the thorax and abdomen by inductance plethysmography , and arterial oxygen saturation at the index finger .
sleep data were analyzed according to criteria of rechtschaffen and kales ( 1968 ) by an experienced sleep technician who was blinded to group identity .
apneas and hypopneas were scored when an airflow reduction of 50%90% ( hypopnea ) or > 90% ( apnea ) was detected for at least 10 seconds , except when such airflow reductions occurred after arousals or during wake .
an ahi>15 per hour is the cutoff for obstructive sleep apnea syndrome ( epstein et al . , 2009 ) .
an ahi>10 is also considered clinically relevant and used as an exclusion criterion in sleep studies ( engdahl , eberly , hurwitz , mahowald , & blake , 2000 ; mellman , nolan , hebding , kulick - bell , & dominguez , 1997 ) .
we compared both indices between groups . in comparisons of mean caps scores between ptsd patients with and without osa , we used the cutoff of ahi>10 .
an anova was used to test group differences when variables were normally distributed . in other cases , a non - parametric mann
correlations were analyzed with a pearson 's test and were controlled for age and bmi .
twenty - three male veterans with ptsd were recruited through the outpatient clinic of the military mental healthcare , utrecht , the netherlands .
ptsd patients with habitual benzodiazepine usage ( n=1 ) and substance abuse were excluded ( n=2 ) .
patients who used benzodiazepines , alcohol , or drugs weekly or monthly were instructed to refrain from these substances on the day of the sleep recordings .
twenty - four trauma controls ( tcs ; veterans without ptsd ) and 17 healthy controls ( hcs ; civilians or service members who were nave for deployment ) were recruited through advertisements in veteran - related magazines and newspapers .
tcs were matched for age , year , and region of deployment with the ptsd group and were excluded when they had a clinician - administered ptsd scale ( caps ) score of 18 or higher .
hcs were matched for age with the ptsd group and were excluded when reporting significant psychotrauma in the past according to the caps .
all control subjects were medically healthy males without a history of psychiatric disorders and without sleep complaints .
written consent was obtained from all participants , after a complete written and verbal description of the study .
the study was approved by the institutional review board of the university medical centre of utrecht , the netherlands .
all veterans were screened for psychiatric illness using the structured clinical interview for dsm iv axis i disorders [ severe combined immunodeficiency ( scid ) ] ( spitzer , williams , gibbon , & first , 1992 ) .
the diagnosis of ptsd ( assessed with the scid ) was confirmed by the caps ( blake et al . , 1995 ) and after consensus by two clinicians ( svl .
trauma controls were included if they met the a1 criterion for ptsd ( the person experienced , witnessed , or was confronted with an event or events that involved actual or threatened death or serious injury , or a threat to physical integrity of self or others ) but had a caps score below 18 and did not meet dsm iv criteria for ptsd or any other life time axis i disorder .
subjects were screened for medical conditions by history taking and physical examination . as a high bmi is associated with ahi , weight and length were determined .
psg recordings were obtained during one night at kempenhaeghe , the netherlands , with brainlab real time , including electrooculography for vertical and horizontal eye movements , electromyography ( chin , left and right m. tibialis anterior ) , electrocardiography , and electroencephalography ( f0-c0 , f3-c3 , p3-o1 , c4-a1 , o2-a2 ) , airflow at the nose ( nasal pressure ) , movement of the thorax and abdomen by inductance plethysmography , and arterial oxygen saturation at the index finger .
sleep data were analyzed according to criteria of rechtschaffen and kales ( 1968 ) by an experienced sleep technician who was blinded to group identity .
apneas and hypopneas were scored when an airflow reduction of 50%90% ( hypopnea ) or > 90% ( apnea ) was detected for at least 10 seconds , except when such airflow reductions occurred after arousals or during wake .
an ahi>15 per hour is the cutoff for obstructive sleep apnea syndrome ( epstein et al . , 2009 ) .
an ahi>10 is also considered clinically relevant and used as an exclusion criterion in sleep studies ( engdahl , eberly , hurwitz , mahowald , & blake , 2000 ; mellman , nolan , hebding , kulick - bell , & dominguez , 1997 ) .
we compared both indices between groups . in comparisons of mean caps scores between ptsd patients with and without osa
differences between the groups were tested in spss 17.0 . an anova was used to test group differences when variables were normally distributed . in other cases , a non - parametric mann
correlations were analyzed with a pearson 's test and were controlled for age and bmi .
ptsd patients ( n=20 ) , tcs ( n=24 ) , and hcs ( n=17 ) did not differ in age or alcohol intake per week .
the mean caps score was 67.50 ( sd=11.01 ) in ptsd patients and 4.00 ( sd=5.12 ) in tcs .
ptsd patients ( n=18 ) had a higher bmi than both tcs ( n=20 ) and hcs ( n=16 ) ( respectively , 27.86 ( sd=4.86 ) ; 24.64 ( sd=2.90 ) and 23.65 ( sd=2.42 ) , ( anova f ( 2,51)= 6.66 , p<0.01 ) .
demographic characteristics notes : bmi , body mass index ; hc , healthy controls ; m , mean ; ptsd , posttraumatic stress disorder ; sd , standard deviation ; tc , trauma controls .
ptsd > tc , ( p=0.036 ) ; ptsd > hc , ( p=0.006 ) ; tc = hc , ( p=1.0 ) .
ptsd = tc , ( p=0.16 ) ; hc < ptsd , ( p<0.001 ) ; hc < tc , ( p<0.001 ) .
the ahi was higher than 15 in 10% of the ptsd patients , 13% of the tcs , and 12% of the hcs ( =0.07 , df=2 , p= n.s . ) .
twenty - nine percent of the ptsd patients , 21% of tcs , and 29% of hcs had an ahi > 10 per hour ( = 0.60 , df=2 , p = n.s . ) .
the mean ahi was 6.79 ( sd=5.58 ) in ptsd patients , 7.18 ( sd=6.50 ) in tcs , and 5.68 ( 5.91 ) in hcs ( anova , f ( 2,58)=0.32 , p = n.s . ) .
the patients with an ahi>10 ( n=6 ) exhibited significantly higher caps scores ( m=77 , sd=9 ) than patients without osa ( m=63 , sd=9 ) ( n=14 ) ( mann whitney utest : z= 2.36 , p<0.05 ) . with a partial correlation ,
controlling for age and bmi , a significant relation between the caps and ahi was found in ptsd patients ( pearson , r=0.46 , p<0.05 , df=14 ) .
the three subscales ( b , c , d ) of the caps were not significantly related to the ahi in ptsd .
however , the correlation between the d - subscale ( hyperarousal ) and ahi was at trend - level significance in ptsd patients ( pearson , r=0.36 , p=0.08 , df=14 ) . relationship between apnea hypopnea index and ptsd severity
. notes : ahi , apnea hypopnea index ; caps , clinician - administered ptsd scale ; ptsd , posttraumatic stress disorder .
the mean nightmare score was 3.33 ( sd=2.56 ) in patients with osa ( ahi>10 , n=6 ) and 3.21 ( sd=2.29 ) in patients without osa ( n=14 ) ( anova , f ( 1 , 19)=0.1 , p = n.s . ) . similarly , there was no relationship between b2 score of the caps and ahi , when corrected for bmi and age ( pearson 's test r=0.076 , p = n.s , df=14 ) .
ptsd patients ( n=20 ) , tcs ( n=24 ) , and hcs ( n=17 ) did not differ in age or alcohol intake per week .
the mean caps score was 67.50 ( sd=11.01 ) in ptsd patients and 4.00 ( sd=5.12 ) in tcs .
ptsd patients ( n=18 ) had a higher bmi than both tcs ( n=20 ) and hcs ( n=16 ) ( respectively , 27.86 ( sd=4.86 ) ; 24.64 ( sd=2.90 ) and 23.65 ( sd=2.42 ) , ( anova f ( 2,51)= 6.66 , p<0.01 ) .
demographic characteristics notes : bmi , body mass index ; hc , healthy controls ; m , mean ; ptsd , posttraumatic stress disorder ; sd , standard deviation ; tc , trauma controls .
ptsd > tc , ( p=0.036 ) ; ptsd > hc , ( p=0.006 ) ; tc = hc , ( p=1.0 ) .
ptsd = tc , ( p=0.16 ) ; hc < ptsd , ( p<0.001 ) ; hc < tc , ( p<0.001 ) .
the ahi was higher than 15 in 10% of the ptsd patients , 13% of the tcs , and 12% of the hcs ( =0.07 , df=2 , p= n.s . ) .
twenty - nine percent of the ptsd patients , 21% of tcs , and 29% of hcs had an ahi > 10 per hour ( = 0.60 , df=2 , p = n.s . ) .
the mean ahi was 6.79 ( sd=5.58 ) in ptsd patients , 7.18 ( sd=6.50 ) in tcs , and 5.68 ( 5.91 ) in hcs ( anova , f ( 2,58)=0.32 , p = n.s . ) .
the patients with an ahi>10 ( n=6 ) exhibited significantly higher caps scores ( m=77 , sd=9 ) than patients without osa ( m=63 , sd=9 ) ( n=14 ) ( mann whitney utest : z= 2.36 , p<0.05 ) . with a partial correlation ,
controlling for age and bmi , a significant relation between the caps and ahi was found in ptsd patients ( pearson , r=0.46 , p<0.05 , df=14 ) .
the three subscales ( b , c , d ) of the caps were not significantly related to the ahi in ptsd . however , the correlation between the d - subscale ( hyperarousal ) and ahi was at trend - level significance in ptsd patients ( pearson , r=0.36 , p=0.08 , df=14 ) . relationship between apnea hypopnea index and ptsd severity .
notes : ahi , apnea hypopnea index ; caps , clinician - administered ptsd scale ; ptsd , posttraumatic stress disorder .
the mean nightmare score was 3.33 ( sd=2.56 ) in patients with osa ( ahi>10 , n=6 ) and 3.21 ( sd=2.29 ) in patients without osa ( n=14 ) ( anova , f ( 1 , 19)=0.1 , p = n.s . ) . similarly , there was no relationship between b2 score of the caps and ahi , when corrected for bmi and age ( pearson 's test r=0.076 , p = n.s , df=14 ) .
our study shows that the occurrence of osa in ptsd patients was 10% and was not increased compared with tcs and hcs , despite a higher bmi of ptsd patients .
furthermore , we found a relationship between ptsd severity and the ahi , whereas nightmare frequency was not related to the occurrence of apneas .
several studies reported high indices of ahi in 69%91% of the ptsd patients ( krakow et al . , 2001b , 2002 , 2004 ; yesavage et al . , 2010 ) .
in these studies , screening instruments for detecting osa may have been more sensitive than in our study , especially because some studies defined a cutoff of five events per hour .
as none of the previous studies included a control group , it can not be concluded that the incidence of osa is elevated in ptsd .
another explanation for the high incidence of osa in some previous studies is that the usage of benzodiazepines was not discontinued before sleep recordings , which increases the occurrence of osa ( dolly & block , 1982 ) . in our study , participants with regular benzodiazepine usage were excluded , and participants with habitual benzodiazepine usage refrained from sleep medication in the sleep laboratory .
lastly , our study group consisted of middle - aged veterans , whereas other studies included predominantly female ptsd patients . the incidence of osa may be different in other populations with ptsd .
our study underlines the importance of controlled studies to determine whether osa is more prevalent in ptsd than in matched controls .
although controlled studies have not been published on this subject before , our results are in concordance with two sleep studies in ptsd patients , which excluded an equal amount of patients and controls due to osa ( breslau et al .
in contrast , engdahl et al . ( 2000 ) reported that in a sample of elderly war veterans , more ptsd patients were excluded due to osa than controls . in our study ,
furthermore , ptsd patients with an ahi>10 exhibited significantly higher caps scores compared with ptsd patients without osa .
this may be due to decreased concentration , depression , and irritability that are common complaints in osa ( saunamki & jehkonen , 2007 ) .
also , previous studies have suggested a relationship between ptsd complaints and the occurrence of osa . in the study of engdahl
et al . ( 2000 ) , four ptsd patients with osa improved on overall well - being after treatment with continuous positive airway pressure ( cpap ) .
the relationship between remitted osa and improvement of ptsd severity was also found in an uncontrolled retrospective study on cpap treatment in ptsd patients ( krakow et al . , 2000 ) .
this contrasts with a study in which breathing interruptions , as witnessed by bed partners , were related to nightmare frequency in ptsd patients ( de groen et al . , 1993 ) .
reports from the bed partner are less reliable for detecting osa than polysomnography that was used in the current study .
possibly , bed partners of patients with nightmares are more alert during the night and therefore better aware of breathing interruptions
. therefore , breathing interruptions may be more frequently reported in patients with nightmares in the study of de groen et al .
the incidence of osa in ptsd patients was 10% in our study ; however , this can not be generalized .
furthermore , the power for detecting correlations between osa and symptom clusters of ptsd may have been too low .
in summary , our results indicate that ptsd is not necessarily associated with the occurrence of osa , as some uncontrolled studies suggested .
furthermore , osa was not related to sleep complaints , while patients with comorbid osa exhibited higher caps scores .
larger controlled studies in different populations need to be performed to provide more reliable estimations of the incidence of osa in ptsd . as comorbid osa in ptsd patients
may possibly lead to an increase in ptsd symptoms , the influence of osa , and treatment of osa , should be further explored , especially in those with refractory complaints to conventional therapeutic strategies .
there is no conflict of interest in the present study for any of the authors . | backgroundobstructive sleep apnea ( osa ) may be highly prevalent in posttraumatic stress disorder ( ptsd ) and may exacerbate ptsd complaints.objectiveour objective was to determine whether the prevalence of osa was high in a sample of dutch veterans with ptsd as compared to age- and trauma - matched controls , and whether osa was associated with more severe ptsd complaints.methodswe determined the apnea hypopnea indices ( ahi ) with polysomnographic registrations in 20 veterans with ptsd , 24 veterans without ptsd , and 17 healthy controls .
ptsd severity and nightmare complaints were assessed with the clinician - administered ptsd scale ( caps).resultsthe prevalence of an ahi>10 was 29% in ptsd , 21% in trauma controls , and 29% in healthy controls ( 2= 0.60 , df=2 , p = n.s . ) .
the mean caps score in patients with osa ( n=6 ) was significantly higher than in patients without osa ( p<0.05 ) , while nightmare severity was similar in ptsd patients with osa as compared to ptsd patients without osa ( p = n.s . ) .
furthermore , there was a significant correlation between ahi and caps score in ptsd patients ( r=0.46 , p<0.05 , df=14).conclusionsour results indicate that ptsd is not necessarily associated with a higher prevalence of osa .
however , ptsd severity was related to osa , which may possibly mean that comorbid osa leads to an increase in ptsd complaints .
however , future research should indicate whether osa exerts a negative influence on ptsd , and treatment of osa alleviates ptsd symptoms . |
a 53-year - old lady , of height 152 centimeters and weighing 45
kilograms diagnosed with rheumatoid arthritis was first seen
by us in april 2003 for a pre - chloroquine workup .
she was seen at six - monthly intervals and advised
amsler grid self - evaluation between her scheduled eye
examinations . in april 2006 ,
the vision in her left eye dropped to 20/30
n8 with no subjective change in vision .
a clinically
detectable rpe disturbance was noted , more prominent in
her left eye [ figures 1 , 2 ] .
amsler grid evaluation was
normal , but hfa 10 - 2 revealed , repeatable superior paracentral defect
in both eyes [ figure 3 ] .
fundus fluorescein angiography
( ffa ) revealed rpe defects in the macular region in both
eyes . a recommendation to the treating internist
four months later , she reported distortion of vision in both
eyes , which was reflected as a small blurred area in the center
of the amsler grid and vision in both eyes of 20/30 n8 optical coherence tomography ( stratus 4 oct ; carl zeiss
meditec , dublin , calif ) revealed a marked retinal thinning of
the parafoveal region [ figure 4 ] .
the internist was informed and hydroxychloroquine was
replaced by methotrexate . since then , her vision has remained
stable at 20/30 n8 .
chloroquine , and more recently , the apparently less toxic
hydroxychloroquine , is used for long periods of time for
treatment of various autoimmune disorders .
cambiaggi
first described the classic rpe changes in 19571 and hobbs
in 1959 established a definite link between long - term use
of chloroquine and subsequent development of retinal
pathology.6 early chloroquine retinopathy though still inadequately
described , is defined as an acquired paracentral scotoma
on threshold visual field testing , with no detectable retinal
findings , while advanced retinopathy has associated parafoveal
rpe atrophy.7 chloroquine and its metabolites have been found
in the pigmented ocular structures at concentrations much
greater than in any other tissue in the body , which may explain
its toxic properties in the eye.3,8 animal studies have
suggested that ganglion cell damage occurs early with choloroquine.4,8 human pathological studies are however limited to patients
with advanced maculopathy.3 detection of resultant thinning of the ganglion cell and
nerve fiber layer ( nfl ) would be useful in demonstrating
the initial stages of toxicity , perhaps at a stage when further
damage can be halted by stopping the drug at this point .
the nfl measurements of the peripapillary region in
patients on antimalarials by scanning polarimetry have
indeed shown a significant thinning of the nfl which was
dose and duration - dependent.4 however , the ganglion cell
population is the densest in the macular region , and hence ,
toxicity of the drug would be greatest at this location .
the
fact that the first functional change is a paracentral scotoma ,
and the first observable rpe changes are seen in this area
supports this assumption.7 thus retinal thickness and volume
measurements in this area may give more accurate and earlier
predictions of chloroquine toxicity , perhaps even before the
scotoma develops .
the oct , with its high resolution provides
measurements of the retinal thickness and volume , both in
the peripappillary area as well as at the macular region .
it
also gives information on the status of the retinal pigment
epithelium , clearly revealing rpe defects .
in addition , the
limitations of the scanning laser polarimeter with respect
to inaccuracies related to corneal and lens birefringence are
not present . a recent study using a research prototype of a high - speed
ultra - high - resolution oct reported discontinuity or loss
of perifoveal photoreceptor inner segment / outer segment
junctions and thinning of the outer nuclear layer in 15 patients
receiving hydroxychloroquine.5 this instrument is however not
available as yet for general clinical use . in our patient
[ figure 2 ] , the fovea ( central circle ) is of
normal thickness while the perifoveal ( inner circle ) and
peripheral ( outer circle ) , are thinned mainly temporally and
inferiorly .
this is in agreement with the universally accepted
early superior paracentral scotoma , also seen in this patient
[ figure 1 ] .
although reports in the older literature on chloroquine
toxicity had suggested that the cumulative dose of chloroquine
was the critical factor for toxicity , current evidence suggests
that cumulative dosage and duration of therapy are relatively
unimportant and that the crucial index is daily dosage
normalized by lean body weight.7,9 in this patient , the
dose of chloroquine she was on clearly exceeded the recommended
daily dose of 4 mg / kg / day.10 this is probably why she
developed maculopathy just 36 months after starting the
chloroquine therapy for her rheumatoid arthritis .
long - term prospective studies may determine when retinal
thinning starts in patients on antimalarials , and whether
irreversible paracentral field defects could be prevented if
antimalarials are stopped on first detection of thinning . | we herein report the optical coherence tomography ( oct )
findings in a case of chloroquine - induced macular toxicity ,
which to our knowledge , has so far not been reported . a 53-
year - old lady on chloroquine for treatment of rheumatoid
arthritis developed decrease in vision 36 months after initiation
of the treatment .
clinical examination revealed evidence of
retinal pigment epithelial ( rpe ) disturbances .
humphrey
field analyzer ( hfa ) , fundus fluorescein angiography ( ffa )
and oct for retinal thickness and volume measurements at
the parafoveal region were done .
the hfa revealed bilateral
superior paracentral scotomas , ffa demonstrated rpe loss and
oct revealed anatomical evidence of loss of ganglion cell layers ,
causing marked thinning of the macula and parafoveal region .
parafoveal retinal thickness and volume measurements may be
early evidence of chloroquine toxicity , and oct measurements
as a part of chloroquine toxicity screening may be useful in early
detection of chloroquine maculopathy . |
the world health organisation has stated that , since 1990 , more people worldwide have died from coronary heart disease ( chd ) than any other cause .
further , they reported that 80% to 90% of people dying from chd had one or more major risk factors associated with lifestyle . in the uk ,
more than 90,000 deaths per year are due to chd , and although death rates are falling they are still among the highest in western europe . cardiac rehabilitation ( cr )
programmes were initiated in the 1960s when the benefits of mobilisation and physical activity ( pa ) following lengthy hospital stays for chd became known . since then , secondary prevention has become an essential aspect of care of the patient with chd .
research has shown that lifestyle change , including pa , a healthy diet , and smoking cessation , alters the course of chd [ 57 ] , and so disease prevention measures have been designed to focus on a range of lifestyle factors .
indeed , cardiac rehabilitation and secondary prevention programmes have developed from focusing on exercise alone to becoming multidisciplinary and encompassing baseline patient assessments , nutritional counselling , risk factor management ( i.e. , lipids , hypertension , weight , diabetes , and smoking ) , psychosocial and vocational counselling , and pa advice and exercise training , in addition to the appropriate use of cardioprotective drugs .
multidisciplinary measures are advocated by governments around the world , and in the uk the national institute for clinical excellence ( nice ) set out a series of guidelines in 2007 for care of patients who had had a myocardial infarction ( mi ) . the guidelines covered secondary prevention in primary and secondary care and were not focused solely on lifestyle interventions .
they did , however , incorporate pa , diet , smoking , and drug therapy and were based on systematic reviews of the best available evidence .
priority recommendations , considered to have the most important effect on patient care and outcomes , included that on discharge from hospital every mi patient should have had a confirmed diagnosis of acute mi , results of investigations , future management plans , and advice on secondary prevention .
also , nice highlighted the importance of advice being given regarding regular pa in the form of 2030 minutes of exercise per day to the point of slight breathlessness .
patients should also be advised to stop smoking , eat a mediterranean style diet rich in fibre , fruit , vegetables , and fish , and follow a treatment regime with a combination of ace ( angiotensin - converting enzyme ) inhibitors , aspirin , beta - blockers , and statins . however , despite the evidence that positive lifestyle changes bring about improved outcomes , results from a number of secondary prevention initiatives have been disappointing . in a systematic review of multidisciplinary secondary prevention programmes mcalister et al .
reported that although some beneficial impact was achieved on processes of care , morbidity , and mortality , questions remained regarding the duration and frequency of interventions and the best combination of disciplines within an intervention .
the euroaspire ( european action on secondary and primary prevention by intervention to reduce events ) surveys by the european society of cardiology have shown that the adoption of cardiovascular disease prevention measures as part of daily clinical practice was wholly inadequate and that unhealthy lifestyle trends are continuing .
the authors commented on the difficulty experienced by adults in changing behaviour despite having a life threatening disease and that continued professional support was imperative if this was to be achieved .
. carried out a systematic review of rcts of secondary prevention interventions , published in 2001 , and analysed 12 studies .
to our knowledge no comprehensive systematic review has been undertaken since 2001 of the effects of diet , exercise , and other lifestyle factors in the secondary prevention of chd .
we therefore performed a systematic review of randomised controlled trials to determine the effectiveness of lifestyle interventions for the secondary prevention of chd .
we included male and female adults of all ages ( aged 18 + ) with a diagnosis of chd .
patients included those who had experienced a myocardial infarction ( mi ) , coronary artery bypass graft ( cabg ) , or percutaneous transluminal coronary angioplasty ( ptca ) and those with angina pectoris and coronary artery disease defined by angiography .
for the purposes of this review we excluded patients who had had a heart transplant , heart valve surgery , or heart failure , unless it was clearly specified that the cause related to chd .
we have included interventions with a lifestyle and/or behaviour change focus designed for the secondary prevention of chd , incorporating one or a combination of exercise and diet .
we have excluded from this review studies which were not randomized controlled trials , focused on primary prevention , involved patients with multiple diseases and/or outcomes which were related to diseases other than chd , involved patients in a hospital in - patient setting , focused on drug therapy , had short ( less than three months ) or no follow - up , or reported outcomes which were not the focus of this review ( see types of outcome measures section ) such as depression , cost - effectiveness , or service delivery .
trials were included in this review if they reported a minimum postintervention follow - up of three months to allow some change to take place .
for the purposes of this review , interventions have been considered to have been delivered in primary care according to the definition of primary care as stated by the committee on the future of primary care at the institute of medicine in the united states : primary care is the provision of integrated , accessible healthcare services by clinicians who are accountable for addressing a large majority of personal healthcare needs , developing a sustained partnership with patients , and practicing in the context of family and community .
interventions are delivered in primary care by clinicians who are generally considered to be physicians , nurse practitioners and physical assistants and a broader array of individuals in a primary care team . in the care of chd patients , the primary care team may include general practitioners , practice nurses , community pharmacists , community and public health nurses , dieticians , occupational therapists , and physiotherapists .
interventions have been included in the review if they have been delivered in primary care or community settings by primary care clinicians or clinicians whose normal roles may be within secondary care , for example , community hospitals , or tertiary care , for example , general hospitals .
we have excluded from this review studies of interventions undertaken primarily within secondary or tertiary care settings by clinicians or care teams whose relationship with patients is not long term or ongoing . in the included studies , the comparators are normal care or
usual care , meaning standard clinical care or standard care given by a general practitioner ( gp ) .
hospital admissions ( cardiac related / and all - cause if available ) . diet ( e.g. , measured by fibre , fruit , and veg quantity ) .
blood pressure ( bp ) ; systolic bp ( sbp ) ; diastolic bp ( dbp ) .
blood lipid levels ( high density lipoprotein cholesterol ( hdl - chol ) , low density lipoprotein cholesterol ( ldl - chol ) ) . health related quality of life ( qol ) .
we searched electronic databases medline , cinahl , and embase for english language randomized trials in humans published since 1990 .
the search method for medline is detailed below ; it was modified as appropriate for cinahl and embase .
reference lists of review articles were also searched . selecting an advanced ovid search , the keyword
lifestyle was inserted and the box ticked to select map term to subject heading . after selecting search
the same process was followed for the keywords exercise , physical activity , and diet .
in each case only the keyword as a subject heading and as a keyword were selected on the mapping display page .
a fifth search was commanded by using all four keywords above separated by the boolean operator or .
box , ticking each of the four boxes relating to the keywords , and selecting combine selections with or . a sixth search term secondary prevention was added as a keyword in same way as above .
chd and four conditions related to it and their abbreviations , where appropriate , all as separate keywords : myocardial infarction ,
mi , coronary artery bypass graft , cabg , percutaneous transluminal coronary angioplasty , ptca , and angina pectoris .
this group incorporating coronary heart disease and related conditions involved a total of nine separate searches , and they were together inserted as a 16th search , with each term separated by or .
this was done , as above , by expanding the search history , selecting the relevant boxes beside keywords and selecting combine selections with or .
the three components of the search were put together in the following way . on the search history page ,
search five was selected by clicking on the box beside it ( lifestyle.mp . or life style / or exercise.mp . or exercise / or physical activity.mp . or diet / or diet.mp . ) .
search six was then selected ( secondary prevention.mp . or secondary prevention/ ) , and search 16 ( coronary heart disease.mp . or coronary disease / or chd.mp . or myocardial infarction.mp . or myocardial infarction / or mi.mp or coronary artery bypass / or coronary artery bypass graft.mp . or cabg.mp . or percutaneous transluminal coronary angioplasty.mp . or angioplasty , transluminal , percutaneous coronary / or ptca.mp . or angina pectoris.mp . or angina pectoris/ )
search 17 was selected by ticking the box , and limits were imposed in an effort to narrow the search . below the search results , within the limits options ,
additional limits was then selected , search 17 was selected , and in the age groups options
then , limit a search was selected . to refine the search further ,
the terms entered were randomized controlled trial.mp . or randomized controlled trial/ , controlled clinical trial.mp . or controlled clinical trial/ , random allocation.mp . or random allocation/ , double blind method.mp . or double - blind method/ , single blind method.mp . or single - blind method .
each of these terms was combined with or , giving search 24 . selecting search 18 and
titles and abstracts were screened by two reviewers ( mc , jc ) , and potentially relevant references were retrieved .
the two reviewers ( mc , jc ) then independently selected trials to be included in the review .
reference lists of relevant systematic reviews were also screened for potential papers to include . after the final selection of trials was agreed upon , data including study characteristics , outcome measures and results were extracted .
in addition , the quality of trials was assessed in relation to randomization method , loss to follow up , intention to treat analyses , and blinding measures .
dichotomous outcomes for each study have been expressed as risk ratios , where appropriate , and 95% confidence intervals ( cis ) .
meta - analyses were performed using random effects models , and data were presented as forest plots using revman software .
we were only able to perform meta - analysis on three outcomes total mortality , cardiovascular mortality , and nonfatal cardiac events .
continuous variables have been expressed as the difference between intervention and control groups at study completion and the standard deviation difference where reported .
figure 1 shows the selection , screening , and identification of studies for this review . of the 266 papers originally identified through searching the three electronic databases
, we identified 39 that were potentially eligible for inclusion , and full text versions of these studies were retrieved and assessed .
twelve of the 39 papers were excluded because they included diseases other than chd , were drug trials , did not focus on lifestyle , were not randomized controlled trials , had a short follow - up period , were conducted within a hospital in - patient setting , had outcomes which were not of interest , or were retrieved more than once from the three search engines .
two papers were added after screening reference lists of relevant systematic reviews , and one trial was added which reported different outcomes of an identical study which emerged in our searches ( figure 1 ) .
table 1 shows summary data of 30 papers from the 21 randomised controlled trials ( rcts ) found to be eligible for this review [ 1645 ] .
we have presented them in the six categories described in the methods section ( exercise , dietary , psychological , educational , multifactorial , and organisational ) . the category which had the largest number of trials was multifactorial ( 10 in total ) .
we also found one which focused on exercise only , one dietary intervention , three which had a psychological approach , four educational , and two organisational .
in all the trials except where stated , patients randomised to the control group received usual care , which was not defined by every study but usually meant standard clinical care or standard care given by a gp .
the studies varied greatly in terms of sample size , duration , and intervention elements ; however all involved patients of a similar age group ( older adults ) with chd . the sample size of trials ranged from 48 to 3241 , and study duration ranged from three months [ 19 , 43 ] to four and five years .
follow - up analyses varied too , ranging from three months to 15 years .
reported a home - based exercise intervention while two studies incorporated initial short residential stays to deliver an intensive educational programme .
conducted a four - week stay at an intervention unit located in a rural part of northern sweden , followed by a structured 11-month programme consisting of self - recording of lifestyle behaviours and contact with the patients ' personal coaches .
organised a week - long residential stay at a hotel to educate patients on diet , exercise , and stress , followed by twice - weekly group support meetings for five years .
the vestfold heartcare study group conducted an initial six - week heart school at a hospital rehabilitation centre , although it was unclear whether this was residential .
heart school involved physiotherapist - led pa , stress reduction education and lifestyle counselling followed by nine weeks of twice - weekly exercise , and then meetings every three months for two years .
delivered nurse - led lectures , lasting up to two hours each , once a month for 16 months .
in addition , eight group meetings were organised throughout this period , six exercise sessions , and three social events .
less intensive programmes , in which patients were encouraged to self - manage their lifestyle behaviour change , included those reported by murphy et al . and
cupples and mcknight [ 22 , 23 ] , in which patients were seen every four months .
another less intensive intervention , the lyon diet heart study [ 32 , 33 ] , involved a one - hour education session with a cardiologist and dietician , followed by a repeat visit at eight weeks and annually thereafter .
the interventions included in this review involved a range of clinical and other healthcare staff including doctors , nurses , physiotherapists , psychologists , dieticians , and social workers .
seven of the 21 trials were led by nurses , mainly based in primary care . de lorgeril et al . [ 32 , 33 ] reported a trial conducted by a cardiologist and dietician .
four studies were gp - led , one conducted by a physiotherapist and one nutritionist - led .
the quality of the trials , in terms of the method of randomisation , whether groups were similar at baseline , losses to follow up , whether intention to treat analyses were used and blinding of outcome assessors , is as reported in the papers . in seven of the 21 original studies
( excluding follow - up papers ) , the randomisation method was not clearly described . in six of these trials ,
the method was not stated at all [ 16 , 25 , 26 , 39 , 43 , 45 ] .
the majority of the remaining studies described various randomisation techniques including the use of computer - generated random numbers , random numbers tables , and sealed envelopes .
lewin et al . used block randomisation , while carlsson allocated patients in groups of 20 .
murphy et al . used cluster randomisation because their intervention was aimed to alter practitioners ' behaviour which could contaminate their interactions with control patients .
some authors included as losses to follow up deaths and withdrawals due to poor health , while others defined it as people who could not be accounted for or contacted at follow - up .
follow - up as a quality marker relates only to original studies and varied considerably for longer - term follow - up studies as would be expected .
thirteen of the 21 studies stated that analyses were conducted using intention to treat principles .
the vestfold heartcare study group used the last recorded value of a variable from the previous visit if there were missing data .
only seven out of 21 studies reported that outcome assessors were blind to group allocation .
overall , the quality of many trials was poor with a majority not having blinded outcome assessors and many not describing the method of randomisation .
overall , four studies showed benefits for intervention patients compared to controls in relation to total mortality .
because of the large differences in follow - up periods , we have used data from the studies which range from 3 to 5 years to allow easier comparison of studies . for all interventions except two ,
murchie et al . reported a significant survival effect for intervention patients compared to controls at their 4.7 year follow - up . within the same study , delaney et al .
reported that , at 10 years , the observed difference was no longer significant between the groups .
hamalainen et al . reported a significant survival effect for intervention patients compared to controls at their 4.7 year follow - up .
the i test for heterogeneity combined with the chi nonsignificant p value suggests that the level of heterogeneity between studies is not important .
delaney et al . reported a 10-year follow - up study but we have presented their data collected at 4.7 years to allow for easier comparison with other studies . for the same reason we have used data reported by hamalainen et al . at three years of follow - up instead of the authors ' results at 15 years .
the three studies reporting significant survival effects were cupples and mcknight , two - year follow - up results from an educational intervention , de lorgeril et al .
, a mediterranean diet study with four - year follow - up , and hamalainen et al . , a trial of a multifactorial intervention .
hamalainen et al . , who reported coronary mortality , also reported significant survival effects for intervention patients compared to controls in their 15-year follow - up study .
delaney et al . reported cv and coronary deaths at both 4.7- and 10-year follow - up points .
however , the mortality figures were combined with nonfatal mi to calculate a proportional hazard ratio .
murchie et al . , a paper from the same study at 4.7 years of follow - up , reported an adjusted hazard ratio of 0.76 for coronary events ( coronary deaths plus nonfatal mis ) ( 0.58 to 1.00 , p = .049 ) .
this was reported by nine studies . in figure 4 , we have presented data for major nonfatal events ( mi , cabg , pci , coronary angioplasty ) .
four studies reported nonfatal events separately [ 21 , 33 , 41 , 44 ] while five reported the number of patients who had an event
results , therefore although they reported figures for various events and mortality they did not report a separate statistic for nonfatal events .
delaney et al . reported coronary events which included coronary deaths and nonfatal mis ; however separate figures for each variable were not reported .
reported nonfatal stroke , heart failure , and angina data but none of the values was significant .
lewin et al . reported the frequency of angina attack in number of episodes per week .
intervention group patients had a reduction of three attacks per week compared to a reduction of 0.4 attacks among control subjects ( p = .016 ) .
in addition to nonfatal mi and coronary revascularisation , de lorgeril et al . reported a number of major and minor secondary endpoints .
they calculated risk ratios for nonfatal ami combined with cv deaths , major secondary endpoints ( periprocedural infarction , unstable angina , heart failure , stroke , pulmonary embolism , peripheral embolism ) combined with primary endpoints , and primary and major secondary endpoints combined with minor secondary endpoints ( stable angina , post - pcta restenosis , and thrombophlebitis ) .
in addition to mi , ptca , and cabg , ornish et al . reported the frequency , duration , and severity of angina chest pain at one year and five years .
one significant value out of three outcomes reported at the two different time points each was reported : chest pain severity ( scale 17 , baseline to one year : intervention group 1.5 ( 1.5 ) to 0.7 ( 1.2 ) ; control group 0.6 ( 0.8 ) to 1.4 ( 1.2 ) ; p < .001 ) .
the i test for heterogeneity indicated that there may be substantial heterogeneity across studies so results of this meta - analysis have to be interpreted with caution .
five studies reported results relating to hospital admissions . while there was an overall trend to reduced admissions in intervention groups , only one of these studies reported a significant reduction in intervention patients .
data relating to lifestyle risk factors for chd diet , pa , and smoking are presented in table 4 .
table 5 shows a summary of lifestyle risk findings from our included studies . for each of the three areas of diet , exercise , and smoking ,
we have presented the number of studies reporting each outcome , the number of outcomes , and the numbers of significant and nonsignificant values .
dietfifteen studies reported diet as an outcome , with a total of 51 outcomes . of these , 39 showed significant benefits for intervention patients compared to controls in relation to dietary consumption .
these included significant improvement in specific food intake , such as fat , fibre , sugar , and cholesterol [ 28 , 30 , 33 , 34 , 39 , 41 ] , diet score , diet knowledge , and habits [ 21 , 27 , 44 , 47 ] , and for concern about dietary habits .
fifteen studies reported diet as an outcome , with a total of 51 outcomes . of these , 39 showed significant benefits for intervention patients compared to controls in relation to dietary consumption .
these included significant improvement in specific food intake , such as fat , fibre , sugar , and cholesterol [ 28 , 30 , 33 , 34 , 39 , 41 ] , diet score , diet knowledge , and habits [ 21 , 27 , 44 , 47 ] , and for concern about dietary habits .
patwenty - one studies incorporated pa , with 37 outcomes . of these , 20 showed significant improvements for intervention patients compared to controls .
these included significant improvements in maximal workload and vo2peak [ 16 , 18 , 27 ] .
eleven studies used validated questionnaires or patient diaries [ 17 , 20 , 2325 , 28 , 30 , 34 , 36 , 39 , 41 ] .
four conducted patient interviews or used questionnaires which were not reported as validated [ 21 , 40 , 43 , 44 ] . of these
, eight studies reported significant improvements [ 20 , 21 , 23 , 27 , 28 , 30 , 34 , 39 ] .
these included significant improvements in maximal workload and vo2peak [ 16 , 18 , 27 ] .
eleven studies used validated questionnaires or patient diaries [ 17 , 20 , 2325 , 28 , 30 , 34 , 36 , 39 , 41 ] .
four conducted patient interviews or used questionnaires which were not reported as validated [ 21 , 40 , 43 , 44 ] . of these , eight studies reported significant improvements [ 20 , 21 , 23 , 27 , 28 , 30 , 34 , 39 ] .
smokingwhile all studies reported proportions of the study populations that smoked , only 13 studies reported smoking as an outcome and five of these reported significant reductions in smoking behaviours in the intervention groups [ 20 , 21 , 27 , 28 , 45 ] .
reported a nonsignificant difference between intervention and control groups at one year , but significant at two and three years .
while all studies reported proportions of the study populations that smoked , only 13 studies reported smoking as an outcome and five of these reported significant reductions in smoking behaviours in the intervention groups [ 20 , 21 , 27 , 28 , 45 ] .
reported a nonsignificant difference between intervention and control groups at one year , but significant at two and three years .
bpthirteen studies reported bp as an outcome , and five reported significant benefits for intervention compared to control patients [ 18 , 20 , 34 , 39 , 45 ] .
giallauria et al . reported significant improvements in sbp and dbp at 12 months and 24 months .
. reported significant difference in sbp among intervention compared to control patients at three months and 12 months .
collected bp data from medical records and classified it as being managed according to british hypertension society recommendations if the last recorded measurement was less than 160/90 mmhg or receiving attention .
the significant difference between intervention and control groups at one year was no longer observed at four - year follow - up .
thirteen studies reported bp as an outcome , and five reported significant benefits for intervention compared to control patients [ 18 , 20 , 34 , 39 , 45 ] .
giallauria et al . reported significant improvements in sbp and dbp at 12 months and 24 months .
. reported significant difference in sbp among intervention compared to control patients at three months and 12 months .
collected bp data from medical records and classified it as being managed according to british hypertension society recommendations if the last recorded measurement was less than 160/90 mmhg or receiving attention .
the significant difference between intervention and control groups at one year was no longer observed at four - year follow - up .
wallner et al . found significant improvements in sbp and dbp in intervention patients compared to controls .
reported significant benefits for intervention compared to control patients relating to sbp and dbp at one , two , and three years but not at six or 10 years .
total cholesterol and/or lipid levelsthese outcomes were reported by 19 studies and 12 demonstrated significant benefits for intervention patients .
seven of these 12 studies reported significant improvements in total cholesterol for intervention patients compared to controls [ 18 , 20 , 21 , 30 , 41 , 42 , 45 ] .
another study found a significant difference in total cholesterol between female intervention and control groups but not male .
these outcomes were reported by 19 studies and 12 demonstrated significant benefits for intervention patients .
seven of these 12 studies reported significant improvements in total cholesterol for intervention patients compared to controls [ 18 , 20 , 21 , 30 , 41 , 42 , 45 ] .
another study found a significant difference in total cholesterol between female intervention and control groups but not male .
low - density lipoprotein cholesterol ( ldl - chol ) five studies reported significant improvements in levels of ldl - chol among intervention patients compared to controls [ 18 , 30 , 39 , 41 , 42 ] .
. found significant improvements between female intervention and control groups but not male .
five studies reported significant improvements in levels of ldl - chol among intervention patients compared to controls [ 18 , 30 , 39 , 41 , 42 ] .
. found significant improvements between female intervention and control groups but not male . only one study , that by giallauria et al .
, found a significant improvement in levels of high - density lipoprotein cholesterol ( hdl - chol ) among intervention patients compared to controls .
qol10 studies reported quality of life , and four reported significant benefits for intervention patients including for physical activity factor , physical function , and emotional .
. reported five significant results out of the eight health status domains ( physical , social , role physical , role emotional , and pain ) .
10 studies reported quality of life , and four reported significant benefits for intervention patients including for physical activity factor , physical function , and emotional .
. reported five significant results out of the eight health status domains ( physical , social , role physical , role emotional , and pain ) .
reported a significantly better score in intervention patients compared to controls relating to self / stress management at six month follow - up and throughout their study .
reported a significantly better score in intervention patients compared to controls relating to self / stress management at six month follow - up and throughout their study .
medicationnine studies reported use of medications , and six reported significant improvements among intervention patients compared to controls .
nine studies reported use of medications , and six reported significant improvements among intervention patients compared to controls .
lewin et al . reported the number of glyceryl trinitrate pills or puffs of sublingual spray taken per day which were self - reported in a diary by patients .
intervention patients reported a significant reduction in doses per week compared to control group patients .
. a significantly greater number of intervention patients at one - year follow - up was taking aspirin compared to controls .
carlsson found significant differences between intervention and control patients in use of statins and cholestyramine ; however it was unclear how this data was collected . in the study by cupples and mcknight [ 22 , 23 ]
trained health visitors assessed the use of prophylactic drugs for angina by interviewing patients . at the two- and five - year follow - ups ,
we have conducted a systematic review of lifestyle interventions for the secondary prevention of chd using cochrane collaboration methodology .
the review indicates that lifestyle interventions have mixed effects with some benefits in relation to total mortality , cv mortality , and nonfatal cv events as well as pa , diet , blood pressure , cholesterol , qol , and medication adherence .
the review was restricted to the period after 1990 because the concept of secondary prevention and methods to promote it are a relatively recent development in healthcare . nonetheless , we found trials of interventions which were heterogeneous , and this was evidenced by the range of categories into which they could be classified .
few trials evaluated a single component of lifestyle , while many assessed the effects of a complex , multifactorial intervention .
other differences between studies included trial quality , intervention setting , intensity , duration , and length of follow - up .
we excluded trials which evaluated the intervention against a different intervention or other programme which could not be defined as usual care .
overall , a small number of studies showed a significant reduction in deaths and nonfatal mis while several reported positive results relating to adherence to lifestyle change .
regarding total mortality , four studies out of the six reporting this outcome showed significant benefits in the relatively short time scale studied ( 35 years ) .
the lyon diet heart study showed a significant impact on all - cause mortality and fewer deaths from cv causes among intervention patients compared to controls .
this was a noteworthy result because patients were seen annually , indicating that they were able to maintain the diet for long periods alone and without professional motivation .
the other trials showing significant benefits were of an educational intervention and two multifactorial [ 36 , 45 ] . only three out of eight studies showed significant results for cardiovascular mortality .
these were a dietary intervention , educational and multifactorial ones , all of which had also shown significant effects on total mortality .
there were significant improvements in five of these trials , which were of dietary , psychological , and multifactorial [ 21 , 39 , 41 ] interventions .
of the five studies which reported hospital admissions , only one , murphy et al . , reported significant benefit for intervention patients compared to controls , both for overall and cv admissions . in terms of risk factors for chd , these were assessed by greatly differing methods which made comparison difficult .
diet , pa , smoking status , blood pressure , and cholesterol were widely reported , with varying results . in relation to diet ,
significant results were reported for the dietary intervention , one psychological , three educational , and six multifactorial interventions .
significant results relating to pa were reported for one exercise trial , one educational , two psychological , and seven multifactorial .
of the five studies which showed significant benefits for intervention patients compared to controls in relation to smoking , four were multifactorial and one psychological .
all five interventions which showed significant results relating to bp were multifactorial . concerning cholesterol and lipid levels ,
the interventions which showed significant results were one psychological , one educational , and eight multifactorial . in relation to quality of life
, one psychological trial reported a significant benefit for intervention patients compared to controls , as did one educational and two multifactorial .
few trials reported medication intake or adherence . however , as appropriate therapy is a key aspect of secondary prevention of chd , this should surely be given greater consideration .
the trials which reported significant results relating to medication were classified as psychological ( one ) , multifactorial ( two ) , and educational ( three ) .
likewise , self - efficacy , a patient 's ability , and confidence to manage their own condition , was only reported in one study .
recent evidence has shown the importance of focusing on lifestyle to effect positive changes relating to chd .
used the impact chd model to examine the decline in chd mortality in ireland between 1985 and 2000 , and possible reasons for this .
the mortality rate fell during this period by some 47% , representing 3673 fewer observed chd deaths .
the authors found that 48% of this decrease was due to a reduction in major risk factors including smoking and cholesterol levels .
conversely , an upward trend was seen in obesity , diabetes , and pa which were said to contribute an additional 500 deaths in 2000 .
, who also used the impact model and investigated the decline in chd mortality in italy between 1980 and 2000 .
they concluded that over half of the mortality fall was due to risk factors , mainly blood pressure and cholesterol , and just under half was due to medical therapies .
the lyon diet heart study was the first clinical trial evidence in support of the mediterranean diet , which comprises a high intake of fruit , vegetables , nuts , legumes , and grains .
other recent evidence has been found supporting the mediterranean diet 's protective effect in the secondary prevention of chd [ 5052 ] .
examined rcts of cardiac rehabilitation with exercise and found a moderate reduction of 20% in total and cv mortality after one year , with a reduced risk maintained for three years after infarction . in a recent review of interventions incorporating exercise as part of a cardiac rehabilitation programme , jolliffe et al . also observed a reduction in total mortality .
exercise - only interventions resulted in a 27% reduction in total mortality and 31% reduction in cardiac mortality while comprehensive rehabilitation reduced mortality to a lesser extent ( 26% ) .
this is in keeping with similar findings in a recent review of 36 rcts of psychological interventions by rees et al . which showed no evidence of effect on total or cardiac mortality , but found small reductions in anxiety and depression .
jolly et al . investigated a programme to improve communication between hospital and gp practices using liaison nurses .
the programme was effective in promoting follow - up care of patients in general practice ; however health outcomes were not improved .
munoz et al . examined an intervention involving postal reminders sent to patients to encourage them to visit their gp .
blood pressure and hdl - cholesterol levels were improved , but no effect was observed on mortality or morbidity .
the relatively small number of studies which included mortality as an outcome , the heterogeneity between trials , and poor quality of reporting all arise from the primary data . further ,
imprecise descriptions of the interventions and the limited data have made it difficult to determine the benefits of various components of the interventions .
the majority of the population of the interventions was male and relatively low risk ; however greater benefit could have been derived from the interventions by higher risk patients who were excluded on the basis of comorbidity .
the broad range of trials included , and the subsequent large number discovered in our search , may have made comparisons difficult .
nonetheless this reflects the current lack of clarity in respect of the optimal components of effective interventions for chd secondary prevention .
we have found that lifestyle interventions promoting regular pa , a healthy diet , and adherence to medication have beneficial effects among patients with chd .
it is therefore reasonable to promote such a healthy lifestyle to patients similar to those included in the rcts we investigated mainly older adults who had suffered a coronary event .
as practitioners endeavour to achieve target levels of blood pressure and cholesterol by altering their patients ' prescribed medication , the potential value of their advice regarding exercise and diet should not be overlooked .
overall , the current evidence suggests that lifestyle interventions have some beneficial effects on total and cardiac mortality , morbidity , and on behaviour change in relation to modifiable cardiac risk factors .
even where little or no effect was observed relating to mortality or morbidity , some trials reported benefits in terms of lifestyle behaviour change . that healthcare education and even small - scale interventions can lead to healthier lifestyle choices , as shown in this review and others , should be an encouragement to professionals in practice .
future studies should be designed carefully , with attention given to aspects of study quality , which we addressed in table 2 .
for example , rcts of a cluster design help to avoid contamination of control patients .
in addition , it is important to incorporate concealment of allocation and blinding of outcomes .
maximal follow - up should be ensured and more consideration given to the underlying theoretical foundation for the intervention . however , the fact that more profound and wide reaching benefits were not seen in our review is surprising considering that all guidelines focus on the importance of lifestyle interventions .
future research should perhaps focus on the components of interventions and what the ideal combination of measures , intervention intensity , and duration should be .
in addition , investigations into the barriers to lifestyle change among patients with chd may shed new light on why some well - designed and executed interventions have not resulted in expected benefits .
lifestyle.mp . or life style/ exercise.mp . or exercise/ physical activity.mp . secondary prevention.mp . or secondary prevention/ coronary heart disease.mp . or coronary disease/ myocardial infarction.mp . or myocardial infarction/ coronary artery bypass / or coronary artery bypass graft.mp .
percutaneous transluminal coronary angioplasty.mp . or angioplasty , transluminal , percutaneous coronary/ angina pectoris.mp . or angina pectoris/ 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15
limit search 17 by english language , humans , 1990-current and adults all ages ( 19 plus ) .
randomized controlled trial.mp . or randomized controlled trial/ controlled clinical trial.mp . or controlled clinical trial/ random allocation.mp . or random allocation/ double blind method.mp . or double - blind method/ single blind method.mp . or single - blind method/ 19 or 20 or 21 or 22 . | the effectiveness of lifestyle interventions within secondary prevention of coronary heart disease ( chd ) remains unclear .
this systematic review aimed to determine their effectiveness and included randomized controlled trials of lifestyle interventions , in primary care or community settings , with a minimum follow - up of three months , published since 1990 .
21 trials with 10,799 patients were included ; the interventions were multifactorial ( 10 ) , educational ( 4 ) , psychological ( 3 ) , dietary ( 1 ) , organisational ( 2 ) , and exercise ( 1 ) .
the overall results for modifiable risk factors suggested improvements in dietary and exercise outcomes but no overall effect on smoking outcomes . in trials that examined mortality and morbidity ,
significant benefits were reported for total mortality ( in 4 of 6 trials ; overall risk ratio ( rr ) 0.75 ( 95% confidence intervals ( ci ) 0.65 , 0.87 ) ) , cardiovascular mortality ( 3 of 8 trials ; overall rr 0.63 ( 95% ci 0.47 , 0.84 ) ) , and nonfatal cardiac events ( 5 of 9 trials ; overall rr 0.68 ( 95% ci 0.55 , 0.84 ) ) .
the heterogeneity between trials and generally poor quality of trials make any concrete conclusions difficult .
however , the beneficial effects observed in this review are encouraging and should stimulate further research . |
hand deformities are common in rheumatoid arthritis ( ra ) , with 45% of patients affected by ulnar drift at the metacarpophalangeal joints ( mcpjs ) after 5 years of disease activity and with 33% no longer able to work .
surgery is often performed to improve hand function and relieve pain resulting from tendon and joint degeneration .
a common procedure is prosthetic metacarpophalangeal joint ( mcpj ) replacement for the treatment of ulnar drift and mcpj subluxation .
many patients are taking a combination of medications , such as analgesia or anti - inflammatory agents in addition to one or more disease - modifying antirheumatic drugs ( dmards ) to manage their systemic disease .
there are well - documented concerns in the general literature regarding the use of nonsteroidal anti - inflammatory drugs ( nsaids ) , steroids , and immunomodulatory medications in the perioperative period , particularly with respect to wound healing problems .
however , specific data pertinent to patients with rheumatoid arthritis is relatively sparse , and recommendations are frequently based upon data from other patient groups , such as in transplant surgery and the management of inflammatory bowel disease .
there are studies assessing postoperative complications with orthopaedic surgery in patients with ra , but with the exception of methotrexate , the studies are usually small and retrospective . in cases where prosthetic devices are inserted
, a wound breakdown or infection can be devastating and potentially lead to the loss of an implant .
however , these concerns must be carefully balanced against the need to maintain disease stability , as a flare of rheumatoid disease activity in the postoperative period may hamper rehabilitation and impair the final outcome .
the effects of anti - inflammatory and immunomodulatory medications on the different stages of the wound healing process can be widespread
. the initial inflammatory phase may be blunted , or the proliferative and remodelling phases may be abnormal or prolonged .
this retrospective analysis of patients with rheumatoid arthritis presenting for metacarpophalangeal joint ( mcpj ) replacement surgery looks at the rate of postoperative complications affecting 140 wounds in 35 hands when the majority of medications were continued .
all have undergone swanson 's metacarpophalangeal joint ( mcpj ) arthroplasties under the care of one surgeon at the pulvertaft hand centre .
the database is part of a multicentre uk / usa prospective trial comparing medical and surgical management of mcpj disease .
this paper is restricted to the united kingdom arm of the study and includes assessment of the database and medical notes for the group that went forward to surgery .
drugs were grouped as nonsteroidal anti - inflammatory drugs ( nsaids ) , biological agents , noncytotoxic or cytotoxic disease modifying antirheumatic drugs ( dmards ) , steroids , or simple analgesics ( see table 1 ) .
a standard operative technique was used under tourniquet control , with access via four dorsal longitudinal incisions to replace four mcp joints in each hand .
the only medication that was routinely altered in the perioperative period was etanercept , an injectable biological anti - tnf agent .
this was stopped 2 or 3 weeks preoperatively and restarted once wounds were healed ( usually at 2 weeks ) .
routine postoperative care included dressing changes and application of dynamic splints with radial bias at the 5th postoperative day ( for daytime use ) with a static night splint to control extension lag and ulnar drift .
sutures were removed at 2 weeks , and patients remained under the care of the hand therapists thereafter unless problems arose .
patients were reviewed by the doctor again at six months , one , two and three years . at each visit ,
twenty eight patients were included in the study , representing 35 hands and 140 wounds in total .
twenty five patients were females ( mean age 63 years , range 5076 years ) , and three cases were males ( mean age 50 years , range 4455 years ) .
two patients were regular cigarette smokers ( 10 per day and 20 per day ) , and none were being treated for diabetes .
all wounds were reviewed at 2 weeks , and sutures were removed at this stage in 34 of 35 cases .
there were no serious complications ; however , four patients encountered minor problems in the postoperative period .
a 51-year - old female , who was taking a cytotoxic dmard ( methotrexate ) and codydramol and smoked 20 cigarettes per day , had slightly delayed wound healing .
half the sutures were left for a total of 3 weeks as the wounds were not considered clinically ready for suture removal in one area at 2 weeks .
a 63-year - old female , who was taking a cytotoxic dmard ( methotrexate ) , an nsaid ( diclofenac ) , and a noncytotoxic dmard ( gold injections ) , developed clinical signs of an early superficial wound infection within one week of surgery .
after the first operation , when she was taking a cytotoxic dmard ( azathioprine ) , a steroid ( prednisolone ) , and high - dose aspirin ( 300 mg ) , she developed a red painful area around the replaced 5th mcp joint 4 weeks after surgery .
ten days after the second operation , when she was still taking the cytotoxic dmard and steroid at the same doses , but a lower dose of aspirin , she developed erythema and pain around her wounds and was admitted for 48 hours of rest , elevation and intravenous flucloxacillin .
senior clinicians ' opinions at the time were that these were episodes of rheumatoid flare rather than infective processes .
none of her antirheumatoid medications were altered perioperatively . during long - term followup , a 52-year - old lady who was taking an nsaid ( naproxen ) and a biological agent ( etanercept ) developed a suture granuloma at 5 months which required surgical excision under local anaesthesia .
current uk guidelines generally make only brief reference to the perioperative management of antirheumatoid medical therapy .
the issue is not mentioned at all in the current american college of rheumatology recommendations on ra medications .
the traditional stance had been that cytotoxic medications should be stopped a few weeks before surgery and should not be recommenced until wounds had healed .
however , in recent years there has been a trend towards continuing medications to reduce the risk of a flare of ra .
whilst there are good data to support the continuation of methotrexate , data for other dmards and the biological agents has been sparse [ 3 , 7 ] .
there are many ways in which wound healing may be affected by the medications used in ra .
systemic corticosteroids affect gene expression and thereby may blunt the inflammatory phase of wound healing , reduce the rate of reepithelialisation , and alter remodelling of a wound .
however , the doses used in ra are usually low ( 2.55 mg daily in our group ) , and at these doses there has been no evidence of increased complications following joint replacement .
in addition , the risk of hypothalamic - pituitary - adrenal axis suppression with higher steroid doses precludes stopping these medications suddenly , and additional supplementation is usually recommended for major surgery .
methotrexate particularly affects macrophages and t lymphocytes and was shown in animal studies to reduce early wound tensile strength , but at the lower doses used in ra , and in combination with folate supplementation , there has been no clinical evidence over the years to support a detrimental effect on wound healing .
the effects of the cytotoxic dmards leflunomide and azathioprine on wound healing are not clear , but limited animal and clinical data show no clear adverse perioperative effects .
likewise , the data on the noncytotoxic dmards sulfasalazine and hydroxychloroquine are limited , but most authors regard the latter as minimally toxic and recommend its continuation [ 3 , 4 ] . the data assessing anti - tnf agents and wound healing are conflicting and comprise a mixture of small retrospective and prospective studies in ra and a larger study in crohn 's disease .
for example , bibbo and goldberg prospectively assessed 31 patients with ra who underwent a variety of elective foot and ankle procedures .
the groups were split into those receiving an anti - tnf agent and those who were not , with all patients continuing all regular medications . despite the anti - tnf group having a six times higher rate of smoking
den broeder et al . conducted a larger cohort study assessing risk factors for infection rates following 1209 varied elective orthopaedic procedures in patients with ra .
there were apparently more wound dehiscences ( 9.8% ) in those continuing an anti - tnf agent compared to those who were not taking one ( 4.4% ) .
although the differences in infection rates were not statistically significant between those who continued anti - tnf therapy ( 8.7% ) , those who stopped it ( 5.8% ) , and those who were never taking an anti - tnf agent ( 4% ) , it is important to note that only a small proportion of procedures were actually undertaken on patients taking these medications ( 196/1219 ) . a recent systematic review assessing
the risk of orthopaedic surgical site infection in patients with ra taking anti - tnf agents concluded that there was insufficient data internationally to reach any conclusion .
the topic of anti - tnf agents is reviewed in detail by pieringer et al . and
is also considered in the guidelines discussed below . in this study , we continued all medications except the anti - tnf agents .
we observed a low perioperative complication rate , with only one episode of minor wound infection ( 1/35 hands ) , one episode of minor delay in wound healing ( 1/35 hands ) , and one patient ( whose medications were not altered ) probably affected by flares of ra activity ( 2/35 hands ) .
there was only one late wound complication of a suture granuloma ( 1/140 wounds ) .
the recent british society for rheumatology dmard therapy guidelines suggest that methotrexate is unlikely to increase the risk of infection or surgical complications if continued .
this is predominantly influenced by one large prospective randomised controlled trial of methotrexate use in 388 rheumatoid patients undergoing elective orthopaedic procedures , which showed no evidence of increased complications with perioperative continuation of methotrexate .
further data analysis suggested increased infection and complication rates with other antirheumatoid medications ; however , these subgroups were smaller , and the trial was designed to assess methotrexate . the 2005 british society for rheumatology ( bsr ) anti - tnf ( tumour necrosis factor ) guidelines base perioperative advice upon drug company recommendations , stating that treatment with infliximab , etanercept , and adalimumab should be withheld for 2 to 4 weeks prior to major surgical procedures .
they recommend restarting treatment when wound healing is satisfactory , and there are no signs of infection .
this was supported by the british society for rheumatology biologics register data which showed a four - fold increase in the rate of skin and soft tissue infections with the use of biological agents in a cohort of 8973 ra patients observed prospectively .
the current bsr anti - tnf guidelines are essentially no different , except that more specific recommendations are made regarding the timing of stopping the different anti - tnf agents before surgery , based upon pharmacological data and clinical expertise .
this retrospective study of 140 wounds supports a policy of continuing all medications used for rheumatoid arthritis , except the injectable biological agents , in the perioperative period .
although the numbers are small , they add to the growing body of literature regarding the use of these medications .
there is unlikely to be any detrimental effect on wound healing in patients undergoing joint replacement in the hand , provided that constant vigilance in the postoperative period is maintained . | introduction . medications used to treat rheumatoid arthritis , such as corticosteroids , disease - modifying agents ( dmards ) , and injectable biological agents ( anti - tnf ) , may have widespread effects on wound healing . in hand surgery , it is important to balance the risks of poor wound healing from continuing a medication against the risks of a flare of rheumatoid arthritis if a drug is temporarily discontinued .
materials and methods . a united kingdom ( uk )
group of 28 patients had metacarpophalangeal joint replacement surgery in 35 hands ( 140 wounds ) .
all medication for rheumatoid arthritis was continued perioperatively , except for the injectable biological agents .
results .
there were no instances of wound dehiscence or deep infection and only one episode of minor superficial infection .
conclusions .
we conclude that provided care is taken to identify and treat any problems promptly , it is appropriate to continue most antirheumatoid medications in the perioperative period during hand surgery to reduce the risk of destabilising the patients ' overall rheumatoid disease control . |
department of health and human services has published statistics presenting total health spending during each year .
the basic aim of these statistics , termed nhe , is to identify all goods and services that can be characterized as relating to health care in the nation , and determine the amount of money used for the purchase of these goods and services ( rice , cooper , and gibson , 1982 ) . in this article
, we present nhe through an accounting structure to describe historical trends in the size , growth , and distribution of health care by type of service , such as hospital care and physician services , matched against the sources that pay the health care bill , such as private health insurance ( phi ) and government programs like medicare and medicaid .
this matrix provides policymakers , researchers , and the public with a system for understanding movement of the aggregate health economy and can serve as a data base from which hypotheses about the causal factors at work can be generated . in the charts that follow ,
we present detailed information on our nation 's health spending , focusing on calendar year ( cy ) 1997 .
the charts are organized into three primary areas : an aggregate overview , the major sources of private and public funding , and the key service areas where our nation 's health dollars were spent .
included in the overview are estimates of total aggregate health spending , health expenditures as a share of gdp , the distribution of health spending by primary funding sources and services , and the basic economic factors accounting for the growth in health spending . the overview is followed by more detailed statistics on each of the primary funding sources including phi , medicare , medicaid , and individual consumer health spending , as well as each of the primary service areas , including hospitals , physicians , prescription drugs , nursing homes , and home health services .
tables 4 - 13 following the charts provide detailed nhe information for selected cys by type of service and funding source .
over the past several years , health care expenditures have shown remarkable moderation in growth . continued excess capacity in hospitals , efforts by employers to hold down cost increases , and the downward impact of bba on medicare costs over the next 5 years should moderate future health care expenditure growth . however , beneath the recent calm waters are potential pressures for increased spending .
private insurers are seeing growth in benefit payments that exceed premiums earned and increasing demands by stockholders to improve eroding operating margins .
calls for increased quality and choice continue to be debated in state legislatures and in congress , with the potential for accelerating the growth in benefit costs if enacted . at the same time ,
consolidation in the insurer and provider marketplaces could dampen the intense competitive pressures that have helped to keep the lid on premium price increases in recent years .
additional pressures will likely come in the form of increased consumer demand ( smith et al . ,
recent strong economic growth has boosted employment and wages while prompting employers to offer health insurance to attract a qualified workforce . with rising incomes , the demand for new technologies and treatments will likely increase , including a continued upward trend in the fastest growing nhe category , prescription drugs .
although few expect a return to the double - digit growth of the past , significant forces for accelerated growth in future aggregate health spending are emerging . | in 1997 health spending in the united states increased just 4.8 percent to $ 1.1 trillion . as a share of gross domestic product ( gdp ) , national health expenditures ( nhe ) absorbed 13.5 percent of the country 's output in 1997a share that has remained relatively constant for 5 years .
despite the relative stability in recent years , signs of changing trends are emerging . |
acute gastroenteritis ( age ) is one of the most common diseases in children , and the second leading cause of morbidity and mortality worldwide . in the united states ,
current recommendations for the treatment of acute gastroenteritis focus primarily on the correction of dehydration and electrolyte abnormalities .
oral rehydration is safe and effective and is the preferred therapy in mild to moderate dehydration , whereas intravenous fluids are recommended in more severe cases .
vomiting is a common symptom in children with gastroenteritis , but its treatment remains controversial . in the clinical practice
a recent retrospective survey retrieved data from 4 national and international databases showed that prescription of antiemetic medication varied considerably .
antiemetics are commonly prescribed by physicians , in particular , between 2% and 23% of children with gastroenteritis received prescriptions for antiemetic medications and many physicians believe that vomiting is a contraindication to oral rehydration therapy ( ort ) .
multiple medications have been used in an attempt to limit vomiting and facilitate oral rehydration but have not gained acceptance , because of their limited success and high rates of complicating adverse effects .
according to previous studies administering promethazine , prochlorperazine , and metoclopramide can cause extrapyramidal reactions , lethargy , respiratory depression , and cardiac dysrhythmias .
ondansetron is a 5-hydroxytryptamine subtype 3 ( 5-ht3 ) receptor antagonist which has been documented to be an effective anti - emetic in preventing and treating postoperative nausea and vomiting ( pnov ) with few side effects . orally disintegrating ondansetron has been shown to be effective in preventing chemotherapy induced nausea and vomiting .
although it has shown great effectiveness in reducing vomiting when compared with other antiemetic drugs or placebo , it causes an increased incidence of diarrhea .
literature documenting ondansetron usage in gastroenteritis is limited and most studies focus on its application as an intravenous medication .
the aim of this study was to determine the effect of oral ondansetron in decreasing the vomiting due to acute gastroenteritis in children .
our study was double - blind , randomized , placebo - controlled clinical trial to compare the effect of a single dose oral ondansetron and placebo in decreasing vomiting due to acute gastroenteritis in children .
we attempted to determine whether the use of oral ondansetron would facilitate ort tolerance in dehydrated vomiting children with gastroenteritis and decrease the need for intravenous rehydration therapy .
this investigation was conducted in the emergency department of children 's hospital in rasht , iran between september 1 , 2010 and march 1 , 2011 .
we hypothesized that patients receiving ondansetron would tolerate ort by oral rehydration solution ( ors ) better than patients receiving placebo and lower proportion of them would require intravenous hydration therapy .
we enrolled a sample of patients with the clinical diagnosis of acute gastroenteritis and dehydration .
specific inclusion criteria were : age between 1 and 10 years , clinical presentation of simple acute gastroenteritis , dehydration , onset of disease in preceding 24 hours prior to the study , at least one episode of vomiting in previous 6 hours , no fever or low grade fever ( less than 38.2 c ) .
exclusion criteria included : any antiemetic drug usage in last 24 hours , any chronic disease , and alarming signs such as headache , abdominal distention , severe dehydration or shock , severe diarrhea more than one episode in one hour , any previous unfavorable response to 5ht3 receptor inhibitor drugs and any other situation in which ort is not recommended .
after enrollment demographic features and clinical data like patient 's name , age , sex , weight , height and telephone number were recorded .
we used the world health organization protocol ( the treatment of diarrhea , a manual for physicians and other senior health workers ) to classify severity of dehydration and treat patients with ors which was one of the main differences in our study because of extent of application in many developing and developed countries , our local policy and its feasibility for outpatient setup ( table 1 ) .
approximate amount of oral rehydration therapy to give in the first 4 hours the patients were randomized in two groups by computer randomization using a block of two , to receive ondansetron or placebo .
the oral ondansetron and the placebo tablets were 4 mg tablets provided by tehran chemie pharmaceutical company and dissolved in 2 cc water .
drug dosing was weight based in which children less than 15 kg received 2 mg ( tablets ) , children between 15 to 30 kg received 4 mg ( 1 tablet ) and more than 30 kg received 6 mg ( 1.5 tablets ) .
enrollment and outcomes thirty minutes after the medication was given , ort was initiated and continued based on the who recommendations on ort . if they tolerated the ors and vomiting did not occur during oral rehydration therapy , treatment was considered successful , but if vomiting occurred and continued , treatment was considered unsuccessful and patient was hospitalized and underwent treatment with intravenous fluids based on hospital protocol . again after 4 hours , the physician evaluated those who tolerated oral rehydration therapy and discharged them based on who guidelines .
after 48 hours , investigator called the childs family and asked whether the child had any vomiting episode , was brought to an emergency department , had received intravenous fluid treatment or had any additional symptoms .
our primary outcome was the proportion of subjects receiving ondansetron versus placebo who had vomiting episodes during 4 hours of ort initiation and also 48 hours after discharge .
we hypothesized that because of stopped vomiting , subjects receiving ondansetron would tolerate ort better than subjects receiving placebo and then lower proportion of them would require intravenous rehydration therapy in 4 and 48 hours .
comparative analysis entailed calculating the relative risk plus 95% confidence interval for dichotomous outcomes , the mean difference ( plus 95% confidence interval ) for normally distributed continuous outcomes , and the median difference plus 95% confidence interval ) for skewed continuous variables . to account the number of comparisons 99% confidence intervals were used for subgroup analyses .
during the study period , 202 patients aged 1 to 10 years were approached for study enrollment and 176 eligible patients were randomly assigned to two treatment groups .
1 ) . eleven cases of 176 patients did not contribute to 48-hour follow up .
therefore , 165 patients were included in data analysis as an intention - to - treat analysis .
of the 176 patients , 58.5% were males and 11 cases of 176 patients who did not contribute 41.5% were females with a mean age of 3.12 years ( 2.30 ) ( table 2 ) .
demographic variables of participants in ondansetron and placebo groups
table 3 shows primary outcome of the participants in this study in ondansetron and placebo . according to primary outcome , 10 patients in ondansetron and 14 in placebo group had persistent vomiting during ed stay .
twenty two and 31 patients in ondansetron and placebo groups experienced persistent vomiting during 48 hours follow up respectively .
failure of ort in ondansetron and placebo groups was respectively 13.6% and 28.9% , which showed significant relation in both groups ( p=0.04 ) .
furthermore , results showed significant relation between failure of re - hydration therapy during 6 hours after diarrhea and vomiting initiation in both groups ( p=0.03 ) and secondary outcomes in both groups showed no significant difference ( table 4 ) .
primary outcomes of the study of the study in the ondansetron and placebo groups secondary outcomes of the study in the ondansetron and placebo groups according to binary logistic regression test , sex , age , weight , first vomiting and diarrhea had no effect on failure of ort ( table 5 ) .
more ever in ondansetron and placebo group , no evidence of complications such as headache , vertigo , dermal rash were reported and there were no significant differences with regard to diarrhea and constipation between the two groups .
effect of confounding factors on failure of oral rehydration therapy by regression logistic ci : confidence interval ; se : standard error
ondansetron is a 5-ht3 receptor antagonist which has been documented to be an effective anti - emetic in preventing and treating pnov with few side effects . orally disintegrating ondansetron has been shown to be effective in preventing chemotherapy induced nausea and vomiting . in 1997 , cubeddu was the first to demonstrate the antiemetic effect of ondansetron in age .
although in developed communities children with severe dehydration are routinely admitted for intravenous therapy and ondansetron 's efficacy as an intravenous antiemetic is well documented , its effect as an oral antiemetic drug is controversial yet . in our study results showed that ondansetron decreases failure in ort that was similar to freedman et al results which showed that a single dose of ondansetron improves the success of oral rehydration in dehydrated children with gastroenteritis .
the oral dose was well tolerated and resulted in a reduction of more than 50 percent in both the proportion of children who vomited during oral rehydration and the proportion treated with intravenous fluids .
in addition , roslund demonstrated an improved success rate of ort and a decreased need for intra venous fluid in patients with age treated with a single dose of oral ondansetron .
furthermore , in this survey results showed no difference of vomiting episodes during ed stay and 48-hour follow up in the two groups and no significant reduction in hospital admissions in ondansetron group .
however , roslund et al and freedman et al demonstrated fewer vomiting episodes in the ondansetron group and both demonstrated a slightly lower admission rate in the ondansetron group , .
also , ramoosk found that patients receiving ondansetron had significantly fewer emetic episodes ( p=.001 ) and most of them did not vomit at all during the initial observation period in the emergency department ( 87% versus 65% , p=.004 ) ; on the other hand , a statistically significant difference was not found during a 48-hour follow - up period . in the ondansetron group , there were fewer admissions and shorter length of stay in hospital .
according to previous studies traditional antiemetics such as promethazine , prochlorperazine , and metoclopramide reveal limited success and high rates of complicating adverse effects such as extra pyramidal reactions , lethargy , respiratory depression , and cardiac dysrhythmias .
ondansetron was well tolerated in our study sample , and as mentioned , no evidence of complications was reported in ondansetron group . only diarrhea and constipation was reported in both groups which showed no significant difference between groups during the ed stay and in the 48 hours after ed discharge .
freedman et al reported a statistically significant increase in diarrhea during the ed stay in patients treated with ondansetron but did not evaluate the incidence of diarrhea during follow - up .
furthermore , ramsook et al did not detect a difference in diarrheal episodes during the ed stay but reported a statistically significant increase in diarrheal episodes in the 48 hours after ed discharge .
more diarrheal episodes were also reported in the 24 hours after drug administration in the ondansetron group by cubeddu et al .
neither roslund et al nor reeves et al detected a difference in diarrheal episodes among study groups during five to seven days after ed discharge .
according to results mentioned which revealed decreased failure of ort by administering oral ondansetron in gastroenteritis and no significant difference between groups regarding complications , and while ondansetron tablet is easy to administer , has few side effects , and is safe and effective , it can be a useful therapy in the emergency department for children with vomiting and mild - to - moderate dehydration as a result of gastroenteritis .
| objectivethe aim of this study was to determine the effect of oral ondansetron in decreasing the vomiting due to acute gastroenteritis in children.methodsin a single center , randomized , double blind , controlled trial , the effect of oral ondansetron was compared with placebo on 176 patients between 1 and 10 years old with acute gastroenteritis .
30 minutes after drug administration , oral rehydration therapy ( ort ) was initiated .
severity of vomiting was evaluated during emergency department ( ed ) stay and 48 hours follow up .
data were collected and analyzed by spss16.findingsfifty two of children ( 58.5% ) were males with the mean age of 3.12 ( 2.30 ) years .
ten patients in ondansetron and 14 in placebo group had persistent vomiting during ed stay . after analyzing , there was no significant relation between vomiting in 4 and 48 hours and need for intra venous fluid therapy between the two groups although ondansetron generally decreased ort failure ( p=0.03).conclusionalthough administrayion of oral ondansetron in gastroenteritis could decrease failure of ort , it seems that further well - conducted clinical studies are needed to determine effects of oral ondansetron precisely . |
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